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Purification of Bovine Pregnancy-Associated Proteins by Two-Dimensional Gel Electrophoresis
Abstract
We purified and characterized a bovine pregnancy-associated protein in pregnant cow urine using two-dimensional gel electrophoresis. Urine from cows was collected according to their status of pregnancy and non-pregnancy. Proteins in the cow urine were fractionated with 50% ammonium sulfate prior to two-dimensional gel electrophoresis. Proteins separated on the gels were compared in terms of expression level and new expression by molecular mass and isoelectric point. We localized two pregnancy-associated protein spots on the gels at molecular masses of 24 kDa and 20 kDa and isoelectric points of 5.5 and 5.7, respectively. Likewise, two non-pregnancy specific proteins were localized at 27 kDa and 28 kDa with isoelectric points of 5.7 and 5.9, respectively. To rule out the possibility that environmental or genetic factors might influence the expression of the proteins, we demonstrated the pregnancy-associated expression of the proteins in two-dimensional gels with pregnant urine taken from cows raised in a different institute. The pregnancy-associated protein with molecular mass of 20 kDa and isoelectric point of 5.7, namely spot 2, was microsequenced and found to be highly homologous to the bovine collagen alpha 1 chain.
... This technique has also been used to detect any change in urinary protein in different human and animal disease conditions (Hormaeche et al, 2014;Lamoureux et al, 2013). The bovine pregnancy-associated protein (bPAP) isolated from pregnant bovine urines was characterized using two-dimensional electrophoresis (2-DE) (Hwang, 1999;Pyo et al, 2003). 2D DIGE has also been used to identify potential protein biomarkers for early detection of pregnancy in cow urine (Rawat et al, 2016). ...
Two-dimensional electrophoresis (2-DE) is an important method for studying urinary proteomics, playing an important role in assessing changes in protein concentrations under different physiological conditions. This study aimed to evaluate the different proteins expressed in the urine of normal cycling, healthy cows. We attained the images from 2-DE of urinary proteins in cow urine. Then, we implemented and evaluated an image analysis protocol with 2D ImageMaster software. Using ImageMaster, different spots were identified. On two-dimensional electrophoresis gels, 40 protein spots from anionic fractions and 15 spots from the cationic fractions of urine were identified. These spots corresponded to a molecular weight (MW) of 10-113 kDa for anionic fractions, and a MW of 15-51 kDa in cationic fractions. There were 12 master spot features across both the gels. This dataset can serve as a foundation for further investigations into the functional roles of specific proteins and their implications in the biological system under study. How to cite : Ambika Sharma, Ashish Kumar, Rajesh Nigam and Abhishek Pal (2024) Comprehensive analysis of protein separation using 2D electrophoresis : Insights into anionic and cationic fractions.
... Salam (2016) observed over-expression of 66 kDa protein band in pregnant buffalo as compared to nonpregnant buffalo. In a study on the urine of pregnant HF cows, proteins bands of molecular weight 20 kDa and 24 kDa were observed in the pregnant animals and 27 kDa and 28 kDa in non-pregnant animals (Hwang and Lim 1999). Pyo et al. (2003) isolated 21 kDa protein with 6.1 p.I. from cow urine and named it bovine pregnancy associated protein (bPAP). ...
Early pregnancy diagnosis is necessary to maintain reproductive efficiency in animals. To determine biomarker
for the early pregnancy diagnosis, total protein was estimated and SDS PAGE was performed on urine and serum in
selected 30 Sahiwal cows. Total serum protein concentration was increased in pregnant as compared to non-pregnant Sahiwal cows. A continuously increasing trend of protein was observed in pregnant group after 12 (6.36 ± 0.76 g/dl) to 22 (6.87± 0.39 g/dl) days post-breeding whereas non-specific trend was found in non-pregnant group. In urine, total protein concentration increased significantly in pregnant group as compared to non-pregnant group from 16 to 22 days. An increase in protein concentration from day 0 to 16 followed by decrease till day 22 was observed in both groups. Maximum and minimum protein concentration was observed on day 16 (31.6 ± 0.68 mg/dl) and day 0 (23.32 ± 0.81 mg/dl) respectively, in urine of pregnant group. Upon SDS PAGE analysis of serum, expression of specific proteins of molecular weight between 29 to 43 kDa with over expression of 66 kDa protein were observed in pregnant cows. It was observed that, proteins with molecular weight of 43 kDa and 66 kDa were over-expressed in pregnant cows as compared to non-pregnant cows. These findings suggest that pregnancy specific proteins ranging 29 to 43 kDa of pregnant Sahiwal cows can be used as pregnancy biomarker in near future
... Therefore, not all proteins in the experimental 'PSD fraction' are bona fide members of the PSD in vivo. Determination of the amino acid sequence is the most direct method to identify unknown proteins (Choi and Rhee, 1998;Moon et al., 1998;Oh and Lee, 1998;Hwang and Lim, 1999). In this study we determined the partial amino acid sequence of a 17 kDa protein (PSD-17), which constitutes ~2% of the 1% n-octyl glucoside (NOG)insoluble PSD fraction. ...
The postsynaptic density (PSD), a large protein complex beneath the postsynaptic membrane, is notorious for its 'stickiness'. In order to understand the molecular composition of the PSD fraction, a 17 kDa protein band was isolated by electroelution from SDS-gels, and its partial amino acid sequence was determined from HPLC-purified tryptic peptides of the protein. Surprisingly, the amino acid sequence was identical to that of the previously reported 17 kDa isoform of the myelin basic protein (MBP), an essential protein in CNS myelin formation. Since the protein band represented ~2% of the total proteins in the 1% n-octyl glucoside-insoluble PSD fraction, these results indicate that a significant amount of the 17 kDa isoform of MBP is tightly associated with the PSD during preparation of the PSD fraction.
... Protein concentrations were determined by a modified Bradford procedure (1976) with bovine serum albumin as a standard and the purity of the protein was analyzed as described by Hwang and Lim (1999). ...
The steady-state investigation of the mechanism of D-hydroxyisovalerate dehydrogenase was performed in order to understand this type of kinetic patterns. The initial velocity was measured with various amounts of both substrates, NADPH and 2-ketoisovalerate. Double reciprocal plots gave patterns that conversed on or near the abscissa. Binding studies indicated that NADPH bound first to the enzyme. The product NADP + was found to be a competitive inhibitor with respect to NADPH at a constant concentration of 2-ketoisovalerate. However, it showed non-competitive inhibition against 2-ketoisovalerate at a fixed amount of NADPH. Another product, D-hydroxy-isovalerate, was a non-competitive inhibitor versus NADPH and 2-ketoisovalerate at constant levels of 2-ketoisovalerate and NADPH, respectively. These results were comparable with an ordered bi-bi mechanism, in which NADPH bound first to the enzyme, followed by the binding of 2-ketoisovalerate. NADP + is the last product to be released. The ordered reaction manner of D-hydroxyisovalerate dehydrogenase from 2-ketoisovalerate to D-hydroxy-isovalerate allows the accurate regulation of valine metabolism and it may lead to the regulation of total biosynthesis of enniatins in the Fusarium species.
... In the human population, the food consumed, stress, physiological conditions, environment, genetic background, and psychological status vary from individual to individual. In contrast, in cases of lab animals or live stock animals, the above conditions are almost uniform so that their urine proteins show very similar 2D gel patterns [22,23] . The variations of MALDI-TOF-MS patterns of urinary proteins have made it difficult to localize new biomarkers. ...
Protein analysis in biological fluids, such as urine, by means of mass spectrometry (MS) still suffers for insufficient standardization in protocols for sample collection, storage and preparation. In this work, the influence of these variables on healthy donors human urine protein profiling performed by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was studied. A screening of various urine sample pre-treatment procedures and different sample deposition approaches on the MALDI target was performed. The influence of urine samples storage time and temperature on spectral profiles was evaluated by means of principal component analysis (PCA). The whole optimized procedure was eventually applied to the MALDI-TOF-MS analysis of human urine samples taken from prostate cancer patients. The best results in terms of detected ions number and abundance in the MS spectra were obtained by using home-made microcolumns packed with hydrophilic-lipophilic balance (HLB) resin as sample pre-treatment method; this procedure was also less expensive and suitable for high throughput analyses. Afterwards, the spin coating approach for sample deposition on the MALDI target plate was optimized, obtaining homogenous and reproducible spots. Then, PCA indicated that low storage temperatures of acidified and centrifuged samples, together with short handling time, allowed to obtain reproducible profiles without artifacts contribution due to experimental conditions. Finally, interesting differences were found by comparing the MALDI-TOF-MS protein profiles of pooled urine samples of healthy donors and prostate cancer patients. The results showed that analytical and pre-analytical variables are crucial for the success of urine analysis, to obtain meaningful and reproducible data, even if the intra-patient variability is very difficult to avoid. It has been proven how pooled urine samples can be an interesting way to make easier the comparison between healthy and pathological samples and to individuate possible differences in the protein expression between the two sets of samples.
A proteomic map for human urine on two-dimensional (2-D) gels has been developed. Initial studies demonstrated that the urine proteins prepared by conventional methods showed interference and poor reproducibility in 2-D electrophoresis (2-DE). To address this issue, urine samples were dialyzed to remove any interfering molecules. The dialysis of urine proteins and the concentration by lyophilization without fractionation significantly improved the reproducibility and resolution and likely represents the total urine proteins on a 2-D gel. In addition, removing albumin from urine using Affi-Gel Blue helped to identify the low-abundant proteins. Using the developed method, we prepared proteins from urine collected from healthy females and males. The large inter- and intra-subject variation in protein profiles on 2-D gels made it difficult to establish a normal human urine proteomic 2-D map. To resolve this problem, urinary proteins were prepared from the pooled urine collected from 20 healthy females and males, respectively. The established male and female urine proteomes separated on 2-D gels were almost identical except for some potential sex-dependent protein spots. We have annotated 113 different proteins on the 2-D gel by peptide mass fingerprinting (PMF). We propose that the established total urine proteome can be used for 2-DE analysis, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and identification of novel disease-specific biomarkers.
Bovine pregnancy-associated protein (bPAP) isolated from pregnant bovine urines by two-dimensional electrophoresis (2-DE) was characterized by N-terminal sequencing, internal sequencing, and mass spectrometric analyses using matrix-assisted laser desorption/ionization-time of flight mass spectrometry and tandem mass spectrometry. Database search using the amino acid sequences and the peptide mass profiles showed that the protein is a novel bovine pregnancy-associated protein of which the N-terminus has a high similarity to collagen alpha. The protein has a molecular mass of 21 kDa and a pI of 6.1. The expression profiles of the protein from the urine of 30 pregnant and 20 nonpregnant cows characterized by 2-DE indicated that the expression of bPAP during pregnancy increased to over nmol from the pmol level basal expression of bPAP at the nonpregnant state with < 3% of false negatives and < 10% of false positives. Using the peptide sequence information, polyclonal antibodies against the bPAP protein were generated. The purified polyclonal antibodies against the peptide sequences of bPAP detected the 21 kDa protein on the blots of pregnant cow urine by Western blot analysis. In addition, analysis showed that the expression of bPAP in the urine is associated with pregnancy, but that the urine concentration of bPAP is not correlated with the duration of the pregnancy.
Extracellular phospholipase activity has been detected in urine of patients with acute pyelonephritis (APN). This enzyme required micromolar ion for its maximum activity and showed a broad range of pH (4.5~10) optimum. Urine enzyme hydrolyzed phosphatidylethanolamine (PE) and phosphatidylserine (PS) more effectively than phosphatidylcholine (PC). activity in the urine of patients with APN was about 5-fold higher than that of healthy individuals. When urine was subjected to heparinSepharose column chromatography, phospholipase activity was detected in both heparin-non-binding and binding fractions. Both phospholipase activities were sensitive less than a micromolar calcium concentration and did not react with anti-human 14-kDa group II phospholipase monoclonal antibody, HP-l. These findings suggest that two kinds of novel extracellular phospholipase . which may not belong to the 14-kDa group II phospholipase family, exist in the urine of patients with APN.
One of the essential functions of virus surface proteins is the recognition of specific receptors on target cell membranes, and cellular receptors play an important role in viral pathogenesis. But the earliest steps of hepatitis B virus (HBV) infection, such as hepatocyte receptor interaction with the virus, are poorly understood. Previous work has suggested an important role of the preS1 region of HBV envelope protein in mediating viral binding to hepatocytes. Although hepatitis B virus (HBV) infection appears to be initiated by specific binding of virions to cell membrane structures via one or potentially several viral surface proteins, data showing the identification or isolation of the HBV receptor (s) are not yet available. The receptor-like proteins on the plasma membrane surface of HepG2 cells that bind to PreS1 were separated and identified using affinity chromatography, and the amino-terminal amino acid sequences of the receptor-like proteins were determined.
Thioltransferase, also known as glutaredoxin, was previously purified and characterized from Chinese cabbage (Brassica campestris ssp. napus var. pekinensis). However, in the process of gel filtration on Sephadex G-75, there were two activity peaks. In this study, a second thioltransferase (TTase CC-2) in the minor peak of the Sephadex G-75 elution profile was further purified using affinity chromatography on an S-hexylglutathione-agarose column by eluting with buffer solution containing 2.5 mM S-hexylglutathione. It showed a single band on SDS-PAGE indicating that TTase CC-2 is electrophoretically homogeneous. The molecular weight of TTase CC-2 was estimated to be about 22,000 daltons, and its isoelectric point was determined to be 6.73. Its size appears to be atypical and much larger than that of the first thioltransferase (TTase CC-1) from Chinese cabbage, and it can utilize 2-hydroxyethyl disulfide, S-sulfocysteine, and insulin as substrates. S-sulfocysteine was found to be a superior substrate for TTase CC-2. TTase CC-2 also displayed the reducing activity for non-disulfides such as dehydroascorbic acid. Its optimum pH was 8.5, which was consistent with that of TTase CC-1. TTase CC-2 activity was greatly activated by L-cysteine and reduced glutathione, and was found to be less heat-stable compared with TTase CC-1. Molecular and physiological differences between TTase CC-1 and TTase CC-2 remain to be elucidated. Chinese cabbage is the first plant which is known to contain two kinds of thioltransferases.
The amino acid sequence of 120 residues in the N-terminal region of the alpha1-chain of calf skin collagen (comprising the cyanogen-bromide-derived peptides alpha1-CB2, alpha1-CB4 and alpha1-CB5) has been determined by automated Edman degradation. The lysyl residue in position 87 is completely hydroxylated, while those in positions 99 and 108 partially hydroxylated. Two substitutions are found with respect to the homologous region of the alpha1-chain from rat skin collagen. Positions 101 and 102 of calf skin collagen are occupied by Asp-Ala, in rat skin collagen by Asn-Thr. The extensive homology in this region is remarkable and is not found in other regions of the alpha1 and alpha2-chain.
There have been many attempts to develop a test which can detect a pregnancy between the time of fertilization and the time of implantation. The subject of this review is Morton's "early pregnancy factor", a complex immunological phenomenon which is said to become positive within hours of fertilization. However, many workers have been unable to reproduce these findings and the experimental methods have been severely criticized. Substantial further work is needed before a test of this type can be regarded as suitable for routine clinical practice, if indeed it will ever be possible.
About 5% of rabbit antisera to calf or human collagen contained antibodies which reacted specifically with the N-terminal cyanogen bromide peptide α1-CB (0.1). A considerable cross-reaction was observed between the calf and human collagen peptide. The sequence of calf
Pro-Gly-Pro-Hse. An analogous sequence except for an inserted glycine between position 12 and 13 was suggested for the human peptide (cf. Click and Bornstein, 1970). Inhibition studies with protease-derived fragments localized the human collagen antigenic determinant on the N-terminal pentapeptide. The calf collagen antigenic determinant occupied a different and larger region. Their structural requirements were discussed in relation to the cross-reactions observed.