Novel purification method for mammalian seminal plasma phospholipid-binding proteins reveals the presence of a novel member of this family of protein in stallion seminal fluid.
A family of bull seminal plasma (BSP) phospholipid-binding proteins (BSP proteins), potentiate heparin- and HDL-induced capacitation. The homologous proteins have been purified from stallion and boar seminal plasma, and detected in low concentrations in other mammalian seminal plasma. In this study, we developed a new isolation method for mammalian seminal plasma choline phospholipid-binding proteins wherein they are present in low concentrations. The method is based on the interaction of this family of proteins with egg yolk low-density lipoprotein fraction (LDF). In order to demonstrate the feasibility of the method, we incubated LDF with alcohol precipitates of bull, boar, and stallion seminal plasma. LDF were re-isolated by ultracentrifugation along with bound proteins. LDF with associated proteins were dialyzed, lyophilized, and delipidated. BSP homologous proteins were finally purified by p-aminophenyl phosphorylcholine (PPC)-agarose and/or gelatin-agarose chromatographies, and analyzed by SDS-PAGE. With this new protocol, phospholipid-binding proteins of bull, boar, and stallion seminal plasma were recovered almost 100%. A new 12 kDa stallion seminal plasma protein of the same family was also isolated and partially sequenced. The radio-immunoassay (RIA) data showed that 10 mg of LDF can bind all BSP proteins present in 120 mg of alcohol precipitated BSP proteins. These results confirm the efficiency of the method and that the LDF step could be used for the isolation of all BSP proteins homologs from different mammalian species.
Ménard
Martin
M
Department of Medicine and of Biochemistry, University of Montreal, Quebec, Canada.
Nauc
Veronica
V
Lazure
Claude
C
Vaillancourt
Denis
D
Manjunath
Puttaswamy
P
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D006736:Horses; D008297:Male; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D012661:Semen / Q000737:chemistry*; D029589:Seminal Plasma Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D013552:Swine
D010743:Phospholipids; D029589:Seminal Plasma Proteins