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Isolation and characterization of gelatin-binding proteins from goat seminal plasma.

Villemure M, Lazure C, Manjunath P - Reprod. Biol. Endocrinol. (2003)

Bottom Line: Frozen semen (-80 degrees C) was thawed and centrifuged to remove sperm.The precipitates were dissolved in ammonium bicarbonate and lyophilised.In addition, these BSP homologs bind to hen's egg-yolk low-density lipoproteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Maisonneuve-Rosemont Hospital, Guy-Bernier Research Centre University of Montreal, Montreal, QC, Canada. mvillemure.hmr@ssss.gouv.qc.ca

ABSTRACT
A family of proteins designated BSP-A1, BSP-A2, BSP-A3 and BSP-30 kDa (collectively called BSP proteins for Bovine Seminal Plasma proteins) constitute the major protein fraction in the bull seminal plasma. These proteins interact with choline phospholipids on the sperm surface and play a role in the membrane stabilization (decapacitation) and destabilization (capacitation) process. Homologous proteins have been isolated from boar and stallion seminal plasma. In the current study we report the isolation and preliminary characterization of homologous proteins from goat seminal plasma. Frozen semen (-80 degrees C) was thawed and centrifuged to remove sperm. The proteins in the supernatant were precipitated by the addition of cold ethanol. The precipitates were dissolved in ammonium bicarbonate and lyophilised. The lyophilised proteins were dissolved in phosphate buffer and loaded onto a gelatin-agarose column, which was previously equilibrated with the same buffer. The column was successively washed with phosphate buffer, with phosphate buffer saline and with 0.5 M urea in phosphate buffer saline to remove unadsorbed proteins, and the adsorbed proteins were eluted with 5 M urea in phosphate buffer saline. Analysis of pooled, dialysed and lyophilised gelatin-agarose adsorbed protein fraction by SDS-PAGE indicated the presence of four protein bands that were designated GSP-14 kDa, GSP-15 kDa, GSP-20 kDa and GSP-22 kDa (GSP, Goat Seminal Plasma proteins). Heparin-affinity chromatography was then used for the separation of GSP-20 and -22 kDa from GSP-14 and -15 kDa. Finally, HPLC separation permitted further isolation of each one from the other. Amino acid sequence analysis of these proteins indicated that they are homologous to BSP proteins. In addition, these BSP homologs bind to hen's egg-yolk low-density lipoproteins. These results together with our previous data indicate that BSP family proteins are ubiquitous in mammalian seminal plasma, exist in several forms in each species and possibly play a common biological role.

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SDS-PAGE pattern of gelatin-agarose and heparin-Sepharose eluted fractions. Between 5 to 10 μg of lyophilised proteins from each fraction (except fraction A: 20 μg) were previously reduced and denatured, and loaded on a 15% gel. After migration of the proteins, the gel was stained with Coomassie brilliant Blue R-250. Electrophoresis pattern of the starting material is shown, as well as the pattern of BSP proteins for comparison.
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Figure 2: SDS-PAGE pattern of gelatin-agarose and heparin-Sepharose eluted fractions. Between 5 to 10 μg of lyophilised proteins from each fraction (except fraction A: 20 μg) were previously reduced and denatured, and loaded on a 15% gel. After migration of the proteins, the gel was stained with Coomassie brilliant Blue R-250. Electrophoresis pattern of the starting material is shown, as well as the pattern of BSP proteins for comparison.

Mentions: Figure 1 shows the gelatin-agarose chromatography pattern of proteins precipitated from goat seminal plasma. Column was washed to remove unadsorbed proteins (fraction A), and the adsorbed ones were eluted with 5 M urea (fraction B). The total weight recovered from this chromatography step was ~72%, and the adsorbed proteins constituted almost 50% of it. Each fraction was analyzed by SDS-PAGE (Figure 2), which revealed the presence of four different proteins of apparent molecular masses 14 kDa, 15 kDa, 20 kDa and 22 kDa. We designated those Goat Seminal Plasma proteins, respectively GSP-14 kDa, GSP-15 kDa, GSP-20 kDa and GSP-22 kDa. We observed that the unadsorbed fraction A contained a certain amount of proteins with a molecular mass similar to the GSPs on SDS-PAGE, that did not bind the gelatin-agarose (Figure 2). It is not likely that those proteins are GSPs, but other proteins of same apparent molecular weight contained in seminal plasma, since they again didn't bind to gelatin-agarose after we rechromatographed the unadsorbed fraction on the column (data not shown). Alternatively, a certain amount of GSP proteins could be in aggregated form or associated with phospholipids and thus would not be able to bind the column.


Isolation and characterization of gelatin-binding proteins from goat seminal plasma.

Villemure M, Lazure C, Manjunath P - Reprod. Biol. Endocrinol. (2003)

SDS-PAGE pattern of gelatin-agarose and heparin-Sepharose eluted fractions. Between 5 to 10 μg of lyophilised proteins from each fraction (except fraction A: 20 μg) were previously reduced and denatured, and loaded on a 15% gel. After migration of the proteins, the gel was stained with Coomassie brilliant Blue R-250. Electrophoresis pattern of the starting material is shown, as well as the pattern of BSP proteins for comparison.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC155548&req=5

Figure 2: SDS-PAGE pattern of gelatin-agarose and heparin-Sepharose eluted fractions. Between 5 to 10 μg of lyophilised proteins from each fraction (except fraction A: 20 μg) were previously reduced and denatured, and loaded on a 15% gel. After migration of the proteins, the gel was stained with Coomassie brilliant Blue R-250. Electrophoresis pattern of the starting material is shown, as well as the pattern of BSP proteins for comparison.
Mentions: Figure 1 shows the gelatin-agarose chromatography pattern of proteins precipitated from goat seminal plasma. Column was washed to remove unadsorbed proteins (fraction A), and the adsorbed ones were eluted with 5 M urea (fraction B). The total weight recovered from this chromatography step was ~72%, and the adsorbed proteins constituted almost 50% of it. Each fraction was analyzed by SDS-PAGE (Figure 2), which revealed the presence of four different proteins of apparent molecular masses 14 kDa, 15 kDa, 20 kDa and 22 kDa. We designated those Goat Seminal Plasma proteins, respectively GSP-14 kDa, GSP-15 kDa, GSP-20 kDa and GSP-22 kDa. We observed that the unadsorbed fraction A contained a certain amount of proteins with a molecular mass similar to the GSPs on SDS-PAGE, that did not bind the gelatin-agarose (Figure 2). It is not likely that those proteins are GSPs, but other proteins of same apparent molecular weight contained in seminal plasma, since they again didn't bind to gelatin-agarose after we rechromatographed the unadsorbed fraction on the column (data not shown). Alternatively, a certain amount of GSP proteins could be in aggregated form or associated with phospholipids and thus would not be able to bind the column.

Bottom Line: Frozen semen (-80 degrees C) was thawed and centrifuged to remove sperm.The precipitates were dissolved in ammonium bicarbonate and lyophilised.In addition, these BSP homologs bind to hen's egg-yolk low-density lipoproteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Maisonneuve-Rosemont Hospital, Guy-Bernier Research Centre University of Montreal, Montreal, QC, Canada. mvillemure.hmr@ssss.gouv.qc.ca

ABSTRACT
A family of proteins designated BSP-A1, BSP-A2, BSP-A3 and BSP-30 kDa (collectively called BSP proteins for Bovine Seminal Plasma proteins) constitute the major protein fraction in the bull seminal plasma. These proteins interact with choline phospholipids on the sperm surface and play a role in the membrane stabilization (decapacitation) and destabilization (capacitation) process. Homologous proteins have been isolated from boar and stallion seminal plasma. In the current study we report the isolation and preliminary characterization of homologous proteins from goat seminal plasma. Frozen semen (-80 degrees C) was thawed and centrifuged to remove sperm. The proteins in the supernatant were precipitated by the addition of cold ethanol. The precipitates were dissolved in ammonium bicarbonate and lyophilised. The lyophilised proteins were dissolved in phosphate buffer and loaded onto a gelatin-agarose column, which was previously equilibrated with the same buffer. The column was successively washed with phosphate buffer, with phosphate buffer saline and with 0.5 M urea in phosphate buffer saline to remove unadsorbed proteins, and the adsorbed proteins were eluted with 5 M urea in phosphate buffer saline. Analysis of pooled, dialysed and lyophilised gelatin-agarose adsorbed protein fraction by SDS-PAGE indicated the presence of four protein bands that were designated GSP-14 kDa, GSP-15 kDa, GSP-20 kDa and GSP-22 kDa (GSP, Goat Seminal Plasma proteins). Heparin-affinity chromatography was then used for the separation of GSP-20 and -22 kDa from GSP-14 and -15 kDa. Finally, HPLC separation permitted further isolation of each one from the other. Amino acid sequence analysis of these proteins indicated that they are homologous to BSP proteins. In addition, these BSP homologs bind to hen's egg-yolk low-density lipoproteins. These results together with our previous data indicate that BSP family proteins are ubiquitous in mammalian seminal plasma, exist in several forms in each species and possibly play a common biological role.

Show MeSH
Related in: MedlinePlus