Heparin-sepharose affinity chromatography for purification of bull seminal-plasma hyaluronidase.
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ABSTRACT:
SUBMITTER: Srivastava PN
PROVIDER: S-EPMC1161633 | biostudies-other | 1979 Dec
REPOSITORIES: biostudies-other
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Project description:Metabolites play essential roles in biological systems, but detailed identities and significance of the seminal plasma metabolome related to bull fertility are still unknown. The objectives of this study were to determine the comprehensive metabolome of seminal plasma from Holstein bulls and to ascertain the potential of metabolites as biomarkers of bull fertility. The seminal plasma metabolome from 16 Holstein bulls with two fertility rates were determined by gas chromatography-mass spectrometry (GC-MS). Multivariate and univariate analyses of the data were performed, and the pathways associated with the seminal plasma metabolome were identified using bioinformatics approaches. Sixty-three metabolites were identified in the seminal plasma of all bulls. Fructose was the most abundant metabolite in the seminal fluid, followed for citric acid, lactic acid, urea and phosphoric acid. Androstenedione, 4-ketoglucose, D-xylofuranose, 2-oxoglutaric acid and erythronic acid represented the least predominant metabolites. Partial-Least Squares Discriminant Analysis (PLSDA) revealed a distinct separation between high and low fertility bulls. The metabolites with the greatest Variable Importance in Projection score (VIP > 2) were 2-oxoglutaric acid and fructose. Heat-map analysis, based on VIP score, and univariate analysis indicated that 2-oxoglutaric acid was less (P = 0.02); whereas fructose was greater (P = 0.02) in high fertility than in low fertility bulls. The current study is the first to describe the metabolome of bull seminal plasma using GC-MS and presented metabolites such as 2-oxoglutaric acid and fructose as potential biomarkers of bull fertility.
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Project description:Male fertility is the ability of sperm to fertilize the egg and sustain embryo development. Several factors determine the fertilizing capacity of mammalian sperm, including those intrinsic to sperm and components of the seminal plasma. The present study analyzed the seminal fluid proteome of Bos taurus and potential associations between proteins and fertility scores. Mass spectrometry coupled with nano HPLC allowed the identification of 1,159 proteins in the dairy bull seminal plasma. There were 50 and 29 seminal proteins more abundant in high (HF) low fertility (LF) bulls, respectively. Based on multivariate analysis, C-type natriuretic peptide, TIMP-2, BSP5 and sulfhydryl oxidase indicated relationship with HF bulls. Clusterin, tissue factor pathway inhibitor 2, galectin-3-binding protein and 5'-nucleotidase were associated with LF bulls. Abundance of NAD(P)(+)-arginine ADP-ribosyltransferase, prosaposin and transmembrane protein 2 proteins had the highest positive correlations with fertility ranking. Quantities of vitamin D-binding protein, nucleotide exchange factor SIL1 and galectin-3-binding protein showed the highest negative correlations with fertility ranking. A fertility ranking score was calculated and the relationship with these proteins was significant (Spearman's rho?=?0.94). The present findings represent a major and novel contribution to the study of bovine seminal proteins. Indicators of fertility can be used to improve reproductive biotechnologies.
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Project description:Extracellular vesicles (EVs) are lipid membrane vesicles released by cells. They carry active biomolecules including DNA, RNA, and protein which can be transferred to recipient cells. Isolation and purification of EVs from culture cell media and biofluids is still a major challenge. The most widely used isolation method is ultracentrifugation (UC) which requires expensive equipment and only partially purifies EVs. Previously we have shown that heparin blocks EV uptake in cells, supporting a direct EV-heparin interaction. Here we show that EVs can be purified from cell culture media and human plasma using ultrafiltration (UF) followed by heparin-affinity beads. UF/heparin-purified EVs from cell culture displayed the EV marker Alix, contained a diverse RNA profile, had lower levels of protein contamination, and were functional at binding to and uptake into cells. RNA yield was similar for EVs isolated by UC. We were able to detect mRNAs in plasma samples with comparable levels to UC samples. In conclusion, we have discovered a simple, scalable, and effective method to purify EVs taking advantage of their heparin affinity.
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Project description:The binding of bovine testicular hyaluronidase to AH-Sepharose (1,6-diaminohexane--Sepharose) gels substituted with (1) dermatan sulphate, (2) desulphated dermatan sulphate, (3) heparin and (4) de-N/O-sulphated, re-N-acetylated heparin was investigated. Hyaluronidase was found to bind to (1) and (3), but not (2) and (4). On the basis of these observations a preparative scheme for the purification of testicular hyaluronidase was developed. This consisted of two steps: (i) chromatography on dermatan sulphate-substituted AH-Sepharose 4B; (ii) chromatography on acetylated AH-Sepharose 4B. This procedure gave hyaluronidase with a specific activity of 19.1 units (mumol/min)/mg in high yield. Polyacrylamide-gel electrophoresis at pH 4.3 revealed two components, both possessing hyaluronidase activity. Sodium dodecyl sulphate polyacrylamide-gel electrophoresis likewise revealed two close bands with approximate molecular weights of 61000 and 67200.
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Project description:PDZ domains function in nature as protein-binding domains within scaffold and membrane-associated proteins. They comprise approximately 90 residues and undergo specific, high-affinity interactions with complementary C-terminal peptide sequences, other PDZ domains, and/or phospholipids. We have previously shown that the specific, strong interactions of PDZ domains with their ligands make them well suited for use in affinity chromatography. This unit provides protocols for the PDZ affinity chromatography procedure that are applicable for the purification of proteins that contain PDZ domains or PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We detail the preparation of affinity resins composed of PDZ domains or PDZ domain peptide ligands coupled to solid supports. These resins can be used to purify proteins containing endogenous or genetically introduced PDZ domains or ligands, eluting the proteins with free PDZ domain peptide ligands.
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Project description:The purpose of this study was to identify seminal plasma proteins in Bali bull and their potential as biomarkers of fertility. Semen was collected from 10 bulls aged 5-10 years using an artificial vagina. Fresh semen was then centrifuged (3000× g for 30 min). The supernatant was put into straws and stored in liquid nitrogen. The semen plasma protein concentration was determined using the Bradford method, and the protein was characterized using 1D-SDS-PAGE. Coomassie Brilliant Blue (CBB) was used to color the gel, and the molecular weight of the protein was determined using PM2700. A total of 94 proteins were identified in the seminal plasma of Bali bulls analyzed using LC-MS/MS (liquid chromatography-mass spectrometry). Proteins spermadhesin 1 (SPADH1), C-type natriuretic peptide (NPPC), clusterin (CLU), apoliprotein A-II (APOA2), inositol-3-phosphate synthase 1 (ISYNA1), and sulfhydryl oxidase 1 (QSOX1) were identified as important for fertility in Bos javanicus. These proteins may prove to be important biomarkers of fertility in Bali bulls. These proteins are important for reproductive function, which includes spermatozoa motility, capacitation, and acrosome reactions. This study provides new information about the protein content in seminal plasma in Bali bulls. The LC-MS/MS-based proteome approach that we applied in this study obtained 94 proteins. The identification of these seminal plasma proteins of Bali bulls and their potential as fertility biomarkers may have an impact on the success of future artificial insemination (AI).
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Project description:This study monitored the chemical and biochemical composition of bovine seminal plasma (SP). Freshly ejaculated semen (n = 20) was aliquoted into two parts. The first aliquot was immediately assessed to determine the sperm motion parameters. Another motility measurement was performed following an hour-long co-incubation of spermatozoa with SP at 6 °C. The other aliquot was processed to obtain the SP. Seminal plasma underwent the analyses of chemical composition and quantification of selected proteins, lipids and RedOx markers. Determined concentrations of observed parameters served as input data to correlation analyses where associations between micro and macro elements and RedOx markers were observed. Significant correlations of total oxidant status were found with the content of Cu and Mg. Further significant correlations of glutathione peroxidase were detected in relation to Fe and Hg. Furthermore, associations of chemical elements and RedOx markers and spermatozoa quality parameters were monitored. The most notable correlations indicate beneficial effects of seminal Fe on motility and Mg on velocity and viability of spermatozoa. On the contrary, negative correlations were registered between Zn and sperm velocity and seminal cholesterol content and motility. Our findings imply that seminal plasma has a prospective to be developed as the potential biomarker of bull reproductive health.
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Project description:The purification is described of rat hepatic hexokinase type III and kidney hexokinase type I on a large scale by using a combination of conventional and affinity techniques similar to those previously used for the purification of rat hepatic glucokinase [Holroyde, Allen, Storer, Warsy, Chesher, Trayer, Cornish-Bowden & Walker (1976) Biochem. J. 153, 363-373] and muscle hexokinase type II [Holroyde & Trayer (1976) FEBS Lett. 62, 215-219]. The key to each purification was the use of a Sepharose-N-aminoacylglucosamine affinity matrix in which a high degree of specificity for a particular hexokinase isoenzyme could be introduced by either varying the length of the aminoacyl spacer and/or varying the ligand concentration coupled to the gel. This was predicted from a study of the free solution kinetic properties of the various N-aminoacylglucosamine derivatives used (N-aminopropionyl, N-aminobutyryl, N-aminohexanoyl and N-aminooctanoyl), synthesized as described by Holroyde, Chesher, Trayer & Walker [(1976) Biochem. J. 153, 351-361]. All derivatives were competitive inhibitors, with respect to glucose, of the hexokinase reaction, and there was a direct correlation between the Ki for a particular derivative and its ability to act as an affinity matrix when immobilized to CNBr-activated Sepharose 4B. Muscle hexokinase type II could be chromatographed on the Sepharose conjugates of all four N-aminoacylglucosamine derivatives, although the N-aminohexanoylglucosamine derivative proved best. This same derivative was readily able to bind hepatic glucokinase and hexokinase type III, but Sepharose-N-amino-octanoyl-glucosamine was better for these enzymes and was the only derivative capable of binding kidney hexokinase type I efficiently. Separate studies with yeast hexokinase showed that again only the Sepharose-N-amino-octanoylglucosamine was capable of acting as an efficient affinity matrix for this enzyme. Implications of these studies in our understanding of affinity-chromatography operation are discussed.
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Project description:Analysis of seminal plasma from 17 bulls used for inseminations
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Project description:Fibroblast growth factor-2 (FGF2) is a pleiotropic growth factor exhibiting a variety of biological activities. In this article, we studied the capacity of FGF2 purified with or without heparin affinity chromatography to self-associate. Analyzing the NMR HSQC spectra for different FGF2 concentrations, heparin-affinity purified FGF2 showed perturbations that indicate dimerization and are a higher-order oligomerization state. HSQC perturbation observed with different FGF2 concentrations revealed a heparin-binding site and two dimer interfaces. Thus, with increasing protein concentrations, FGF2 monomers make contacts with each other and form dimers or higher order oligomers. On the contrary, FGF2 purified with ion-exchange chromatography did not show similar perturbation indicating that self-association of FGF2 is eliminated if purification is done without heparin-affinity chromatography. The HSQC spectra of heparin-affinity purified FGF2 can be reproduced to some extent by adding heparin tetra-saccharide to ion exchange chromatography purified FGF2. Heparin-affinity purified FGF2 bound to acceptor and donor beads in a tagged form using His-tagged or GST-tagged proteins, also dimerized in the AlphaScreen™ assay. This assay was further validated using different experimental conditions and competitors. The assay constitutes an interesting tool to study dimerization of other FGF forms as well.
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