Enhancement of in vitro release of FSH from rat pituitaries by a synthetic nonapeptide fragment of human seminal plasma inhibin

A synthetic C-terminal nonapeptide fragment of human seminal plasma inhibin preferentially enhances the basal release of FSH from rat pituitaries incubated in vitro, which indicates a direct action of the peptide on the pituitary. However, in the presence of LHRH, both FSH and LH release was increased particularly at higher doses of the nonapeptide. There was no change in prolactin release at 5 and 50 ng/ml but prolactin release was suppressed significantly at 500 ng/ml.     

Development of a radioimmunoassay for human seminal plasma inhibin.

Using a homogeneous inhibin preparation from human seminal plasma with a molecular weight of about 19 000, a sensitive and specific radioimmunoassay (RIA) for inhibin has been developed. None of the purified hormones tested, such as LH, FSH and prolactin from different species, showed any cross-reaction in this RIA. Steroid hormones such as testosterone, dihydrotestosterone, oestradiol-17 beta and progesterone did not interfere with the assay. The antiserum had an affinity constant (Ka) of 2.379 X 10(9). The assay sensitivity was 10-15 ng per tube and the intra- and inter-assay coefficients of variation were 5-7% (n = 6) and 15% (n = 10) respectively. The recovery for inhibin added to the serum of a castrated man was 95-110%. Using this RIA, inhibin levels in various biological fluids and tissues were measured. Normo-spermic semen contained significantly higher levels of inhibin than did oligospermic semen. Human prostate contained a substantial quantity of inhibin. Monkey semen, rat serum, and bovine, ovine and porcine follicular fluids cross-reacted in the RIA, while ram testicular inhibin and bull semen did not do so. In developing (9-28 days of age) male rats, circulating inhibin levels showed an inverse relationship with serum FSH levels. In female rats of this age endogenous inhibin concentrations changed in parallel with those of serum FSH.     

Suppression of steroidogenesis in mice granulosa cells by a synthetic nonapeptide fragment of human seminal plasma inhibin.

A synthetic nonapeptide, which is C-terminal sequence of 94-amino acid of prostatic inhibin peptide was tested for progesterone and estrogen secretion by mouse granulosa cell cultures. Nonapeptide suppressed the progesterone and estrogen synthesis, the magnitude of suppression was highest at 5 ng dose level for progesterone and 50 ng dose level for estradiol. The study suggests that, nonapeptide exerts its effect by impairing the binding of FSH to granulosa cell receptors.     

Partial amino acid sequence of a human seminal plasma peptide with inhibin-like activity

An extract of human seminal plasma was found to have inhibin-like activity. The active factor was purified to homogeneity by ion exchange chromatography, molecular sieving and high performance liquid chromatography. The purified material has a mass of approximately 5 kDa and is very basic. Amino acid analysis showed the presence of approximately 35 residues while the sequencing data allowed the determination of the N-terminal 31 amino acids. There is a possibility of an additional 2–4 residues at the C-terminus, which could not be determined.     

Phosphorylation of the human interleukin-2 receptor and a synthetic peptide identical to its C-terminal, cytoplasmic domain

The recombinant human interleukin-2 (IL-2) receptor was expressed in mouse mammary epithelial cells following the transfection of these cells with an expression vector containing the human IL-2 receptor cDNA. The recombinant IL-2 receptor in these cells was rapidly phosphorylated in response to phorbol myristate acetate (PMA), but its phosphorylation could not be detected in the absence of PMA or upon addition of human IL-2. The C-terminal, cytoplasmic peptide domain of the IL-2 receptor, Gln-Arg-Arg-Gln-Arg-Lys-Ser-Arg-Arg-Thr-Ile, was synthesized and used as a substrate for protein kinase C. The Km for phosphorylation of the peptide by protein kinase C was 23 microM. The stoichiometry of phosphorylation was 1 mol of phosphate/mol of peptide and serine was the predominant amino acid phosphorylated. Because this peptide was a good substrate for protein kinase C in vitro, it was possible that the same serine (serine 247) was also phosphorylated in the receptor in the cell. The IL-2 receptor gene in the expression vector was therefore altered by site-directed mutagenesis to code for an IL-2 receptor containing an alanine in the place of serine 247. The IL-2 receptor expressed by these cells was not phosphorylated in the presence of PMA. These data suggest that protein kinase C, in response to PMA, phosphorylates the C-terminal serine residue (serine 247) in the human IL-2 receptor.     

Identification of novel semenogelin I-derived antimicrobial peptide from liquefied human seminal plasma.

Semenogelin I (SgI) is one of the most abundant proteins in human seminal plasma. SgI plays a key role in sperm coagulation and spermatozoon immobilization. In addition, SgI and/or its proteolytic fragments are involved in regulating spermatozoon motility, capacitation and inhibin-like activity. However, little is known about the antibacterial activity of SgI-derived peptides. By a combination of ion-exchange, gel filtration and high-performance liquid chromatography, peptides from liquefied human seminal plasma from 40 healthy donors were isolated and characterized. N-terminal amino-acid sequencing and fast atom bombardment mass spectrometry revealed that four isolated peptides were SgI-derived, namely SgI-29 (85-113), SgI-46 (85-130), SgI-47 (85-131) and SgI-52 (85-136). Interestingly, SgI-29, SgI-46 and SgI-47 are newly identified SgI-derived peptides. Antimicrobial activity assay results indicated that synthesized SgI-29 had strong antibacterial activity toward various bacterial strains. Our results indicate that SgI can be digested into small fragments like newly identified SgI-29, SgI-46 and SgI-47 and may have diversified functions.     

Immunosuppressive activity of human seminal plasma. I. Inhibition of in vitro lymphocyte activation.

A high molecular weight fraction prepared from human seminal plasma by gel filtration chromatography suppresses human lymphocyte transformation and DNA synthesis induced by mitogens (PHA, Con A, PWM), antigens (Candida albicans, tetanus toxoid), and allogenic cells. This same fraction also suppresses the stimulated response of mouse lymphocytes to allogenic cells and to various mitogens, including T cell-dependent and T cell-independent mitogens. The induction, but not the expression, of cell-mediated cytotoxicity is also suppressed. Similar high molecular weight fractions suppress the in vitro humoral response of mouse spleen cells to both a T cell-dependent (SRBC) and a T cell-independent (DNP-F) antigen. The high m.w. fraction exhibited in vitro suppressive activity at concentrations of 0.1 to 1.0 mg/ml which corresponds to a 1/50 or greater dilution of human seminal plasma. These observations support the concept that a local immune response against sperm in the female reproductive tract is actively suppressed by a component in seminal plasma.     

Characterization of a protein kinase from human seminal plasma.

Localization of creatine kinase in isolated cardiac cells nuclei has been studied. Carefully purified cardiac nuclei preparations contain creatine kinase electrophoretically similar to the mitochondrial creatine kinase isoenzyme. Histochemical electron microscopic investigations have shown that nuclear creatine kinase is localized inside nuclei closely to chromatin.     

Ion-channel properties of mastoparan, a 14-residue peptide from wasp venom, and of MP3, a 12-residue analogue.

Mastoparan, a 14-residue peptide, has been investigated with respect to its ability to form ion channels in planar lipid bilayers. In the presence of 0.3-3.0 microM mastoparan, two types of activity are seen. Type I activity is characterized by discrete channel openings, exhibiting multiple conductance levels in the range 15-700 pS. Type II activity is characterized by transient increases in bilayer conductance, up to a maximum of about 650 pS. Both type I and type II activities are voltage dependent. Channel activation occurs if the compartment containing mastoparan is held at a positive potential; channel inactivation if the same compartment is held at a negative potential. Channel formation is dependent on ionic strength; channel openings are only observed at KCl concentrations of 0.3 M or above. Furthermore, raising the concentration of KCl to 3.0 M stabilizes the open form of the channel. Mastoparan channels are weakly cation selective, PK/Cl approximately 2. A 12-residue analogue, des-Ile1,Asn2-mastoparan, preferentially forms type I channels. The ion channels formed by these short peptides may be modelled in terms of bundles of transmembrane alpha-helices.     

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.     

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.     

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.     

Inhibition of p53 activity in vitro and in living cells by a synthetic peptide derived from its core domain

Much effort was expended to develop anti-cancer drugs that restore the function of the p53 tumor suppressor protein. However, the p53 activity might be harmful to the organism by amplifying side effects of chemotherapy. Therefore, under certain conditions, inhibition of p53 can serve to prevent inappropriately triggered apoptosis in normal tissues. We have identified a short 22-mer peptide derived from the p53 core domain (peptide 14), which can inhibit p53 specific DNA binding. Upon introduction in living cells, peptide 14 inhibited the ability of p53 to transactivate a reporter gene. Moreover, peptide 14 blocked p53-induced apoptosis in two different cell lines. Peptide 14-mediated inhibition of p53 activity appears to operate via the binding of peptide to the core and/or C-terminal domains of the p53 protein. Our findings provide a basis for the development of a novel approach aimed at the inhibition of p53. This could be essential for the protection from cell death in tissues thus suppressing for example neurodegenerative process or side effects of radio- or chemotherapy.     

Crystal structure of a prostate kallikrein isolated from stallion seminal plasma: a homologue of human PSA

Prostate-specific kallikrein, a member of the gene family of serine proteases, was initially discovered in semen and is the most useful serum marker for prostate cancer diagnosis and prognosis. We report the crystal structure at 1.42A resolution of horse prostate kallikrein (HPK). This is the first structure of a serine protease purified from seminal plasma. HPK shares extensive sequence homology with human prostate-specific antigen (PSA), including a predicted chymotrypsin-like specificity, as suggested by the presence of a serine residue at position S1 of the specificity pocket. In contrast to other kallikreins, HPK shows a structurally distinct specificity pocket. Its entrance is blocked by the kallikrein loop, suggesting a possible protective or substrate-selective role for this loop. The HPK structure seems to be in an inactivated state and further processing might be required to allow the binding of substrate molecules. Crystal soaking experiments revealed a binding site for Zn(2+) and Hg(2+), two known PSA inhibitors.     

Isolation and characterization of a basic carboxypeptidase from human seminal plasma

A carboxypeptidase which cleaves the C-terminal arginine or lysine from peptides was purified by a two-step procedure; gel filtration on Sephacryl S-300 and affinity chromatography on arginine-Sepharose. The activity increased 280% after the first step, indicating the removal of an inhibitor from the crude starting material. The activity in the crude seminal plasma eluted from the Sephacryl S-300 column with an apparent Mr 98,000 and after purification with an Mr 67,000, indicating that it binds to another protein in the crude seminal plasma. When analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a single band at Mr 53,000 was seen which was converted to two smaller bands (Mr 32,000 and/or 26,000) after reduction. The seminal plasma carboxypeptidase has a neutral pH optimum, is inhibited by o-phenanthroline and by the inhibitor of carboxypeptidase B-type enzymes, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, and can be activated by cobalt. The purified enzyme has a high specific activity (67.8 mumol/min/mg) with the ester substrate benzoyl (Bz)-Gly-argininic acid and readily cleaves Bz-Ala-Lys, Bz-Gly-Arg, and Bz-Gly-Lys. It also hydrolyzes biologically active peptides such as bradykinin (Km = 6 microM, kcat = 43 min-1), Arg6-Met5-enkephalin (Km = 103 microM, kcat = 438 min-1), and Lys6-Met5-enkephalin (Km = 848 microM, kcat = 449 min-1). The seminal plasma carboxypeptidase did not cross-react with antiserum to human plasma carboxypeptidase N; other properties distinguish it from the blood plasma enzyme as well as from pancreatic carboxypeptidase B and granular, acid carboxypeptidase H (enkephalin convertase). The carboxypeptidase could be involved in the control of fertility by activating or inactivating peptide hormones in the seminal plasma. In addition it could contribute to the degradation of basic proteins during semen liquefaction.     

Purification, biochemical properties and active sites of N-acetyl-beta-D-hexosaminidases from human seminal plasma.

Two isoenzymes of N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30) (Hex A and Hex B) from human seminal plasma were purified to homogeneity with specific activities of 26 and 60 units/mg of protein respectively. N-Acetyl-beta-D-glucosaminidase activity was inseparable from N-acetyl-beta-D-galactosaminidase activity in both Hex A and Hex B by various conventional chromatographic procedures. Although Km values of N-acetyl-beta-glucosaminidase activity of Hex A and Hex B were similar (1.33 mM), those of N-acetyl-beta-galactosaminidase activity were 0.14 mM for Hex A and 0.40 mM for Hex B. However, pH optima and temperature optima were identical for N-acetyl-beta-glucosaminidase and N-acetyl-beta-galactosaminidase activities of both isoenzymes; Hex A was far more heat-sensitive than Hex B. Thiol-reactive compounds such as silver salts, mercuric salts, p-chloromercuribenzoate and thimerosal strongly inhibited the N-acetyl-beta-glucosaminidase activities of both isoenzymes. GSH protected the enzyme activities from inactivation caused by these reagents, confirming the presence of thiol groups at the active centres. Inhibitions of N-acetyl-beta-glucosaminidase activities of both isoenzymes by metal salts and organic anions were comparable; acetate and arsenite were effective inhibitors for both isoenzymes. In contrast, inhibitions of N-acetyl-beta-glucosaminidase activities of the two isoenzymes by iodoacetic acid, iodoacetamide and ethylmaleimide were not comparable; Hex B was more susceptible to inhibition by these agents at 20 mM concentration. The N-acetyl-beta-glucosaminidase activities of both isoenzymes are strongly inhibited, in decreasing order, by N-acetyl-galactosamine, mannosamine, disaccharic acid lactone, N-acetylglucosamine and gluconolactone. The Ki values of the N-acetyl-beta-glucosaminidase and N-acetyl-beta-galactosaminidase activities for N-acetylhexosamines and results from mixed-substrate kinetics indicated that the activities for the two substrates are located at different sites in Hex A and at the same site in Hex B. The Mr values of Hex A and Hex B were determined to be 195,000 and 210,000 respectively by gel filtration through Sephadex G-200. SDS/polyacrylamide-gel electrophoresis revealed that Hex A and Hex B are each composed of four subunits corresponding to Mr about 50,000 each. No further polypeptide chain was obtained after reduction and alkylation of Hex A and Hex B with 10 mM-dithiothreitol and 10 mM-iodoacetamide.     

Effects of active immunization against a synthetic peptide sequence of the inhibin alpha-subunit on plasma gonadotrophin concentrations, ovulation rate and lambing rate in ewes.

Thirty adult Mule (Blue-faced Leicester x Swaledale) ewes were actively immunized against a synthetically produced peptide corresponding to the N-terminus of the alpha-subunit of bovine inhibin conjugated to tuberculin purified protein derivative (PPD). Primary immunization in the late anoestrous period was followed by two booster injections at 5 week intervals. Control groups were either not immunized (n = 15) or received PPD only (n = 15). Ten days after the second booster, oestrus was synchronized using progestagen sponges and ovulation rate was assessed by laparoscopy on days 9-10 of the cycle. Blood samples were taken at the time of each immunization and immediately before laparoscopy. Ewes were mated with fertile rams in mid-November and the resulting conception, pregnancy and lambing rates monitored. All inhibin-immunized ewes generated antibodies that bound 125I-labelled native bovine inhibin (M(r) 32,000), and their plasma follicle-stimulating hormone (FSH) concentrations after the second booster were significantly higher than the preimmunization values (30%; P less than 0.001) and the corresponding value in the controls (25%; P less than 0.025). Inhibin immunization was associated with a 90% increase in ovulation rate (P less than 0.005) and had no adverse effect on conception rate (100%), pregnancy rate (100%) or length of gestation (146 days). However, only a 37% increase (P less than 0.05) in lambing rate was recorded for inhibin-immunized ewes, indicating a higher incidence of wastage of ova, or embryos, or both, in these ewes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Missense mutations define a restricted segment in the C-terminal domain of phytochrome A critical to its regulatory activity.

The phytochrome family of photoreceptors has dual molecular functions: photosensory, involving light signal perception, and regulatory, involving signal transfer to downstream transduction components. To define residues necessary specifically for the regulatory activity of phytochrome A (phyA), we undertook a genetic screen to identify Arabidopsis mutants producing wild-type levels of biologically defective but photochemically active and dimeric phyA molecules. Of eight such mutants identified, six contain missense mutations (including three in the same residue, glycine 727) clustered within a restricted segment in the C-terminal domain of the polypeptide. Quantitative photobiological analysis revealed retention of varying degrees of partial activity among the different alleles--a result consistent with the extent of conservation at the position mutated. Together with additional data, these results indicate that the photoreceptor subdomain identified here is critical to the regulatory activity of both phyA and phyB.