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A simple method for the preparation of 32 P-labelled adenosine triphosphate of high specific activity
Abstract
A method is described for preparing /sup 32/P-labeled ATP of high
specific activity by an excbange reaction. The method is simple and requires
only substrates and enzymes that are available commercially. Hydrolysis of the
labeled ATP with heavy meromyosin indicates that 98 to 99% of the /sup 32/P is in
the 1 -phosphate group. (auth)
... The enzyme was incubated with Mg buffer for 1 h at 23°C. [γ-32 P]ATP was prepared as described previously (Glynn and Chappell, 1964). The specific activity of the [γ-32 P]ATP used was 10 6 -10 7 counts min -1 nmol -1 . ...
F1-ATPase is a universal multisubunit enzyme and the smallest-known motor that, fueled by the process of ATP hydrolysis, rotates in 120o steps. A central question is how the elementary chemical steps occurring in the three catalytic sites are coupled to the mechanical rotation. Here, we performed cold chase promotion experiments and measured the rates and extents of hydrolysis of preloaded bound ATP and promoter ATP bound in the catalytic sites. We found that rotation was caused by the electrostatic free energy change associated with the ATP cleavage reaction followed by Pi release. The combination of these two processes occurs sequentially in two different catalytic sites on the enzyme, thereby driving the two rotational sub-steps of the 120o rotation. The mechanistic implications of this finding are discussed based on the overall energy balance of the system. General principles of free energy transduction are formulated, and their important physical and biochemical consequences are analyzed. In particular, how exactly ATP performs useful external work in biomolecular systems is discussed. A molecular mechanism of steady-state, trisite ATP hydrolysis by F1-ATPase, consistent with physical laws and principles and the consolidated body of available biochemical information, is developed. Taken together with previous results, this mechanism essentially completes the coupling scheme. Discrete snapshots seen in high-resolution X-ray structures are assigned to specific intermediate stages in the 120o hydrolysis cycle, and reasons for the necessity of these conformations are readily understood. The major roles played by the “minor” subunits of ATP synthase in enabling physiological energy coupling and catalysis, first predicted by Nath's torsional mechanism of energy transduction and ATP synthesis 25 years ago, are now revealed with great clarity. The working of nine-stepped (bMF1, hMF1), six-stepped (TF1, EF1), and three-stepped (PdF1) F1 motors and of the α3β3γ subcomplex of F1 is explained by the same unified mechanism without invoking additional assumptions or postulating different mechanochemical coupling schemes. Some novel predictions of the unified theory on the mode of action of F1 inhibitors, such as sodium azide, of great pharmaceutical importance, and on more exotic artificial or hybrid/chimera F1 motors have been made and analyzed mathematically. The detailed ATP hydrolysis cycle for the enzyme as a whole is shown to provide a biochemical basis for a theory of “unisite” and steady-state multisite catalysis by F1-ATPase that had remained elusive for a very long time. The theory is supported by a probability-based calculation of enzyme species distributions and analysis of catalytic site occupancies by Mg-nucleotides and the activity of F1-ATPase. A new concept of energy coupling in ATP synthesis/hydrolysis based on fundamental ligand substitution chemistry has been advanced, which offers a deeper understanding, elucidates enzyme activation and catalysis in a better way, and provides a unified molecular explanation of elementary chemical events occurring at enzyme catalytic sites. As such, these developments take us beyond binding change mechanisms of ATP synthesis/hydrolysis proposed for oxidative phosphorylation and photophosphorylation in bioenergetics.
... Gill returned with an unlabeled sample of the azido-ATP, and we confirmed that it could compete with GDP and inhibit proton conductance in the dark. 30 Some years earlier, Glynn and Chappell had published a method to exchange high specific activity [ 32 P]phosphate into the γposition of ATP, 31 and we adapted this to label the azido-derivative to high specific activity. With the BAT mitochondria in a thin film on a petri dish, we added the 31 P-azido-ATP, irradiated with intense UV, prepared submitochondrial particles by sonication, solubilized them, ran the PAGE, dried the gel and exposed it to film. ...
Exactly 50 years ago, I was a post-doc in the laboratory of Olov Lindberg in Stockholm measuring fatty acid oxidation by mitochondria isolated from thermogenic brown adipose tissue, when we noticed a curious non-linearity in the respiration rate. This initiated a convoluted chain of experiments revealing that the mitochondria were text-book demonstrations of the then novel and highly controversial 'chemiosmotic hypothesis' of Peter Mitchell, and that thermogenesis was regulated by a proton short-circuit, mediated by a 32kDa 'uncoupling protein', UCP1, activated by fatty acid. This review is a personal account of the research into the bioenergetics of isolated brown adipocytes and isolated mitochondria, which led, after fifteen years of investigation, to what is still accepted as the 'canonical' UCP1-mediated mechanism of non-shivering thermogenesis, uniting whole animal physiology with mitochondrial bioenergetics.
... The reagents were purchased from Sigma Aldrich (St. Louis, MO, USA), Cripion Biotecnologia LTDA (Andradina, SP, Brazil), LGC Biotecnologia (Cotia, SP, Brazil), GE Healthcare Biosciences (Pittsburgh, PA, USA), Thermo Fisher (Waltham, MA, USA), Zymo Research (Irvine, CA, USA) and Santa Cruz Biotechnology (Dallas, TX, USA). Water used in preparation of all solutions was purified with a four-stage Milli-Q system (Millipore Corp., Bedford, MA, USA), [γ-32 P] ATP was prepared as described by Glynn and Chappell [23]. ...
Infections caused by Leishmania amazonensis are characterized by a persistent parasitemia due to the ability of the parasite to modulate the immune response of macrophages. It has been proposed that ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDases) could be able to suppress the host immune defense by reducing the ATP and ADP levels. The AMP generated from E-NTPDase activity can be subsequently hydrolyzed by ecto-nucleotidases, increasing the levels of adenosine, which can reduce the inflammatory response. In the present work, we provide new information about the role of E-NTPDases on infectivity and virulence of L. amazonensis. Our data demonstrate that not only the E-NTPDase activity is differentially regulated during the parasite development but also the expression of the genes ntpd1 and ntpd2. E-NTPDase activity increases significantly in axenic amastigotes and metacyclic promastigotes, both infective forms in mammalian host. A similar profile was found for mRNA levels of the ntpd1 and ntpd2 genes. Using parasites overexpressing the genes ntpd1 and ntpd2, we could demonstrate that L. amazonensis promastigotes overexpressing ntpd2 gene show a remarkable increase in their ability to interact with macrophages compared to controls. In addition, both ntpd1 and ntpd2-overexpressing parasites were more infective to macrophages than controls. The kinetics of lesion formation by transfected parasites were similar to controls until the second week. However, twenty days post-infection, mice infected with ntpd1 and ntpd2-overexpressing parasites presented significantly reduced lesions compared to controls. Interestingly, parasite load reached similar levels among the different experimental groups. Thus, our data show a non-linear relationship between higher E-NTPDase activity and lesion formation. Previous studies have correlated increased ecto-NTPDase activity with virulence and infectivity of Leishmania parasites. Based in our results, we are suggesting that the induced overexpression of E-NTPDases in L. amazonensis could increase extracellular adenosine levels, interfering with the balance of the immune response to promote the pathogen clearance and maintain the host protection.
... AT32P was synthesized by the method of Glynnand Chappell (9). ...
Yeast cells grown under conditions of glucose repression (5.4% glucose) exhibit a lower ATPase activity than cells grown on 0.8% glucose. The ATPase activity of the mitochondria increases during derepression, and this increase can be shown to be accompanied by an increase in the F1 (ATPase) content of the mitochondrial membranes. The increase of ATPase in the mitochondrial fraction during derepression is prevented by chloramphenicol. Under these conditions, however, there is an accumulation of soluble ATPase in the postribosomal supernatant. The soluble ATPase has been partially purified, and its properties indicate it to be identical to F1. Cycloheximide also prevents the increase of ATPase activity in the mitochondrial fraction during derepression. There is no accumulation of ATPase in the postribosomal fraction of cells incubated in a derepression medium containing cycloheximide. These results are interpreted to indicate that F1 is synthesized by the cytoplasmic-ribosomal protein-synthesizing system of the yeast cell.
... [y-32P]-ATP was synthesized from z2Pi by the method of Glynn and Chappell (9). Microsomes of Eledrophorus electricus electroplax were prepared as previously described (1) and stored in liquid nitrogen. ...
Electrophorus microsomal (Na⁺ + K⁺)-ATPase is inhibited by arsenite. The arsenite concentration which produces 50% inhibition is reduced from 6 mm to 0.1 mm by the addition of equimolar 2,3-dimercaptopropanol. An excess of this dithiol reverses the inhibition. Neither 2-mercaptoethanol nor dithiothreitol potentiate arsenite inhibition to a comparable extent.
The microsomes are phosphorylated by AT³²P: the extent of phosphorylation is Na⁺-dependent and reduced by K⁺. In the presence of equimolar arsenite and 2,3-dimercaptopropanol, the concentration of Na⁺ which produces half-maximal phosphorylation is decreased from 4 mm to 0.8 mm. K⁺ does not decrease the phosphorylation of the inhibited enzyme. The effects of British anti-Lewisite-arsenite on phosphorylation are accompanied by activation of Na⁺-dependent ATP-ADP transphosphorylation similar to that previously observed to attend the inhibition of (Na⁺ + K⁺)-ATPase by N-ethylmaleimide. These observations can be interpreted in terms of the existence of two states of the enzyme with different ligand affinities.
... When alkaline phosphatase was present in the DNA sample to be assayed, 3 3 This error probably resulted because the rotor temperature was higher than had been anticipated. 25,1967 J. W. Little 681 mM potassium phosphate, pH 7.0, was added to inhibit its action during kinase treatment. ...
λ exonuclease exhibits a much greater rate of attack on native deoxyribonucleic acid than on denatured DNA, the difference in rate being 350-fold for T7 phage DNA. The attack is exclusively exonucleolytic, and the only acid-soluble products are deoxyribonucleoside 5‘-monophosphates. In contrast to the known Escherichia coli exonucleases, its initial site of attack is at the 5‘ termini of a DNA molecule, with a strong preference for termini bearing a phosphoryl residue as compared with those bearing a free hydroxyl group.
Immune complex kinase assays in the simian virus 40 system were performed by incubation of immunoprecipitates containing tumor antigens with [gamma-32P]ATP, followed by analysis of any phosphoacceptor proteins. These assays yielded mainly the viral large T-antigen and, in particular, the associated cellular p53 as endogenous substrates. The nature of these substrates was confirmed by proteolysis techniques. Under specific conditions, casein could be used as an exogenous substrate as well. The kinase reactions showed preference for ATP and MgCl2 instead of GTP or MnCl2. Both phosphoserine and phosphothreonine, but in no case phosphotyrosine, were detected after an immune complex kinase reaction. Apparently, several in vivo phosphorylation sites were recognized in vitro in both large T-antigen and p53, but the presence of some artifactual sites could not be completely excluded. Although contaminating kinases were detectable in the immune complexes, at least the p53 molecules were phosphorylated in vitro in a more specific way. This followed from several characteristics of the immune complex kinase reactions and especially from the strong inhibition of p53 phosphorylation by two anti-large-T monoclonal antibodies. It was shown that large T-antigen showed associated kinase activity, although none of our results could unambiguously demonstrate an intrinsic kinase activity of this protein. Finally, anti-p53 monoclonal antibodies only slightly affected in vitro phosphorylation reactions, whereas a p53 molecule from a simian virus 40-free, chemically transformed human cell line was not phosphorylated in vitro under any condition tested. Thus, it is highly unlikely that the p53 molecule per se carries intrinsic or even associated kinase activities.
A procedure is described for a 450-fold purification of 5-phosphoribosyl α-1-pyrophosphate (PRPP) synthetase from cells of Salmonella typhimurium LT-2. A sedimentation coefficient (s20, w) of 17.7 was estimated for the enzyme by sucrose density gradient centrifugation. Some general properties of the enzyme are described.
PRPP synthetase has a specific and apparently absolute requirement for high levels of inorganic phosphate for PRPP synthesis. Saturation curves for phosphate are bimodal, which suggests multiple effects of the anion on the system. Phosphate appears to have a role in maintaining the structural integrity of the enzyme, since removal of phosphate by dialysis or dilution inactivates the enzyme.
The enzyme requires a divalent cation such as Mg⁺⁺ or Mn⁺⁺ for activity. Kinetic studies indicate that the active substrate is the Mg-ATP complex, and that the divalent cation also activates the system in some other way, possibly by activating the enzyme.
The PRPP synthetase reaction was demonstrated to be reversible. The equilibrium constant for the reaction in the direction of PRPP synthesis at pH 7.5 and 37° was determined to be 28.6, which corresponds to a standard free energy (ΔF‡) of −2.0 ± 0.5 kcal per mole for the reaction. The standard free energy of hydrolysis of PRPP to ribose 5-phosphate and inorganic pyrophosphate was estimated to be −7.0 ± 0.5 kcal per mole.
The Escherichia coli DNA polymerase, in the absence of DNA, contains a single binding site for which all the deoxyribonucleoside 5′-triphosphates complete. Dissociation constants of the enzyme-triphosphate complexes range from 1 × 10⁻⁵ to 2 × 10⁻⁴M.
Various analogues of the deoxyribonucleoside 5′-triphosphates also bind to the same site on DNA polymerase. The principal structural requirement for detectable binding at this site appears to be possession of a triphosphate group.
Binding of triphosphates is inhibited when the concentration of potassium phosphate is increased, and is inhibited, but to a lesser extent, by increasing potassium chloride concentration. In addition, binding is abolished in the absence of Mg⁺⁺ and in the presence of 9 mM EDTA. The strength of binding decreases with increasing temperature.
Binding measurements were made with equilibrium dialysis microcells, which require only 20 µl of protein solution and in which equilibrium with triphosphates is attained within 90 min at 22°.
A cyclic AMP-adenosine binding protein from mouse liver has been purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis in the absence and presence of sodium dodecyl sulfate and by analytical ultracentrifugation. The binding protein had a Stokes radium of 48 A based on gel chromatography. Both the purified binding protein and the binding activity in fresh cytosol sedimented as 9 S on sucrose gradient centrifugation. The homogeneous protein had a sedimentation coefficient (S20, w) of 8.8 x 10-13 s, as calculated from sedimentation velocity experiments. By use of the Stokes radius and S20, w', the molecular weight was calculated to be 180,000. The protein was composed of polypeptides having the same molecular weight of 45,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and thus appeared to consist of four subunits of equal size. The isoelectric point, pI = 5.7. The binding capacity for cyclic AMP increased by preincubating the receptor protein in the presence of Mg2+ ATP. This process, tentatively termed activation, was studied in some detail and was shown not be be be accompanied by dissociation, aggregation, or phosphorylation of the binding protein. Cyclic AMP was bound to the protein with an apparent dissociation constant (Kd) of 1.5 x 10-7 M. The binding of cyclic AMP was competitively inhibited by adenosine, AMP, ADP, and ATP whose inhibition constants were 8 x 10-7 M, 1.2X 10-6 M, 1.5 X 10-6 M, and higher than 5 x 10-6 M respectively. A hyperbolic Scatchard plot was obtained for the binding of adenosine to the activated binding protein, indicating more than one site for adenosine. The binding of adenosine to the site with the highest affinity (Kd=2 x 10-7 M) for this nucleoside was not suppressed by excess cyclic AMP and was thus different from the aforementioned cyclic AMP binding site. Cyclic GMP, GMP, guanosine, cyclic IMP, IMP, and inosine did not inhibit the binding of either cyclic AMP or adenosine. The binding protein had no cyclic AMP phosphodiesterase, adenosine deaminase, phosphofructokinase, or protein kinase activities, nor does it inhibit the catalytic subunit of the cyclic AMP-dependent protein kinase.
The 95,000-dalton polypeptide in human red blood cell membranes constitutes about 25% of the membrane protein. Previous labeling studies have shown that different regions of this polypeptide are exposed to the inside and outside of the cell and have suggested a role for the protein in anion exchange across the membrane. This polypeptide has been fragmented by chymotrypsin digestion of intact red cells and by treatment of purified polypeptide with 2-nitro-5-thiocyanobenzoic acid, hydroxylamine, and N-bromosuccinimide. The sites of cleavage by each of these reagents have been located relative to the NH-2 and COOH-terminals of the intact 95,000-dalton polypeptide. Polypeptide obtained from cells labeled with 1-isothiocyanate-4-benzene [35S]sulfonic acid (an inhibitor of anion transport), 125I and lactoperoxidase, or 32P has been similarly fragmented and these labels have been assigned to specific regions of the polypeptide. There are at least two sites of phosphorylation of the polypeptide; the major sites lies within 10,000 daltons of the NH2-terminal requiring that this portion of the polypeptide lie inside the cell. Sites of chymotrypsin cleavage and 125I and lactoperoxidase labeling are in a 7,000-dalton region toward the COOH-terminal of the polypeptide; this region must lie outside the cell. Between these two regions the polypeptide must traverse the lipid bilayer an odd number of times. 1-Isothiocyanate-4-benzenesulfonic acid also labels the protein near the site of chymotrypsin cleavage.
Phosphoglucomutase from rabbit skeletal muscle has been labeled with ³²P by incubation with 1-³²P-labeled 1,3-diphosphoglycerate. The product was not retained on columns of carboxymethyl cellulose and could thus be separated from nonphosphorylated species. Incubation with 1,3-diphosphoglycerate caused an increase in the proportion of phosphoenzyme as judged chromatographically. The evidence that the labeling occurred at the enzyme's active site is that (a) the incorporation was inhibited by glucose 1-phosphate; (b) the incorporated label was not removed by boiling in N acid but was labile to weak alkali; (c) incubation of the reisolated enzyme with glucose-1-P released the label as organic phosphate; and (d) in all studies, the labeled enzyme behaved the same as that labeled by exchange with glucose phosphates. Phosphoryl transfer from 1,3-diphosphoglycerate to phosphoglyceratemutase, phosphorylase b, or the phosphoenzyme form of phosphoglucomutase was not observed.
The K⁺ activation of the p-nitrophenylphosphatase activity of Electrophorus electric organ (Na⁺-K⁺)-ATPase is characterized by a synergistic activation by Na⁺. This phenomenon does not require the presence of nucleotides, and it is markedly enhanced by dimethylsulfoxide. Analysis of the activation data leads to the conclusion that K⁺ activation in the absence of Na⁺ involves two different sets of binding sites, one of which regulates substrate accessibility to the phosphatase site and one of which acts to increase its catalytic potential. Na⁺ can replace K⁺ in the first instance, but is only 3% as effective as a catalytic activator. Na⁺ appears to exert its regulatory action through a distinct set of Na⁺ specific sites.
The nucleoside triphosphate specificity of Escherichia coli succinic thiokinase (succinate:coenzyme A ligase (ADP), EC 6.2.1.5) was studied. The results showed that ATP, GTP and ITP (in decreasing order of effectiveness) were good substrates, while UTP and CTP were relatively ineffective. A nucleoside triphosphate ⇄ Pi exchange reaction stimulated by succinate and CoA was observed with each nucleoside triphosphate tested. A nucleoside diphosphate kinase activity of the enzyme was observed and found to be different from the ubiquitous nucleoside diphosphate kinase (ATP:nucleoside diphosphate phosphotransferase, EC 2.7.4.6) in its substrate specificity and its marked tendency to be stimulated by succinyl-CoA. Thus, with ATP as the phosphoryl group donor, GDP and IDP were good acceptors, UDP was a relatively poor acceptor, and CDP exhibited almost negligible activity in this respect. The nucleoside diphosphate kinase activity of succinic thiokinase was inhibited at ATP concentrations in excess of 0.5 mm. A comparison of the NDP-kinase to thiokinase activity ratio has revealed that the NDP-kinase activity does not represent a minor catalytic capability of the enzyme.
The DNA-joining reaction catalyzed by the Escherichia coli DNA ligase is markedly enhanced by low concentrations of monovalent cations, NH4⁺ being most effective. The Km for NH4⁺ is about 1 mm, and at saturating concentrations it increases the true Vmax by 20-fold. Under these conditions the Km for diphosphopyridine nucleotide is 7 µm, the Km for single strand breaks is 0.03 to 0.06 µm, and the turnover number is 25 sealing events per min.
The DNA-joining reaction obeys ping-pong kinetics, thus providing kinetic evidence for the participation of a covalent intermediate, presumably ligase-adenylate. The rate of the ligase-catalyzed diphosphopyridine nucleotide-nicotinamide mononucleotide exchange reaction is unaffected by NH4⁺, indicating that the activation occurs at a step subsequent to the formation of ligase-adenylate; furthermore, the exchange reaction is faster than the rate of DNA-joining, demonstrating that ligase-adenylate can be formed at a rate sufficient to be an intermediate in the over-all reaction. The rate of release of adenosine 5′-monophosphate from synthetic DNA-adenylate in the absence of NH4⁺ is greater than the rate of DNA-joining, as expected for a kinetically significant intermediate. However, in the presence of NH4⁺, the rate of adenosine 5′-monophosphate release is less than that of the over-all reaction. Since NH4⁺ markedly increases the apparent rate of dissociation of ligase-adenylate from DNA, the result may reflect reversal of the reaction to form ligaseadenylate from ligase and DNA-adenylate and dissociation of this form of the enzyme from the DNA.
1. Well coupled mitochondria isolated from six hepatomas differing widely in growth rate and degree of differentiation were found to exhibit little or no 2,4-dinitrophenol-stimulated ATPase activity in sharp contrast to normal liver mitochondria. Well coupled mitochondria from L1210 and Ehrlich ascites tumor cells were also found to have markedly reduced uncoupler-stimulated ATPase activity when compared with normal liver mitochondria.
2. When intact mitochondria from one of the hepatomas (7800) were studied in greater detail, it was found that normal uncoupler-dependent ATPase activity could not be demonstrated under a variety of conditions. These included (a) the use of uncouplers more potent than 2,4-dinitrophenol; (b) alteration of osmotic and ionic conditions; (c) variation of temperature between 20 and 40°; (d) variation of pH between 6.9 and 9.5; and (e) inclusion of either defatted bovine albumin or oxidizable substrate in the assay medium.
3. Neither forward nor reverse energy-dependent reactions in hepatoma 7800 mitochondria are altered, nor is their sensitivity to uncoupling agents. In the forward direction, hepatoma mitochondria were found to exhibit normal acceptor control and P:O ratios sensitive to uncouplers, and to exhibit uncoupler-stimulated respiration. In the reverse direction, the ATP-Pi exchange rate was found to be the same in hepatoma 7800 and control liver mitochondria and inhibited almost completely in both cases by 2,4-dinitrophenol. In addition, the capacity for ATP-supported Ca²⁺ accumulation was similar for hepatoma 7800 and control liver mitochondria.
4. Unlike hepatoma mitochondria, mitochondria isolated from regenerating liver 24, 48, and 72 hours after partial hepatectomy exhibit normal levels of uncoupler-stimulated ATPase activity.
5. These results are interpreted most simply by assuming that hepatoma mitochondria, and perhaps mitochondria from L1210 and Ehrlich ascites tumor cells, contain two principal sites of action for uncoupling agents, one of which may become expressed during the normal to neoplastic transition.
Inorganic phosphate, glucose 1-phosphate, divalent metal ions, EDTA, m-propoxybenzamidine, and AMP were found to be competitive inhibitors of the reaction catalyzed by phosphorylase phosphatase. By a comparison of kinetic studies with the substrates, phosphorylase a and the phosphorylated peptide, it was deduced that inorganic phosphate affected the reaction by binding to the catalyst, whereas glucose-1-P inhibited by binding to the substrate, phosphorylase. Inhibition by divalent metal ions and m-propoxybenzamidine was explained by binding to a single site on phosphorylase phosphatase. This site is presumed to be the one that interacts with the arginyl function of the substrate. EDTA inhibition appears to occur at a different locus on the phosphatase. AMP changes the phosphatase reaction by binding to phosphorylase a. The phosphorylase a-AMP complex is poorly recognized, if at all, by phosphorylase phosphatase.
Activation of K⁺-dependent p-nitrophenylphosphatase activity occurs concurrently with inhibition of Na⁺-K⁺-ATPase activity when either glycerol or dimethyl sulfoxide is added to the reaction medium. The effects on both activities are reversible. The Na⁺-dependent ADP-ATP phosphotransferase reaction and K⁺-dependent acetyl phosphatase activities are also inhibited by dimethyl sulfoxide. The effects of these solvents are consistent with a decreased interaction of phosphokinase and phosphatase sites with the phosphoryl acceptor site and an increased accessibility of p-nitrophenylphosphate to the phosphatase site.
The molecular weight of tryptophanyl-tRNA synthetase of Escherichia coli is 74,000 by Sephadex gel filtration and 81,000 by sucrose gradient centrifugation. Both sodium dodecyl sulfate polyacrylamide gel electrophoresis and agarose gel filtration in 8 m urea reveal subunits of 37,000 in molecular weight. In addition only one protein band is observed after polyacrylamide disc gel electrophoresis in the presence if 8 m urea. By these criteria the enzyme is a dimer composed of subunits identical in mass and charge.
The enzyme catalyzes synthesis of the unusual product, tryptophanyl adenosine triphosphate ester (tryptophanyl-ATP). In attempts to form complexes with tryptophan, ATP, and enzyme we recovered the enzyme in a complex with tryptophanyl-ATP, 1.7 to 1.8 moles of tryptophanyl-ATP per mole of active enzyme. A symmetric dimer model was supported by studies with 5,5′-dithiobis(2-nitrobenzoic acid). The reagent detected two sulfhydryl groups per mole of enzyme, and both thiols reacted with the same first order rate constant. The sulfhydryl groups were essential for enzymatic activity and were protected from 5,5′-dithiobis(2-nitrobenzoic acid) by tryptophan and ATP together but not individually.
A complex of tryptophanyl-tRNA-enzyme (0.9:1.0) is isolable by sucrose gradient centrifugation.
A suspension of intact guinea pig polymorphonuclear leukocytes hydrolyzed added ATP, AMP, and p-nitrophenyl phosphate under physiologically appropriate conditions. These enzymatic activities were not due to artifacts such as breakage of the cells during the incubation period. It thus seemed possible that the hydrolyses were being catalyzed by ecto-enzymes, i.e. enzymes on the plasma membrane with their active sites facing the external medium. Three types of experiment were designed to test this hypothesis.
First, the activities of intact cells were compared to those of homogenates, sonicates, and cells treated with detergent. Disruption of cells resulted in an approximately 2-fold increase in maximal ATPase and p-nitrophenyl phosphatase activities, suggesting that the plasma membrane was acting as a permeability barrier to the substrates involved. Disruption did not increase AMPase activity, leaving open the possibility that an ecto-enzyme is the only protein in polymorphonuclear leukocytes capable of hydrolyzing AMP.
Second, the products of ATPase, AMPase, and p-nitrophenyl phosphatase activities of intact cells were localized by using radioactively labeled substrates. The concentration of inorganic phosphate produced by these reactions was 18 to 100 times greater in the extracellular medium than in the intracellular milieu. This suggests that the substrates are cleaved outside the cells, or that they are cleaved inside and the products are transported out. The latter possibility was militated against by the following experiment. Cells were loaded with inorganic [³³P]phosphate, then allowed to hydrolyze substrates labeled with ³²P. The distributions of the two isotopes were compared. Almost all of the inorganic [³²P]phosphate was found outside of the cells, while 90% of the inorganic [³³P]phosphate remained inside.
Third, the cells were treated with the diazonium salt of sulfanilic acid, a reagent known not to penetrate into intact erythrocytes. This treatment rapidly and dramatically inhibited the intact-cell ATPase, AMPase, and p-nitrophenyl phosphatase, while lactate dehydrogenase, a soluble cytoplasmic enzyme, was unaffected. Control experiments demonstrated that in sonicates lactate dehydrogenase was as susceptible to inhibition by the diazonium salt as were the other three activities.
The major in vitro RNA transcripts synthesized by T7 RNA polymerase with T7 and T3 DNA templates have been resolved by electrophoresis on polyacrylamide gels. Six discrete size classes of T7 RNAs are found, designated I to VI. The apparent molecular weights, estimated from their electrophoretic mobilities, span a broad range, from 2 x 10⁵ to 5 x 10⁶. At least two minor RNA species, with molecular weights greater than 5 x 10⁶, are also detected.
The six major T7 RNA species are synthesized in approximately equimolar amounts, with the exception of species III, which is made in about twice this amount. It is likely that species III is a mixture of two RNAs (“IIIa” and “IIIb”) transcribed from separate regions of the T7 genome. Hence, the six major T7 RNA species are tentatively identified with seven late transcription units on the phage chromosome, which are read with equal efficiences.
The six (or seven) transcription products are initiated independently with guanosine triphosphate at the 5′ terminus, and are elongated at a rate of 230 nucleotides per s under standard in vitro conditions.
When T7 RNA polymerase is used to transcribe T3 DNA, a single RNA transcript is found, with a molecular weight similar to that of T7 RNA species III. This suggests an explanation for the reduced rate of T3 RNA synthesis by T7 RNA polymerase in vitro, and implies that T3 promoter sites read by the T3 RNA polymerase are heterogeneous in nature.
The phosphorylation of specific membrane proteins by an endogenous protein kinase has been studied in purified membrane fractions from rat adipocytes using trichloroacetic acid precipitation and sodium dodecyl sulfate polyacrylamide disc gel electrophoresis. The endogenous phosphorylation of two specific membrane proteins is completely dependent on the presence of cyclic adenosine 3′:5′-monophosphate (cyclic AMP) and magnesium ions. This phosphorylation occurs very rapidly at 24°, reaching maximal levels at 1 min. The number of sites specifically phosphorylated in the presence of cyclic AMP probably does not exceed 50,000 per fat cell, requiring the use of very high specific activity (10 to 50 Ci per mmole) [γ-³²P]ATP for these studies.
The minimal molecular weights of the two specifically phosphorylated proteins are about 22,000 and 16,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. The same two proteins are phosphorylated when intact fat cells are exposed briefly to low concentrations of exogenous ATP, a process which results in the suppression of the insulin-stimulated rates of d-glucose transport. At least 95 % of the cyclic AMP-dependent ³²P incorporated into trichloroacetic acid-precipitable protein is in the form of protein-bound phosphoserine. The concentration of cyclic AMP required for maximal stimulation of phosphorylation is about 1.5 µm. Cyclic guanosine 3′:5′-monophosphate has no significant effect unless its concentration is increased to 10⁻⁴m. Phosphorylation is inhibited by calcium ions. The cyclic AMP-stimulated phosphorylation is inhibited by phloretin and by 5′-adenylyl-β, γ-methylene triphosphonate.
The specific cyclic AMP-dependent membrane phosphorylation is also found in membranes from cells of obese rats, an insulin-resistant animal. This phosphorylation system, however, cannot be detected in membranes from guinea pig fat cells. The lack of phosphorylation of specific membrane proteins in guinea pig fat cell membranes is correlated with the insulin insensitivity of this tissue to glucose transport (but not lipolysis) and with the inability of ATP to inhibit the slight stimulation which insulin exerts on glucose transport.
The possibility is considered that this specific cyclic AMP-dependent phosphorylating system is involved in the hormonal regulation of glucose transport and the modulation of insulin sensitivity.
Purification of the phospho-protein phosphatase of skeletal muscle which promotes the conversion of the phospho- (D or b) form to the dephospho- (I or a) form of glycogen synthetase (UDPG:glycogen α-4-glucosyltransferase, EC 2.4.I.II) was aided by the use of buffers which include manganese chloride. The Mn²⁺ (5 mm) appeared to stabilize the enzyme and to insure greater recovery from DEAE-cellulose chromatography than reported previously. The enzyme was purified more than 1,000-fold from the 78,000 x g supernatant of rabbit muscle homogenate. It was essentially free of phosphorylase and synthetase. Phosphatase activity was measured by two methods: (a) as the rate of conversion of glycogen synthetase-D to the I form (D to I conversion) and (b) as the rate of orthophosphate release from ³²P-labeled synthetase-D (³²Pi release).
Inhibition of the phosphatase reaction (more than 50%) was found in the presence of F⁻ (10 mm), Na2SO3 (1 mm), and Pi or PPi (0.2 mm). More than a 2-fold increase in phosphatase activity was found in the presence of divalent metal cations: Mn²⁺ > Ca²⁺ > Mg²⁺ (half-maximal concentration was 0.6 to 1.2 mm).
Glucose-6-P (up to 1 mm) had no effect on the rate of ³²Pi release, but increased about 2-fold the rate of D to I conversion (at 0.1 mm), as did galactose-6-P and glucosamine-6-P to a lesser extent. A glycogen concentration of 0.5 to 1.5 mg per ml was found to be optimal compared to the slower reaction rates found with greater or lesser concentrations.
A histone phosphatase activity found in this preparation, measured using ³²P-labeled histone phosphate as substrate, had properties similar to and was copurified with glycogen synthetase-D phosphatase. However, the above carbohydrate-type effectors of synthetase and synthetase-D phosphatase reactions were without effect on the rate of histone dephosphorylation. These findings suggest that these two phosphatase activities may reside in the same enzyme protein.
Sarcoplasmic reticulum vesicles prepared from rabbit skeletal muscle catalyze a rapid Pi ⇄ HOH exchange in the presence of Mg²⁺ and absence of ATP and Ca²⁺. The capacity for oxygen exchange is about 14 times the potential capacity for ATP cleavage in the presence of Ca²⁺. No detectable exchange is found without added MgCl2. The exchange is unaffected by oligomycin, 2, 4-dinitrophenol, or ouabain, but strongly inhibited by low concentrations of Ca²⁺ in the medium. The Ca²⁺ ion concentration giving a half-maximum inhibition is 2.0 µm in the presence of 5 mm MgCl2. A Hill plot of the Ca²⁺ inhibition gives a straight line with a Hill coefficient of 1.8. The Ca²⁺ inhibition is competitively overcome by additional Mg²⁺. The Pi ⇄ HOH exchange is almost completely inhibited by the detergent Triton X-100 at low concentrations in which the ATPase activity is not disturbed. Sarcoplasmic reticulum vesicles are phosphorylated by Pi in the presence of Mg²⁺ and absence of Ca²⁺ under conditions similar to those for the Pi ⇄ HOH exchange. The phosphorylation requires Mg²⁺ and is strongly inhibited by low concentrations of Ca²⁺. The response of the phosphorylation to Ca²⁺ is quite similar to that of the Pi ⇄ HOH exchange; the Ca²⁺ ion concentration giving a half-maximum inhibition is 2.0 µm in the presence of 5 mm MgCl2, and the Hill coefficient is about 2.0. The various properties of the exchange give strong support to the probability that it results from reversal of steps in the over-all process associated with Ca²⁺ transport driven by ATP cleavage.
In order to investigate the effects of hormones on the adenosine 3′,5′-monophosphate (cAMP)-dependent protein kinase of adipose tissue, it was necessary to study the characteristics of the enzyme in crude tissue extracts. There was an apparent activation of the enzyme by NaF, but this effect was probably due to inhibition of adenosine triphosphatase activity. The activity of phosphodiesterase in the extracts was low under the conditions of the experiments.
Two procedures to determine the state of activation of the protein kinase in crude extracts are described. Both procedures are based on the amounts of inactive protein kinase (holoenzyme) and active catalytic subunit present. In the first procedure the activity ratio of the protein kinase is measured, i.e. the ratio of activity in the absence to activity in the presence of cAMP. At 0° in the presence of 0.5 m NaCl the protein kinase activity ratio was not affected by dilution of the extracts. Under these conditions, when cAMP was removed by gel filtration, the activity ratio decreased only very slowly but decreased rapidly in the absence of NaCl. The salt probably inhibits association of the regulatory and catalytic subunits of the protein kinase.
In the second procedure for determining the activation state of the protein kinase, crude adipose tissue extracts were chromatographed on Sephadex G-100 columns and the areas under the curves of the two peaks of enzyme activity corresponding to holoenzyme and catalytic subunit were estimated. Sodium chloride (0.5 m) at 0° inhibited the association of the protein kinase subunits during this procedure. Sephadex G-100 profiles of the protein kinase in crude extracts of heart, brain, and liver were also determined.
Sodium chloride (0.5 m) was also found to increase the activity ratio of the protein kinase in the presence of cAMP. At 0° this increase occurred relatively slowly and was accompanied by an increase in cAMP binding and a decrease in the cAMP requirement for enzyme activity.
The biochemical properties and template specificity of the T7 phage-specific DNA-dependent RNA polymerase have been studied. The enzyme shows the same absolute requirements for DNA, nucleoside triphosphates, and a divalent metal ion as are found for bacterial RNA polymerases, however, the conditions for optimal RNA synthesis are somewhat different. The T7 RNA polymerase initiates T7 RNA chains rapidly with GTP; each enzyme molecule can initiate several T7 RNA chains in the course of the reaction. Hence, an efficient termination mechanism is present in the in vitro system.
Of the helical DNAs tested, only T7 DNA, T3 DNA, and salmon sperm DNA have appreciable template activity with T7 RNA polymerase. A variety of single-stranded and denatured DNAs support RNA synthesis but at a reduced rate. Of the helical synthetic polynucleotides tested only d(I)n·d(C)n and d(G)n·d(C)n were active; both support the synthesis of poly[r(G)]. In contrast, both single-stranded poly[d(C)] and poly[d(T)] served as templates. It is suggested that T7 RNA polymerase requires a specific promoter site on DNA for effective transcription; this site is different from that used by bacterial RNA polymerase and may be rich in cytosine and thymidine residues.
The incorporation of ribonucleotides into DNA catalyzed by Escherichia coli DNA polymerase I in the presence of Mn⁺⁺ has been studied with two synthetic DNA's of defined sequence. In general agreement with the findings of Berg et al. ((1963) in Symposium on Informational Macromolecules, p. 467, Academic Press, New York), CMP and GMP could be incorporated at rates comparable to their deoxy analogs. AMP was incorporated only slowly and UMP was not incorporated at all. In studies of the fidelity of incorporation, misincorporation was observed at 37° in the presence of both GTP and CTP. The misincorporation was also observed at 10° in the presence of GTP but not in the presence of CTP. The sequence G-G was found to slow down subsequent nucleotide incorporation in the presence of GTP. The DNA's containing CMP or GMP were selectively cleaved by alkali or specific ribonucleases and expected products were thus obtained.
Phosphoenzyme and dephosphoenzyme from the purified (sodium + potassium)-activated adenosine triphosphatase from Squalus acanthias were reduced with sodium [³H]borohydride followed by acid hydrolysis and treatment with NaOH. Standard homoserine and α-amino-δ-hydroxyvaleric acid were added, and amino acid analysis was carried out in an amino acid analyzer. In a modified buffer, homoserine and α-amino-δ-hydroxyvaleric acid were clearly separated without any overlapping of any of the other amino acids. There was a strong radioactive peak coinciding with the homoserine peak. The radioactivity in the homoserine peak from the dephosphoenzyme was less than 5% of that from the phosphoenzyme. No radioactivity was associated with any other amino acid. The yield of radioactivity from the ³²P-labeled acyl phosphate recovered as radioactive homoserine ranged from 19 to 34%. In the complete amino acid analysis, the remainder of the tritium radioactivity was eluted with neutral material and in a small peak emerging at about 25 min. There was no difference in these peaks between dephospho- and phosphoenzyme. Since reduction of an aspartyl β-phosphate residue gives rise to homoserine and reduction of a glutamyl γ-phosphate residue gives rise to α-amino-δ-hydroxyvaleric acid, which can be recoverd after acid hydrolysis of the protein and alkaline treatment, we conclude that the acyl phosphate residue in the (sodium + potassium)-activated adenosine triphosphatase is an aspartyl β-phosphate residue. Under conditions of standard protein hydrolysis in acid for 36 hours, 13% of the α-amino-δ-hydroxyvaleric acid was quantitatively converted to proline. The remainder of the α-amino-δ-hydroxyvaleric acid was recovered unchanged.
Incubation of testis seminiferous tubules with follicle-stimulating hormone (FSH) results within 5 min in an activation of soluble adenosine 3′ : 5′-monophosphate (cAMP)-dependent protein kinase, as indicated by a conversion of the inactive holoenzyme to the active catalytic subunit. Maximal stimulation (3-fold) is achieved by 20 min. Moreover, this increased kinase activity can be directly correlated with increased intracellular accumulation of cAMP. Activation of protein kinase in isolated tubules is specific for FSH and is dependent upon time and temperature of incubation. This response to FSH is also dependent upon the age of the animal and disappears at approximately 30 days. However, sensitivity to gonadotrophin can be restored in adult animals by hypophysectomy or by addition of 1-methyl-3-isobutylxanthine to the incubation medium. These results suggest that the appearance of an active phosphodiesterase may be responsible for the decreased response to FSH during spermatogenesis. Increased protein kinase activity in response to the continued presence of FSH exhibits a half-life time of 2 to 4 hours. Furthermore, bound FSH can be recovered following treatment of the tissue of acid pH and this hormone retains the ability to activate protein kinase in fresh tissue suggesting that FSH may not be degraded while attached to testicular receptors. Excellent temporal correlation exists between binding of FSH and activation of protein kinase. Maximal enzyme activation occurs at a lower concentration of FSH than is necessary to saturate testicular binding sites.
The proteins of rat liver ribosomal subunits were phosphorylated by cyclic adenosine 3′:5′-monophosphate-activated protein kinases and the activity of the particles assessed. While we did not examine all of the possibilities, we did not find a ribosome function that was appreciably and consistently altered by phosphorylation. The synthesis of polyphenylalanine at high concentrations of magnesium (12.5 mm), which was dependent on elongation factors EF-1 and EF-2, was not changed if the ribosomes were phosphorylated. The synthesis of polyphenylalanine at low concentrations of magnesium (6 mm), which required the initiation factors EIF-1 and EIF-2 as well as elongation factors, was increased (18%) if the concentrations of phosphorylated ribosomes were limiting but not if initiation factors were limiting. The phosphorylation of the 40 S subunit increased only slightly (13%) the EIF-1-catalyzed binding of Phe-tRNA to the particle. Finally, there was no appreciable difference in the ability of phosphorylated and nonphosphorylated ribosomes to translate encephalomyocarditis virus RNA (which required all three initiation factors).
Glycogen synthetase was extensively purified from swine kidney by a procedure involving adsorption to calcium phosphate gel, ammonium sulfate fractionation, precipitation with ethanol, and chromatography on DEAE-cellulose. The enzyme was purified more than 10,000-fold to a final specific activity of 9.1 µmoles of glucose transferred from UDP-d-glucose to glycogen per min per mg of protein at 37°. Yields of 15 to 30% of the activity present in the crude extracts were consistently obtained by this isolation procedure. The purified enzyme was converted to an inactive form by washed kidney particulate preparations. When a step involving incubation in the presence of these particulate fractions, ATP, and magnesium ion was included in the purification procedure, only the form of glycogen synthetase dependent on the presence of glucose 6-phosphate for activity was isolated. The stability of the enzyme at all stages of purification was greatly increased in the presence of 0.3 m sucrose and 20 mm 2-mercaptoethanol.
The final preparation was essentially homogeneous as determined by polyacrylamide gel electrophoresis, elution profiles from DEAE-cellulose, Bio-Gel A1.5 and Sepharose 6B columns, and sucrose density gradient centrifugation. The purified preparation was free of protein kinases which convert glycogen synthetase to an inactive form. The enzyme was also completely free of phosphofructokinase, phosphorylase kinase, phosphorylase, and protein phosphatases when measured with phosphorylase a, phosphoglucomutase, casein, phosvitin, and glycogen synthetase labeled with ³²P as substrates.
The molecular weight of kidney glycogen synthetase calculated from data obtained by sucrose density centrifugation and chromatography on Sepharose 6B was 370,000. Analysis of the amino acid composition indicated a high ratio of acidic to basic residues, which may account in part for the tight binding of the enzyme to DEAE-cellulose columns. Sedimentation equilibrium analysis of the denatured enzyme in 4 m guanidine hydrochloride indicated that four polypeptide chains were present in the native enzyme. These results were confirmed by polyacrylamide gel electrophoresis in 0.1% sodium dodecyl sulfate which showed that the enzyme contained 4 subunits with molecular weights of 92,000 ± 3,000.
Some of the kinetic properties of the active and inactive forms of kidney glucogen synthetase were examined. Glucose-6-P stimulated the activity of the inactive form of the enzyme. There was no activity in the absence of glucose-6-P and the Km for UDP-glucose was 1.1 x 10⁻⁴m at 1 mm glucose-6-P and 6.7 x 10⁻⁵m at 11 mm glucose-6-P. The Km of the active form of the enzyme for UDP-glucose was 8.7 x 10⁻⁴m, and the activity was increased only slightly in the presence of glucose-6-P. The Km of the active form of the enzyme for UDP-glucose was about 15 times greater than that of the inactive form. The purified enzyme preparation was completely free of glycogen, and no activity was observed in the absence of this primer. Increasing concentrations of glycogen affected the activity of both forms of the enzyme in a similar manner.
A cyclic adenosine 3′ : 5′-monophosphate (cyclic AMP)-independent protein kinase was purified 116-fold from cellfree extracts of Neurospora crassa mycelium. The molecular weight of the enzyme as determined by gel filtration was 60,000. The sedimentation coefficient by sucrose density gradient centrifugation was 3.8 S. The enzyme has a broad pH optimum (7.0 to 8.5). Activity was maximal at an ionic strength of 0.15. The Eact was 10.7 Cal.
Kinetic studies have shown that the actual substrate for the reaction is the MgATP²⁻ complex. Free ATP is inhibitory. The Km for MgATP²⁻ at saturating casein is approximately 3 x 10⁻⁵m. The Km for casein at saturating Mg-ATP²⁻ is approximately 1.60 mg per ml. Phosvitin is the only other protein substrate tested that could replace casein. Co²⁺ and Mn²⁺ could partially substitute for Mg²⁺ in the reaction. Neither MgUTP²⁻ nor MgGTP²⁻ was a substrate. Both nucleotides were competitive inhibitors with respect to MgATP²⁻.
Reciprocal plots of 1/v versus 1/[MgATP²⁻] at different fixed concentrations of casein and 1/v versus 1/[casein] at different fixed concentrations of MgATP²⁻ were apparently parallel. However, product inhibition studies (MgADP⁻) and dead end inhibition studies (MgUTP²⁻, casein peptides) yielded results consistent with a random mechanism in which the binding of one substrate greatly decreases the affinity of the enzyme for the other substrate (Kma >> Kia, Kmb >> Kib, or in alternate terms, α >> 1).
Dihydroxyacetone phosphate was converted to a lipid by guinea pig liver mitochondria in the presence of fatty acids, coenzyme A (CoA), ATP, and Mg⁺⁺. The isolated product was identified as acyl dihydroxyacetone phosphate by its chemical and chromatographic properties. The structure of the product was confirmed by comparing the infrared spectra of synthetic and biosynthetic palmitoyl dihydroxyacetone phosphate. The requirement for fatty acid, CoA, and ATP could be replaced by acyl-CoA. When the rate of esterification was examined with different fatty acids, the highest rate was obtained with palmitic acid. At concentrations above 0.1 mM, unsaturated acids such as oleic and linoleic were incorporated at a much lower rate than were palmitic and stearic. Similar results were obtained when the rates of esterification with palmitoyl-, stearoyl-, and oleoyl-CoA were compared. Palmitoyl dihydroxyacetone phosphate was synthesized by mitochondria of brain, kidney, and heart, as well as liver. Biosynthesis of acyl dihydroxyacetone phosphate was also catalyzed by the liver microsomal fraction, but the selective incorporation of saturated fatty acids was not seen.
Aspartokinase, partially purified from extracts of Bacillus polymyxa, is inhibited by combinations of l-threonine and l-lysine at concentrations below 1 mm and by the amino acids separately at much higher concentrations.
At 25°, the presence of the feedback inhibitors leads only to a decrease in the maximal velocity of the aspartokinase reaction. However, at 37° the affinity for l-aspartate is also markedly reduced by the inhibitors. Sigmoid substrate kinetics are not observed under any conditions.
At 37°, but not at 25°, the apparent Km for l-aspartate is highly dependent on enzyme concentration, increasing from 0.4 mm to about 50 mm as the enzyme concentration is decreased from 13.4 to 0.17 units per ml. At 25°, the apparent Km for l-aspartate remains constant at about 1.5 mm over the same range of enzyme concentration. The presence of dioxane also leads to an increase in the apparent Km for l-aspartate, but at 25°, higher concentrations of the solvent are required to exert an effect than at 37°.
It is suggested that at 37°, but not at 25°, the active form of aspartokinase dissociates into lower molecular weight units which have a markedly lower affinity for l-aspartate than the native enzyme.
Hydrolysis of ³²P-labeled adenosine triphosphate by skeletal muscle microsomes occurs through a protein-bound phosphate intermediate. The steady state concentration of intermediate is influenced by the ionic milieu, temperature, and pH of the incubation medium. Treatment of microsomes with phospholipase C causes the inhibition of ATPase activity and Ca⁺⁺ transport, with an increase in the concentration of phosphorylated intermediate. Restoration of ATPase activity and Ca⁺⁺ transport with synthetic lecithin is accompanied by a decline of the phosphorylated intermediate concentration.
Hydroxylamine inhibits the ATPase activity, Ca⁺⁺ transport, and formation of phosphorylated intermediate in similar concentration. On the basis of its pH stability and sensitivity to hydroxylamine the phosphorylated intermediate is probably an acyl phosphate. A ³²P-labeled peptide was separated by high voltage electrophoresis from a pepsin digest of ³²P-labeled microsomes.
Ribonucleic acid polymerase directed by a native deoxyribonucleic acid template shows an initially rapid rate of ribonucleic acid synthesis which subsequently slows and finally establishes a plateau. It has been suggested that these kinetics result from the inhibition of RNA polymerase by the RNA formed during the reaction. The kinetics of the polymerase reaction under conditions where product RNA does not accumulate have been studied. By determining the release of ³²P-labeled pyrophosphate from γ-³²P-labeled ribonucleoside triphosphates, it is possible to assay RNA polymerase in the presence of pancreatic ribonuclease and ribonuclease T1. When the RNases are added to DNA-directed RNA polymerase reactions, there is a stimulation of ³²PPi release and the kinetics approach linearity. The addition of the nucleases to RNA polymerase reactions which had reached a plateau markedly stimulates RNA polymerase-dependent ³²PPi formation.
C-Formylglycinamide ribonucleotide (FGAR) and γ-³²P-ATP (or ¹⁴C-ATP) bind to FGAR amidotransferase to form a complex, which may be isolated by Sephadex gel filtration and characterized. The formation of this complex is dependent on the presence of three components, FGAR, ATP, and magnesium ions, but does not require glutamine. The binding sites for FGAR and ATP on the enzyme molecule may be distinguished from that of glutamine. The binding ratio of either FGAR or ATP to the enzyme expressed in molar quantity was approximately 0.7 under the conditions used at pH 6.5. The ratio of FGAR to ATP bound to the enzyme was unity. The analysis of the radioactive materials obtained from the complex of FGAR and γ-³²P-ATP with the enzyme indicated that the terminal phosphoanhydride bond of ATP was cleaved to ADP and a phosphate moiety, which remained bound to the enzyme presumably in ester linkage. Furthermore, when the enzyme was incubated with ¹⁴C-ATP in the presence of MgCl2, an amount of ¹⁴C-ADP nearly equivalent to the moles of enzyme present was obtained. FGAR did not affect this reaction. These results and others including observations on an ATP-ADP exchange reaction indicate strongly that the enzyme itself is phosphorylated in a step of the over-all reaction catalyzed by FGAR amidotransferase.
Azaserine and iodoacetate, both of which reacted at the glutamine-binding site, reduced and enhanced, respectively, the stability of the FGAR-ATP-enzyme complex. However, these compounds did not affect the true binding ratios of the substrates.
Although both compounds inhibited the reaction in which glutamine was the nitrogen donor, azaserine stimulated and iodoacetate partially inhibited the reaction with ammonium chloride as the nitrogen donor. A mechanism of the reaction catalyzed by FGAR amidotransferase has been discussed on the basis of the above information. The over-all reaction has been broken down into partial reactions, which, however, operate in a highly coordinated manner with one another.
1. DNA-dependent RNA polymerase has been isolated and purified from Saccharomyces cerevisiae. The yeast enzyme resembles the polymerase from Escherichia coli and other sources in its absolute requirements for a DNA primer, a divalent cation, and all four nucleoside triphosphates.
2. Yeast RNA polymerase differs fundamentally from other RNA polymerases in its marked preference for a denatured template. With a wide variety of DNA preparations, the extent of RNA synthesis is more than 10-fold greater with a denatured than with a native template.
3. In contrast to E. coli polymerase, RNA chains formed with yeast polymerase from denatured calf thymus and T2 DNA have long average chain lengths of 1500 to 2000 nucleotides.
4. The effects of yeast and E. coli polymerases on the retention of native DNA by nitrocellulose membranes, on the partitioning of DNA by polyethylene glycol-dextran, and on the rate of RNA synthesis from dAT copolymer at various temperatures have been compared. These studies suggest that the yeast enzyme has a decreased ability to cause localized denaturation of native DNA compared to the E. coli enzyme.
Binding of DNA and d(A-T) oligomers to DNA polymerase was studied by sucrose density gradient centrifugation. A single molecule of d(A-T) oligomer is bound by the enzyme, suggesting only one binding site for DNA. The binding is reversible, does not require Mg⁺⁺, and is inhibited by high concentrations of phosphate. Polymerase binds at multiple sites along single-stranded DNA, but binds to helical DNA only at nicks or at ends of molecules.
Microsomes prepared from electric organ of Electrophorus electricus or cat brain and exposed to ouabain will react with orthophosphate to form a phosphorylated protein. The electrophoretic mobilities of the phosphopeptides cleaved by peptic and Pronase digestion of the microsomes after interaction with phosphate are indistinguishable from those previously known to be formed from microsomes and ATP. The chemical stability of the bound phosphate is also similar to that of the phosphate formed from ATP. N-Ethylmaleimide treatment does not prevent ouabain binding, but does prevent the subsequent incorporation of orthophosphate.
The hydrolytic activity associated with homogeneous preparations of Escherichia coli DNA polymerase, previously designated exonuclease II, hydrolyzes polydeoxyribonucleotides from the 5′ as well as the 3′ terminus. 3′-Phosphate-terminated DNA is hydrolyzed only from the 5′ terminus. The principal products of exhaustive hydrolysis of 3′-phosphate-terminated DNA are 5′-mononucleotides and oligonucleotides terminating in a 3′-phosphate group; the latter are not hydrolyzed from either end of the chain. The rates of hydrolysis from either end of a DNA chain are similar with native DNA as substrate.
The ability of DNA polymerase to recognize and degrade from the 5′ terminus (5′ → 3′) as well as the 3′ terminus (3′ → 5′) of a DNA chain is considered in relation to possible models for DNA replication and to the active center of the enzyme.
Initiation and growth of RNA chains in the DNA-dependent RNA polymerase reaction have been studied by measuring the incorporation of γ-³²P-labeled nucleoside triphosphates and ¹⁴C- (or α-³²P) nucleoside triphosphates into RNA. The ratio of total nucleotide of RNA synthesized to total initiation is a measure of the average chain length of the RNA produced in the polymerase reaction. The length of RNA chains synthesized is independent of nucleoside triphosphate concentration and of enzyme concentration after extensive RNA synthesis, since both initiation and synthesis increase proportionally. However, the average RNA chain length is dependent on DNA template concentration. The rate of RNA synthesis increases more rapidly than RNA chain initiation as the DNA concentration is elevated. The specificity of initiation of chains with either ATP or GTP is independent of the above three parameters.
The nucleotide penultimate to the nucleoside triphosphate end has been determined with Escherichia coli and T4 DNA as templates. All four nucleotides are found in this position in a nonrandom distribution, although there is a preponderance of pyrimidine over purine nucleotides in this second position of a growing RNA chain.
Of a number of inhibitors that have been examined for their differential effects on initiation and chain growth, actinomycin D hardly affects initiation of RNA chains but markedly inhibits the synthesis of RNA, thus leading to a marked decrease in the average chain length of the RNA products.
With denatured DNA as template the average chain length is markedly decreased, since the number of initiations increases as much as 10-fold and synthesis is slower. With either denatured DNA or tobacco mosaic virus RNA as primer, initiation with GTP predominates over that with ATP.
From the known molecular weight of RNA polymerase, it can be calculated that approximately 1 RNA chain is formed by 1 molecule of enzyme. With denatured DNA, reinitiation can occur such that several chains are formed per molecule of enzyme.
Several nonpolar amino acids protect aspartokinase from inactivation by the cationic detergent trimethyloctadecylammonium chloride and by heat. The active agents are L-leucine, L-phenylalanine, L-methionine, L-tryptophan, L-alanine, glycine, L-valine, and L-isoleucine. D-Amino acids and other L-amino acids are inactive.
The same nonpolar L-amino acids also reverse the inhibition of aspartokinase caused by the feedback inhibitors, L-threonine and L-lysine. They differ in their specificity toward the feedback inhibitors. Amino acids of Group I (L-tryptophan, L-methionine, and L-norleucine) counteract the inhibition by L-lysine only, amino acids of Group II (L-leucine, L-valine, L-isoleucine, and L-norvaline) counteract the inhibition by L-threonine only, and those of Group III (L-phenylalanine, L-alanine, glycine, and allylglycine) counteract the inhibition by both L-lysine and L-threonine.
Cooperativity is not observed between amino acids belonging to different groups in overcoming the inhibition by combinations of the feedback inhibitors or in protecting the enzyme from inactivation by trimethyloctadecylammonium chloride.
Group I amino acids antagonize the ability of those of Groups II and III for overcoming the inhibition by L-threonine. Similarly, Group II amino acids counteract those of Groups I and III in overcoming the inhibition by L-lysine. Group I amino acids act cooperatively with L-threonine to inhibit aspartokinase, and those of Group II act cooperatively with L-lysine. There is no correlation between the ability of an amino acid to overcome feedback inhibition and its ability to antagonize the effects of amino acids belonging to other groups.
These results can be accounted for by a model that assumes a single binding site for all nonpolar L-amino acids and different patterns of nonexclusive binding of these amino acids to the site in four allosteric states of the enzyme.
Some biological implications of the effect of nonpolar L-amino acids on the feedback inhibition of aspartokinase are discussed.
The (sodium + potassium)-activated adenosine triphosphatase (NaK ATPase) in Lubrol extracts of NaI-treated bovine brain microsomes has been purified 30 to 50 times over that in the microsomes by salt and isoelectric precipitation, zonal centrifugation, and a new ammonium sulfate fractionation procedure. The final step in the purification procedure renders the NaK ATPase insoluble. The partially purified enzyme has been examined by polyacrylamide gel electrophoresis after solubilization in either phenol-acetic acid-urea or sodium dodecyl sulfate-mercaptoethanol. The former system is unsatisfactory in that a considerable proportion of the protein, including the NaK ATPase, remains at the origin; however, all of the protein penetrates the gel with the latter system. The purification procedure removes the majority of proteins seen in the microsomal fraction. With the partially purified enzyme, one prominent band, two less prominent bands, and two to three rather minor bands are seen with the sodium dodecyl sulfate system. The most prominent band has been identified as the phosphorylated subunit of the NaK ATPase by labeling its glutamyl-γ-phosphate residue with ³²P by prior incubation of the partially purified enzyme with ATP-γ-³²P in the presence of magnesium and sodium. Its apparent molecular weight is 94,000. Scanning of the protein-stained gel indicates that 25 to 40% of the protein applied to the gel can be accounted for by this 94,000 molecular weight subunit. The protein, phospholipid, and carbohydrate content of the preparation remain remarkably constant as purification proceeds, being approximately 50%, 25%, and 2 to 3%, respectively, of the dry weights of the preparations after correction for bound Lubrol. The protein-bound Lubrol at the final stage of purification is 17%. The cholesterol content falls with purification. Ninety-nine per cent of the ATPase in the partially purified enzyme is ouabain-sensitive. Ouabain-sensitive, potassium-activated p-nitrophenylphosphatase activity parallels NaK ATPase. All detectable cholinesterase in the microsomes is removed by purification. With the method of purification reported here, as much as 1.2 kg of bovine brain cortex can be worked up weekly giving 30 to 50 mg of final enzyme with a specific activity of 450 to 750 µmoles of ATP split per mg of protein per hour. Based on the gel scans and on calculations from the picomoles of phosphorylated subunit and molecular weight of the native enzyme, the best preparations are judged to be approximately half pure. The partially purified enzyme is quite stable on storage at 0°, retaining full activity for two months. Electron microscopy of the enzyme preparation after zonal centrifugation shows rods of about 60 A in diameter, spherical protein, and aggregated protein. The ammonium sulfate fraction shows chains of protein averaging 60 A in diameter, which are believed to be end-to-end aggregates of the rods seen in the zonal fraction. Many chains form ringlike structures. Sheets of aggregated protein are also seen. Resolubilization of the ammonium sulfate enzyme with Lubrol to protein ratios of 20:1 shows predominantly protein subunits averaging 60 A in diameter and some aggregates of subunits.
A Mg²⁺-dependent enzyme which will phosphorylate protamines and histones has been isolated and purified approximately 30-fold from rainbow trout testis. The enzyme transfers the terminal phosphoryl group from ATP (but not cytidine, guanosine, or uridine triphosphates) into O-phosphoseryl linkages in the acceptor molecule. The enzyme is dependent on the addition of thiol compounds for maximal activity and cyclic 3',5'-AMP stimulates both protamine and histone phosphorylation. At a high ionic strength (0.3 m NaCl), the rate of protamine phosphorylation is enhanced approximately 3-fold while histone phosphorylation is suppressed. KCl, ammonium chloride, and sodium acetate have the same effect as NaCl.
Mg²⁺ is essential for enzyme activity and can also satisfy the requirement of a high ionic strength for maximal activity.
In a low ionic strength medium the rate of protamine phosphorylation is slightly higher than that of unfractionated histones, but in the presence of 0.3 m NaCl the ratio of protamine to histone phosphorylation rises to 13.5. Of different histone fractions, the slightly lysine-rich fraction is most readily phosphorylated by the enzyme while both the lysine-rich and the arginine-rich fractions are poor substrates. Casein and free serine are phosphorylated to a small extent.
Enzymatically phosphorylated protamine can be separated from unphosphorylated protamine by starch gel electrophoresis.
IntroductionChemistry of Adenosine TriphosphateSynthesis of Adenosine Triphosphate in Biological SystemsConcluding Remarks
- J M Lowenstein
- R L Metzenberg
Lowenstein, J. M. & Metzenberg, R. L. (1960). Biochem.
Prep. 7, 5.
- E Racker
Racker, E. (1961). Advanc. Enzymol. 23, 323.
- H Mueller
- S V Perry
Mueller, H. & Perry, S. V. (1962). Biochem. J. 85, 431.