Purification of bovine angiotensin converting enzyme

A change has been made in the commonly used lisinopril affinity gel procedure for purifying angiotensin converting enzyme. The new method greatly decreases the time required and greatly increases the yield of pure enzyme. All of the enzyme in various bovine tissues was extracted with 0.5% triton X-100 and applied to the affinity column; 70% was trapped and all of the trapped enzyme was released as the apoenzyme by EDTA. The holoenzyme was recovered by dialysis against zinc containing buffer. The turnover numbers were precisely the same for enzyme from lung, atrium, kidney, striatum and blood. The tissue concentrations of ACE were very different but the final specific activities were the same.     

Complete amino-acid sequence of bovine seminal ribonuclease, a dimeric protein from seminal plasma

The complete amino-acid sequence of BS-RNAse, a dimeric ribonuclease isolated from bovine seminal plasma, was determined. The reduced and S-carboxymethylated subunit chain of the enzyme was cleaved by trypsin and chymotrypsin. The resulting peptides, purified by cation-exchange chromatography were sequenced by dansyl-Edman, subtractive Edman degradation and carboxypeptidase A and B digestion. Chymotryptic peptides were used for the alignment. Automated Edman degradation of the native protein, through the N-terminal 41 amino-acid residues, completed the sequence information. The subunit chain of BS-RNAse, composed of 124 amino-acid residues, with a molecular mass of 13,610 Da, is highly homologous (81%) to pancreatic ribonuclease A. A good degree of homology (31%) was also found with human angiogenin. No N-linked carbohydrate-attachment sites, such as Asn-X-Ser/Thr, were found in the protein.     

Purification and properties of beta-galactosidase from chicken seminal plasma

1. beta-Galactosidase (EC 3.2.1.23) from chicken seminal plasma was purified approx. 111-fold to homogeneity. 2. pH optimum of the enzyme ranged from 3.6 to 4.0 and its Km was 0.65 mM with p-nitrophenyl-beta-D-galactoside as substrate. 3. The enzyme was unstable at its optimal activity pH and was activated by Cl- ions. 4. The enzyme had pI value of 4.0. 5. The active enzyme had Mr approx. 100,000 by Sephacryl S-300 chromatography. SDS electrophoresis in the presence of beta-mercaptoethanol showed four bands corresponding to Mr of approx. 90,000, 75,000, 65,000 and 13,000.     

Characterization of bovine seminal plasma by proteomics

Previous investigations of bovine seminal plasma (BSP) have revealed the identities of the three major proteins, BSP-PDC109, BSP-A3 and BSP-30 kDa, which together constitute about half of the total protein, as well as about 30 of the minor proteins. Analyses of BSP by 2-DE have revealed about 250 protein spots, suggesting that much of the BSP proteome remains undescribed. In this study, BSP has been analyzed by 2-D LC-based and SDS-PAGE-based proteomic methods. Ninety-nine proteins were identified, including 49 minor proteins that have not previously been described in seminal plasma of any species.     

Purification and properties of alkaline phosphatase from boar seminal plasma

An alkaline phosphatase was purified from boar seminal plasma using adsorption to calcium phosphate gel, gel filtration, and ion-exchange chromatography. The preparation gave a single band on SDS polyacrylamide electrophoresis. The enzyme was a non-specific alkaline phosphatase that hydrolysed pyrophosphate slowly and had no phosphodiesterase activity. The pH optimum was 10 and the Km was approximately 0.2 mM with p-nitrophenyl phosphate as substrate. The enzyme was a zinc metalloenzyme as indicated by the loss of activity when treated with o-phenanthroline and the restoration of activity by zinc and magnesium ions. It also lost activity when treated with thiols. Molecular weight estimates from SDS polyacrylamide gel electrophoresis and gel filtration suggest that the enzyme is a tetramer of identical subunits, each of which has a molecular weight of 68,000.     

Identification of heparin-binding proteins in bovine seminal plasma

A group of four similar proteins, BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa, represent the major acidic proteins found in bovine seminal plasma (BSP). These proteins are secretory products of the seminal vesicles; they bind to spermatozoa upon ejaculation and could represent decapacitation factors. It has been shown that the glycosaminoglycans present in the female reproductive tract are involved in the capacitation of spermatozoa. Therefore, it was of interest to investigate whether BSP-A1, -A2, -A3, and -30-kDa proteins of bovine seminal fluid interact with heparin. Chromatography of alcohol precipitates of bovine seminal fluid on a heparin-Sepharose column resolved these proteins into three peaks. Peaks 1 and 2 (retarded proteins) were eluted upon extensive washing of the column with 0.05 M phosphate buffer, pH 7.4 (equilibrating buffer), and accounted for approximately 25% of the applied proteins. Proteins in peak 3 represented adsorbed proteins and were eluted with phosphate buffer containing 1 M NaCl. Proteins in each peak were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Peak 1 contained proteins with molecular weights ranging from 8 to 350 kDa, peak 2 contained a single protein with a molecular weight of 14 kDa, and peak 3 contained proteins with molecular weights of 15.5, 16, 25, and 30 kDa. The proteins in peak 3 were further resolved into unadsorbed (peak 4) and adsorbed (peak 5) proteins on a gelatin-Agarose column. Separation of the proteins of peak 3 and peak 5 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and reducing agents followed by transfer to nitrocellulose and probing with antibodies against the previously well-characterized BSP proteins indicated the presence of BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biological characteristics of the peptides α and β isolated from bovine seminal plasma

The bull seminal plasma peptides α andβ have been examined for their biological properties. While both the peptides were able to inhibit the human chorionic gonadotropin-dependent uterine response in the mouse, α alone exhibits the property of suppressing post-castrational rise in gonadotropin in appropriate animal models. This suggests that the peptideβ must be acting directly on the ovary to suppress estrogen production and, consequently, the uterine weight increase. Such a possibility was confirmed when α andβ were examined by the coupled bioassay which is capable of discriminating between pituitary feedback factors and those acting directly on the gonad. In a test system designed to examine chronic effects, both α andβ showed evidence of acting directly on the ovary to inhibit human menopausal gonadotropin-induced estrogen production. Such a direct action could not be correlated with the relative potencies of these peptides when examined for their follicle stimulating hormone-receptor binding inhibitor and lutinizing hormone-receptor binding inhibitor activities.     

Kininase II from human seminal plasma. Isolation and properties

A rapid and highly efficient procedure for purification of kininase II from human seminal plasma is described. After ultra centrifugation, the enzyme was purified by gel filtration on Sepharose 6B CL and ion exchange chromatography followed by affinity chromatography of EDTA-inhibited enzyme on bradykinin-Sepharose. The enzyme was specifically inhibited by Captopril and BPP9a but not by phosphoramidon. PAGE in the presence of sodium dodecyl sulfate under reducing conditions resulted in two major protein bands with apparent molecular masses of about 55 kDa and 65 kDa and two faint protein bands at higher molecular masses. Antibodies raised against the major protein bands showed full cross reactivity with all four protein bands. The presented data indicate that kininase II consists of subunits.     

Purification of four gelatin-binding proteins from bovine seminal plasma by affinity chromatography

Bovine seminal plasma contains three similar acidic proteins, which we have previously designated as BSP-A1, BSP-A2, and BSP-A3. These proteins contain two homologous domains that are similar to type II structures present in the gelatin-binding domain of fibronectin. The present data have revealed that these proteins, like fibronectin, also form complexes with gelatin, a denatured collagen. Based on this property, a single step affinity purification method has been developed. In addition to these three proteins BSP-A1, -A2 and -A3, another protein with an apparent molecular weight of 30,000 dalton (named BSP-30-kDa) also bound to the gelatin-agarose column. Elution of these proteins from affinity columns using a linear gradient of either urea or arginine gave essentially the same pattern with a high yield of 90–95%. The purified proteins were homogeneous by SDS-polyacrylamide gel electrophoresis, amino acid composition and HPLC. Chromatography of bull seminal vesicular fluid also exhibited an elution pattern similar to that obtained for bull seminal plasma. The availability of these purified proteins should aid in understanding the physiology of these gelatin-binding proteins.     

Antibacterial activity of seminalplasmin, a basic protein from bovine seminal plasma

Seminalplasmin, a 6,000 dalton antimicrobial protein present in bovine seminal plasma, is shown to inhibit growth and/or RNA synthesis in several bacterial species. In only one strain out of twenty one belonging to fourteen species, did both RNA synthesis and growth appear to be resistant to seminalplasmin. The antibacterial activity of seminalplasmin, in the case of E. coli, was also studied as a function of its concentration and of time; the minimal concentration of the protein required for 100% bactericidal activity was only about twice that required for 100% bacteriostatic activity. The killing of E. coli cells proceeded in two phases, a slow phase and then a rapid one, and required several hours for completion. Several bacterial species tested secreted proteases into the medium that destroyed seminalplasmin.     

Sphingomyelinases in human, bovine and porcine seminal plasma

The seminal plasma of man, boar and bull was found to have a sphingomyelinase (SMase) activity hydrolysing [N-methyl-14C]sphingomyelin. The human and porcine enzymes had an acid pH optimum and were not influenced by divalent metal ions or chelating agents. They were closely similar with the lysosomal enzyme in many tissues. The bovine seminal plasma SMase was partially purified. The enzyme was a glycoprotein with pH optimum at 6.5, a broad pI 4.2-4.8 and molecular mass of 160 and 60 kDa, respectively, in native and SDS-PAGE. The enzyme was activated by Co greater than Mn greater than Cd greater than Ni and inhibited by chelating agents, Cu, Fe, Pb and Zn. The enzyme was clearly distinct from the acid lysosomal SMase and the previously described neutral Mg2+-dependent and independent activities. It had a wide distribution in the bull reproductive tissues.     

Purification and properties of phospholipase A2 from human seminal plasma

The soluble Ca2+-dependent phospholipase A2 (EC 3.1.1.4) was purified 6500-fold with a yield of about 20% from human seminal plasma. The successive purification steps comprised gel filtration, affinity chromatographies and micropartition. The final preparation consisted of two proteins in about equal quantities with molecular weights of 12000 and 14000, according to SDS-polyacrylamide slab gel electrophoresis. As yet these two proteins can not be separated without complete loss of activity. Apparent kinetic parameters have been determined for the purified preparation with different substrates (Vmax = 494 U/mg, and Km = 1.25 X 10(-4) M long-chain phosphatidylethanolamine; Vmax = 7.4 U/mg, and Km = 2.5 X 10(-5) M long-chain phosphatidylcholine; Vmax = 7196 U/mg and Km = 8.32 X 10(-4) M dioctanoylphosphatidylcholine). The enzymatic activity was not affected by diisopropylfluorophosphate and thiol reagents but it was inhibited by higher concentrations of nonionic and ionic (except taurocholate) detergents and by the alkylating reagent p-bromophenacyl bromide. Although the seminal enzyme functionally strongly resembles the pancreatic phospholipase A2, no immunochemical relationship was observed; anti-pancreatic phospholipase A2 IgGs did not inhibit seminal phospholipase A2. Similarly, partially purified phospholipase A2 from horse seminal fluid was not affected by antibodies raised against horse pancreatic phospholipase A2.     

On multiple forms of bovine seminal plasma inhibin.

Out of a possible minimum of four, three distinct molecular species of bovine seminal plasma inhibin-differing either in Mr or in pI--have been purified to homogeneity. All three molecules exhibit the same proportion of alpha-helicity and beta-form when examined for their CD-spectra in a non-aqueous solvent medium. The implication of this finding for an induced conformation at the receptor-binding site for these hormonal peptides is briefly discussed.     

The effect of bovine seminal plasma on the function and integrity of bovine neutrophils

Spermatozoa, by virtue of their high content of polyunsaturated fatty acids and limited antioxidant activity, are highly sensitive to lipid peroxidation. Sources of reactive oxygen species in the female genital tract include neutrophils recruited in response to insemination or those contained within the ejaculate, or even spermatozoa themselves. This study was undertaken to investigate the effect of seminal plasma of bulls on bovine neutrophils. Seminal plasma was removed from ejaculates of 6 healthy bulls by centrifugation. Neutrophils were isolated from jugular venous blood samples obtained from lactating Holstein cows. Seminal plasma was found to decrease measured Superoxide anion production by activated neutrophils (P = 0.008) in a concentration-dependent manner over the range tested (0 to 16%). This effect may reflect Superoxide scavenging by the seminal plasma or direct inhibition of the neutrophils. It has the potential physiological advantage of protecting spermatozoa from oxidative damage. Seminal plasma was found to reduce homotypic neutrophil aggregation dramatically (P < 0.0001). At a seminal plasma concentration of 0.2% neutrophil aggregation was virtually abolished. Homotypic neutrophil aggregation depends on the CD 11b/CD 18 (Mac-1) receptor, which also functions as the C3bi receptor (known as complement receptor-3 or CR3). If this receptor is down-regulated, the ability of neutrophils to phagocytose complement-opsonized microorganisms would be compromised. Finally, seminal plasma was found to increase neutrophil permeability to trypan blue in a concentration and time dependent fashion (P < 0.0001), indicating a direct cytotoxic effect. Taken together, these findings indicate a profound inhibition of neutrophil function by bovine seminal plasma. While these mechanisms are likely to protect spermatozoa from oxidative damage or phagocytosis, they also have the potential to diminish defense against pathogenic microorganisms.     

Isolation and properties of a phosphatidylcholine-specific phospholipase C from bull seminal plasma

A phospholipase C which hydrolyzes [14C]phosphatidylcholine has been purified 1782-fold from 70% ammonium sulfate extract of bull seminal plasma. Purification steps included acid precipitation, chromatography on DEAE-Sephacel, concanavalin A, octyl-Sepharose 4B and Ultrogel AcA 34. The final step provided homogeneous phospholipase C as determined by polyacrylamide gel electrophoresis. The enzyme comprised two subunits, Mr 69,000 and Mr 55,000, respectively. The enzyme had an optimum at pH 7.2 and pI 5.0. EDTA, Cd2+, Pb2+, Ni2+, Fe2+, and Zn2+ inhibited phospholipase C activity. Km and Vmax on p-nitrophenyl phosphorylcholine and phosphatidylcholine substrates were 20 mM and 17 mumol/min/mg of the purified enzyme and 100 microM and 18 mumol/min/mg of the purified enzyme, respectively. The enzyme appeared to be localized in the acrosome as judged by the binding of anti-phospholipase C to the acrosome. This phospholipase C, unlike other known phospholipases (C), did not hydrolyze [1-14C]phosphatidylinositol. The testicular extract of the guinea pig contained inactive phospholipase C which was activated on incubation with acrosin and trypsin but not chymotrypsin.     

Heparin-binding proteins from seminal plasma bind to bovine spermatozoa and modulate capacitation by heparin

Bovine spermatozoa that have been exposed to seminal plasma possess more binding sites for heparin than sperm from the cauda epididymis that have not been exposed to accessory sex gland secretions. Seminal plasma exposure enables sperm, following incubation with heparin, to undergo zonae pellucidae-induced exocytosis of the acrosome. In this study, the regulatory role of seminal plasma heparin-binding proteins in capacitation of bovine spermatozoa by heparin was investigated. Plasma membranes from sperm exposed to seminal plasma in vivo or in vitro contained a series of acidic 15-17 kDa proteins not found in cauda epididymal sperm. Western blots of membrane proteins indicated that these 15-17 kDa proteins bound [125I]-heparin. Heparin-binding proteins were isolated by heparin affinity chromatography from seminal plasma from vasectomized bulls. Gel electrophoresis indicated that the heparin-binding peaks contained 14-18 kDa proteins with isoelectric variation, a basic 24 kDa protein, and a 31 kDa protein. Western blots probed with [125I]-heparin confirmed the ability of each of these proteins to bind heparin. Each of these proteins, as well as control proteins, bound to epididymal sperm. The seminal plasma proteins were peripherally associated with sperm since they were removed by hypertonic medium and did not segregate into the detergent phase of Triton X-114. Seminal plasma heparin-binding proteins potentiated zonae pellucidae-induced acrosome reactions in epididymal sperm. However, seminal plasma proteins that did not bind to the heparin affinity column were unable to stimulate zonae-sensitivity. Control proteins, including lysozyme--which binds to both heparin and sperm, were ineffective at enhancing zonae-induced acrosome reactions. These data provide evidence for a positive regulatory role of seminal plasma heparin-binding proteins in capacitation of bovine spermatozoa.