Isolation and characterization of the major form of beta-glucuronidase from human seminal plasma

Human seminal plasma contain two forms of beta-glucuronidase (beta-D-glucuronidase glucuronosohydrolase, EC 3.2.1.31) which are present in the ratio of 4:1. The major form of beta-glucuronidase with a slow moving band in electrophoresis was purified to homogeneity as revealed by polyacrylamide gel electrophoresis, double immunodiffusion and immunoelectrophoresis. The major form of beta-glucuronidase shows dual optimum pH at 4.3 and 4.7 with a dip in the activity at pH 4.5. The Km of this form of beta-glucuronidase is dependent on pH and was found to be 0.95, 3.08 and 0.67 mM at pH 4.4, 4.5 and 4.7, respectively. The major form of beta-glucuronidase from seminal plasma is stable at 55 degrees C for 30 min but it denatures at 65 degrees C. Heat denaturation is faster at acidic pH (4.7) than at alkaline pH (7.8). However, the activity of enzyme increased linearly with increase in temperature up to 70 degrees C during incubation with substrate. Cu, Ag, Hg and Ni salts inhibited enzyme activity significantly at 0.1 and 1.0 mM concentration, but the inhibition of HgCl2 was protected by cysteine. 1,4-D-Saccharic acid lactone and ascorbic acid inhibited seminal beta-glucuronidase competitively, yielding Ki values of 1.7 . 10(-3) mM and 10.3 mM, respectively. Though fructose and mannose also showed significant inhibition of beta-glucuronidase at 10-100 mM, glucose did not show any effect. The molecular weight of the major form of beta-glucuronidase was found to be 279 000, and it appears to be composed of four subunits each having a molecular weight of 74 000.     

Isolation, characterization and amino-acid sequence of gamma-seminoprotein, a glycoprotein from human seminal plasma

gamma-Seminoprotein (gamma-SM), a glycoprotein from human seminal plasma, was isolated in highly purified form by ion-exchange chromatography on a Mono Q column. The main form, fraction M, was homogeneous by PAGE at pH 8.3 and by SDS-PAGE. The complete amino acid sequence of gamma-SM was determined with the aid of fragments generated by cleavages with cyanogen bromide, clostripain, chymotrypsin and Staphylococcus aureus V8 protease. The fragments were aligned with overlapping sequences. The single polypeptide chain of gamma-SM contains 237 amino acids with a calculated Mr of 26079. A single N-linked carbohydrate side chain is attached to Asn45. The complex structure of this oligosaccharide has been determined recently [van Halbeek H. et al. (1985) Biochem. Biophys. Res. Commun. 131, 507-514]. Sequence comparison with serine proteases shows a high degree of homology, especially with the kallikrein family. The residues in the vicinity of the active site of serine proteases are also highly conserved in gamma-SM, indicating the participation of His41, Asp96 and Ser189 in its active site. gamma-SM hydrolyzed M-casein with a pH optimum at 8.0, but failed to hydrolyze any of the synthetic substrates tested. This proteolytic activity could be inhibited with diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, Zn2+ or Hg2+ ions.     

Isolation, molecular cloning, and partial characterization of a novel carboxypeptidase B from human plasma

A novel plasminogen-binding protein has been isolated from human plasma utilizing plasminogen-Sepharose affinity chromatography. This protein copurified with alpha 2 antiplasmin when the plasminogen affinity column was eluted with high concentrations of epsilon-aminocaproic acid (greater than 20 mM). Analysis by sodium dodecyl sulfate suggests this protein has an apparent Mr of 60,000. The amino-terminal amino acid sequence showed no similarity to other protein sequences. Based on the amino-terminal amino acid sequence, oligonucleotide probes were designed for polymerase chain reaction primers, and an approximately 1,800 base pair cDNA was isolated that encodes this Mr 60,000 protein. The deduced amino acid sequence reveals a primary translation product of 423 amino acids that is very similar to carboxypeptidase A and B and consists of a 22-amino acid signal peptide, a 92-amino acid activation peptide, and a 309-amino acid catalytic domain. This protein shows 44 and 40% similarity to rat procarboxypeptidase B and human mast cell procarboxypeptidase A, respectively. The residues critical for catalysis and zinc and substrate binding of carboxypeptidase A and B are conserved in the Mr 60,000 plasminogen-binding protein. The presence of aspartic acid at position 257 of the catalytic domain suggests that this protein is a basic carboxypeptidase. When activated by trypsin, it hydrolyzes carboxypeptidase B substrates, hippuryl-Arg and hippuryl-Lys, but not carboxypeptidase A substrates, and it is inhibited by the specific carboxypeptidase B inhibitor (DL-5-guanidinoethyl)mercaptosuccinic acid. We propose that the Mr 60,000 plasminogen-binding protein isolated here is a novel human plasma carboxypeptidase B and that it be designated pCPB.     

Isolation of a new actin-binding protein from human seminal plasma

Interaction of a protein of human seminal plasma with actin was detected by agar gel immunoelectrophoresis. A major actin-binding protein was isolated from human seminal plasma using an actin-Sepharose 4B column followed by fast-performance liquid chromatography with an anion-exchange Mono-Q column. The protein showed a single band under reduced conditions on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a position corresponding to a molecular mass of 20 kDa. This 20 kDa polypeptide was detected in saliva and extracts of the submandibular gland and seminal vesicles as well as seminal plasma by the method of immunoblotting using monospecific antibody against the 20 kDa antigenic component of human seminal plasma. The protein might be called secretory actin-binding protein (SABP).     

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

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°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.     

Human plasma carboxypeptidase N. Isolation and characterization.

Human plasma carboxypeptidase N has been purified 2,600-fold from pooled, outdated plasma in a 30% yield. Isolation was accomplished by chromatography on DEAE-cellulose and on a p-aminobenzoyl-L-arginine-Sepharose 6B affinity column. Carbohydrate accounts for 17% of the weight calculated from its amino acid and carbohydrate composition. The enzyme appears to consist of three subunits of Mr = 83,000, 55,000, and 49,000 and contains a significant amount of bound zinc. Purified enzyme preparations are very sensitive to proteolytic degradation but are stable for at least 3 months at 4 degrees.     

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 and characterization of ribonucleases from human seminal plasma.

Four ribonucleases (RNAases I-IV) have been purified to homogeneity from human seminal plasma by precipitation with 40-75%-satd. (NH4)2SO4, followed by chromatographies on concanavalin A-Sepharose 4B, DEAE-cellulose phosphocellulose, agarose-5'-(4-aminophenylphospho)uridine 2'(3')-phosphate (RNAase affinity column) and Sephadex G-75 or G-100. The homogeneity of these RNAases was confirmed by polyacrylamide-gel electrophoresis. Mr values for these purified RNAases were 78 000, 16 000, 13 300 and 5000 as estimated by gel filtration. Enzyme activities of RNAases I, III and IV were inhibited by Mn2+, Zn2+ and Cu2+ and activated by Na+, K+, Ba2+, Mg2+, Fe2+ and EDTA, whereas that of RNAase II was inhibited by Ba2+, Mg2+, Fe2+, Mn2+, Zn2+ and Cu2+ and activated by Na+, K+ and EDTA. RNAases I, II and IV demonstrated a higher affinity for poly(C) and poly(U) or yeast RNA, whereas RNAase III preferentially hydrolysed poly(U) over poly(C) and yeast RNA. In the presence of 5 mM-spermine, RNAase I was dissociated to a low-Mr (5000) enzyme with an increase in total RNAase enzymic activity. Xenoantiserum to each RNAase was raised and evaluated by immunoprecipitation and immunohistochemical methods. Anti-(seminal RNAase III) antiserum showed no immunological cross-reaction with RNAases of other human origin, whereas anti-(seminal RNAase I), -(RNAase II) and -(RNAase IV) antisera exhibited indistinguishable immunological reactions with serum RNAase and other human RNAases, except that anti-(seminal RNAase I) and -(RNAase antisera IV) did not react with pancreatic RNAases. Seminal RNAases I and IV were identical immunologically as shown by anti-(RNAase I) and anti-(RNAase IV) in immunodiffusion. Immunohistochemical study revealed that, among human tissues examined, only prostate expressed seminal RNAase III. These results suggested that human seminal RNAase I may be an aggregated molecule of RNAase IV and that seminal RNAases II and IV are similar to serum RNAases, whereas seminal RNAase III is a prostate-specific enzyme.     

Isolation of basic acrosin inhibitor from bull seminal plasma (BUSI II).

An acrosin inhibitor was isolated from bull seminal plasma by gel filtration on Sephadex G-50 fine and ion-exchange chromatography on CM-Sephadex. The inhibitor is a basic polypeptide (pl greater than or equal to 10.5) of molecular weight 6 200 (calculated from amino acid composition). Its N-terminal amino group is blocked. The inhibitor is not strictly specific in its effect since it also inhibits trypsin and to a lesser degree chymotrypsin, in addition to bull and boar acrosin.     

Isolation, partial characterization and biological activity of mannosyl glycopeptides from seminal plasma

Affinity chromatography on Concanavalin-A Sepharose, followed by gel filtration and hydrophobic interaction chromatography, permits the isolation of low molecular weight N-glycosidically linked oligomannosidic glycopeptides (MGp) from the autoproteolysis products of human seminal plasma. The monosaccharide composition of MGp showed only mannose, N-acetylglucosamine and a small amount of galactose. Structural studies were carried out by methylation analysis and chromium trioxide oxidation, and results were consistent with the structures accepted for high-mannose N-glycans. MGp was capable of inhibiting the sperm acrosomal exocytosis mediated by sperm-surface receptors. These data suggest that MGp act as a decapacitation factor preventing premature sperm exocytosis.     

Purification and characterization of human seminal plasma aminopeptidase

A 50.4-fold purification of aminopeptidase is achieved by alcohol precipitation, DEAE-cellulose, CM-cellulose and finally Sephadex G-200 chromatography. On polyacrylamide gel electrophoresis of the purified enzyme after molecular sieving on Sephadex G-200, only one band was obtained, suggesting that the enzyme preparation was obtained almost homogeneous by three steps of column chromatography. Aminopeptidase showed highest activity at pH 7.0, using a buffer system, of 70 mM Na-phosphate. The enzyme was found to be active at 40 degrees C, even at 60 degrees C (80% activity), suggesting that the human seminal plasma enzyme is fairly thermostable. Amongst the various aminoacyl derivatives evaluated as substrates in the present study, L-alanine beta-naphthylamide hydrochloride was found to have the highest rate of hydrolysis. Ovalbumin showed effective cleavage in comparison to that of other natural substrates. The Km value for the purified seminal plasma aminopeptidase towards L-alanine beta-naphthylamide hydrochloride was 4 x 10(-4) M. Hg+2 showed highest inhibitory effect than other metal ions tested in the present study. Concentration causing 50% inhibition of the enzyme (I50) by Hg2+ was 4.7 x 10(-6) M. Inhibition by EDTA at 1 mM concentration in the incubation system was higher than by EGTA and sodium azide, suggesting that the enzyme contains a metallo group at the active site. A 50% inhibition of the enzyme by EDTA was obtained at 5.11 x 10(-3) M. The Ackerman and Potter plot for EDTA inhibition suggests that EDTA is a reversible inhibitor of seminal plasma aminopeptidase. A single molecular form of aminopeptidase was found to be present in human seminal plasma as shown by polyacrylamide activity gel electrophoresis.     

Isolation and characterization of a microbial Arg/Lys carboxypeptidase, carboxypeptidase F

Carboxypeptidase F was isolated from a fungal strain F-33 and characterized. The enzyme has the ability to release arginine and lysine from the carboxy terminus of peptides, and showed high specific activity against arginine (140 units mg^-1 protein). Optimal temperature and pH for the enzyme reaction were 55°C and pH 8.5, respectively. The enzyme possessed a high thermal stability. Native molecular weight was estimated to be approximately 450000. Enzymatic activity was inhibited by Co²⁺, Cd²⁺, chelating agents and thiol inhibitors.     

Isolation and characterization of wound-inducible carboxypeptidase inhibitor from tomato leaves

In a previous report [Mol. Gen. Genet. 228 (1991) 281], carboxypeptidase inhibitor protein (CPI) mRNA was found to accumulate in leaves of wounded tomato plants, but CPI protein could not be detected. In contrast, we found that CPI protein does accumulate in tomato leaves in response to wounding, and also in response to treatment with either systemin, methyl jasmonate (MeJ), oligogalacturonic acid, or chitosan. Identification of CPI protein was confirmed by its inhibition of metallo-carboxypeptidase A (CPAase), which was used as an assay during purification of the inhibitor from leaves of MeJ-treated tomato plants. Amino acid sequence analysis and mass spectroscopic analyses of the pure protein confirmed its identity as CPI. The pure protein inhibited CPAase in a 1:1 stoichimetric interaction. Time course analyses of the induction of CPI mRNA in tomato leaves in response to wounding indicated that the gene is a member of the group of "late genes" that code for defensive proteins synthesized in leaves in response to herbivore attack.     

Purification and characterization of human seminal plasma acid phosphatase

A 81-fold purification of human seminal plasma acid phosphatase was obtained by a three-step procedure, involving ammonium sulfate precipitation, DEAE-cellulose chromatography and Sephadex G-200 gel filtration. Homogeneity of the preparation during purification steps was tested by polyacrylamide gel electrophoresis and only one major band was obtained after the final step. The pH optimum for the activity of the purified enzyme was 5.6 and thermal stability was obtained even up to 40 degrees C. PNPP was the most specific synthetic substrate. The Km of purified seminal acid phosphatase towards PNPP was 1.5 X 10(-3) M. Among the metal ions tested, Hg+2 showed an I50 value of 4.2 X 10(-7) M. Studies with PCMB, PMSF and EDTA did not show any inhibition, whereas NaF and L(+)tartrate, at 1 mM concentration, inhibited the enzyme by 95% and 85%, respectively.     

Purification and partial chemical characterization of a glycoprotein with antifertility activity from human seminal plasma

The preparation of a highly purified antifertility factor from human seminal plasma is described, and some of its biochemical properties have been determined. Purification was achieved by ultracentrifugation of particle-free human seminal plasma, followed by chromatography of the precipitate using carboxymethyl cellulose, concanavalin A, and Sepharose 6B columns. An occasional contaminant was further removed by preparative isoelectric focusing. During the purification procedures, the activity of the fractions was monitored by mixing them with capacitated mouse spermatozoa for 20 min before adding an aliquot to intact mouse oocytes, and determining fertilization after 24 h. The I50 (amount causing a 50% reduction in fertilization as compared to the control) of the final purified factor was 45 micrograms protein. Purity was established by standard and sodium dodecyl sulfate disc gel electrophoresis, isoelectric focusing, high-pressure liquid chromatography, and sedimentation analysis. These methods, as well as Sepharose gel filtration, were also used for the molecular weight estimations; good agreement was obtained between the various techniques. The factor appears to be a glycoprotein with a molecular weight of about 200,000. It consists of two subunits with molecular weights of about 125,000 and 72,000 and s-20,w of 6.2 s and 4.3 s. The factor contains relatively high amounts of aspartic acid and glutamic acid residues as well as leucine and serine, but only small amounts of tryptophan and no methionine was detected. The carbohydrate fraction is particularly rich in galactose and N-acetylgalactosamine but also contains mannose and N-acetylglucosamine and small amounts of fucose and sialic acid.