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K H Scheit

Publications and source records attributed to K H Scheit.

At least 73 records · Page 4Linked to original sources
PubMed1986

The major protein of bull seminal plasma is a secretory product of seminal vesicle.

We isolated the major protein with apparent molecular weight, Mr, 15,000-16,000 from seminal plasma as well as from seminal vesicle secretion of bull and proved by amino acid analysis and tryptic peptide mapping that the two proteins were identical. An antiserum against this major protein was employed to quantitate and identify the major protein in seminal plasma as well as in seminal vesicle secretion. The antiserum did not cross-react with proteins from bovine or human plasma or follicular fluid, respectively. Cell-free translation of poly(A+)RNA isolated from seminal vesicle tissue resulted in formation of one major species with apparent Mr 18,000. Using the anti-major protein antiserum, this major species was specifically immuno absorbed. We thus provided evidence that the major protein component of bull seminal plasma is a secretory protein of seminal vesicles. Furthermore, it appeared that the isolated major protein may be closely related to the protein PDC109, purified from bull seminal plasma and sequenced by Esch et al. (Biochem. Biophys. Res. Commun. 113, 861-867 (1983).

Animals↗
PubMed1986

Demonstration of mRNAs for oxytocin and prolactin in porcine granulosa and luteal cells. Effects of these hormones on progesterone secretion in vitro.

The relative levels of mRNAs for relaxin, prolactin, inhibin and oxytocin have been measured in porcine granulosa as well as luteal cells by hybridisation to single-stranded synthetic DNA. The likelihood of a paracrine function of oxytocin and prolactin in the porcine ovary was inferred from the in vitro effects of both hormones on progesterone secretion of ovarian cells. Both hormones were found to inhibit progesterone secretion of luteal cells. In contrast, only prolactin but not oxytocin stimulated progesterone secretion in granulosa cells.

Animals↗
PubMed1986

The major basic proteins of bull seminal vesicle secretion.

We have employed HPLC on reversed phase columns to analyse the major basic proteins from bull seminal vesicle secretion. The identification of proteins was achieved by comparison with authentic protein samples from bull seminal plasma as well as immunological characterisation using antisera directed against the latter proteins. The major basic proteins from bull seminal plasma: bull seminal proteinase inhibitor II (BUSI II), the seminal ribonuclease BS1, the protein P6 as well as the antimicrobial protein were also identified as the main constituents of the fraction of basic proteins derived from seminal vesicle secretion. FPLC using Mono S HR columns was also found to resolve the mixture of basic proteins and proved to be especially useful with respect to the isolation of the antimicrobial protein from basic proteins of seminal vesicle secretion. The identity of the antimicrobial protein from bull seminal plasma with the respective protein from seminal vesicle secretion was confirmed by amino-acid analysis and comparison of tryptic peptide patterns by HPLC. The antimicrobial protein was isolated from seminal vesicle secretion with a yield of 3 mg/ml of secretion.

Amino Acids↗
PubMed1986

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.

Animals↗
PubMed1985

Incorporation of the antimicrobial protein seminalplasmin into lipid bilayer membranes.

The interaction between seminalplasmin, an antimicrobial protein from bull semen, and lipid bilayers has been investigated. The fluorescence of the single tryptophan residue of the protein was measured. In the presence of phosphatidylcholine or phosphatidic acid bilayer vesicles the fluorescence maximum was shifted to shorter wavelengths, indicating transfer of the tryptophan to a more apolar environment. Circular dichroism spectra show an increased alpha-helical content for the protein in the presence of lipid. Quenching experiments clearly show the incorporation of the protein with the tryptophan localized near the bilayer surface. The shift of the tryptophan fluorescence emission was used to monitor the lipid phase transition in phosphatidylcholine membranes.

Animals↗
PubMed1985

Seminalplasmin, an antimicrobial protein from bull seminal plasma, inhibits growth, and synthesis of nucleic acids and proteins in S. cerevisiae.

Seminalplasmin, an antimicrobial protein present in bovine seminal plasma, is shown to inhibit the growth of, as well as nucleic acid and protein synthesis in, wild-type Saccharomyces cerevisiae SM202, when used at concentrations greater than 200 micrograms/ml, in contrast to 20 micrograms/ml that is sufficient for Escherichia coli. An osmotically labile strain of S. cerevisiae VY1160 is 1-2 orders of magnitude more sensitive to seminalplasmin than the wild-type strain. RNA synthesis in protoplasts and nuclei of S. cerevisiae SM 202 was also about as sensitive to seminalplasmin as in E. coli and S. cerevisiae VY1160. The RNA polymerases I and II from S. cerevisiae were strongly inhibited by seminalplasmin in vitro, while DNA and protein syntheses were not affected by seminalplasmin in cell-free systems, unlike in the whole cells. It is concluded that seminalplasmin acts in S. cerevisiae by entering the cells and inhibiting transcription.

Anti-Bacterial Agents↗
PubMed1984

Effect of seminal plasmin on rRNA synthesis in Saccharomyces cerevisiae.

Seminal plasmin, the highly basic, antimicrobial protein, isolated from bull semen, was found to inhibit the transcription of ribosomal RNA in yeast. Protein synthesis and processing of rRNA remained unaffected. Seminal plasmin appears to be useful for studies of the biosynthesis of yeast rRNA in pulse-chase experiments.

Anti-Infective Agents↗
PubMed1984

The effect of nucleotide analogs on cell-free gene expression.

The effects of the following pyrimidine nucleoside 5'-triphosphates: f5 UTP, br5 UTP, rTTP, s2 UTP, s4 UTP and s2 CTP on cell-free expression of the beta-galactosidase gene in lambda h80dlac DNA as well as the galactokinase gene in plasmid 01-14 were investigated. Only rTTP could substitute UTP in cell-free gene expression without restriction. Combinations of the other analogs with their respective natural congeners led to inhibition of gene expression. All analogs were found to inhibit transcription. Whereas br5 UTP and s4 UTP did not affect translation, mRNA containing s2 UMP or s2 CMP residues respectively was found to function poorly in translation. Only in the case of f5 UTP could ambiguitive behaviour be demonstrated. Whether mispairing of f5 UMP residues, responsible for this ambiguity takes place in transcription or in translation, could not be decided.

Cell-Free System↗
PubMed1984

Seminal plasmin, an antimicrobial protein from bull semen, inhibits gene expression in E. coli.

RNA synthesis in Escherichia coli was immediately inhibited after addition of seminal plasmin, an antimicrobial protein from bull semen. RNA synthesis progressively decreased within 12 min and then ceased completely. In contrast, protein synthesis was not affected within the first 12 min, but thereafter became progressively inhibited. Inhibition of RNA synthesis by seminal plasmin in E. coli interfered with induction of beta-galactosidase by isopropyl-beta-D-thiogalactoside (IPTG). This implied inhibition of beta-galactosidase mRNA synthesis by seminal plasmin in vivo. The sensitivities of total in vivo RNA synthesis and beta-galactosidase mRNA synthesis against seminal plasmin were found to be similar. Seminal plasmin had no effect on the uptake of the inducer IPTG by E. coli cells.

Animals↗
PubMed1984

Immunological identification of seminalplasmin in tissue extracts of sex glands of bull.

Using immunoglobulin G (IgG) antibodies raised against highly purified, homogeneous seminalplasmin, an antimicrobial protein of bovine seminal plasma, it has been shown that bovine ampullae, gland vesicularis and corpus prostate, but not testes and epididymis, contain seminalplasmin. The content as estimated by radioimmunoassay employing 125I-seminalplasmin was: ampullae, 267 +/- 13; gland vesicularis, 275 +/- 14; and corpus prostate, 445 +/- 22 micrograms per g wet weight of the tissue. Seminalplasmin, as characterized by high-performance liquid chromatography and in vivo inhibition of RNA synthesis in E. coli, was isolated from gland vesicularis. The seminalplasmin content of bovine seminal plasma was shown to be 1%. A chymotryptic peptide of seminalplasmin comprising residues 1-13 from the amino terminus was found to compete with 125I-seminalplasmin for binding to anti-seminalplasmin IgG.

Animals↗
PubMed1984

Characterization of basic proteins of bull seminal plasma.

We have employed high-performance liquid chromatography on reversed phase columns to analyse the major basic proteins from bull seminal plasma. The proteins were separated preparatively and characterized with respect to molecular mass, amino-acid composition as well as by means of immunodiffusion against specific antisera. The following proteins could be identified: bull seminal proteinase inhibitor II (BUSI II), two seminal RNAases, the seminal antimicrobial protein and proteolytic fragments, derived from it, and a hitherto unknown protein P6 of molecular mass 20 000 Da. Another unknown protein, P5, found to be formed during preparation of the basic protein fraction turned out to be a proteolytic fragment of protein P6 with a molecular mass of 8 750 Da for the polypeptide chain. Antisera against the isolated proteins were raised in rabbits and their specificity established. Single radial immunodiffusion was used to determine the concentration of the above basic proteins in bull seminal plasma: BUSI II (0.25 mg/ml), seminal RNAases (6.5 mg/ml) and protein P6 (2.9 mg/ml).

Amino Acids↗
PubMed1983

Amino acid sequence of seminalplasmin, an antimicrobial protein from bull semen.

Analytical ultracentrifugation of highly purified seminalplasmin revealed a molecular mass of 6300. Amino acid analysis of the protein preparation indicated the absence of sulfur-containing amino acids cysteine and methionine. The amino acid sequence of seminalplasmin was determined by manual Edman degradation of peptides obtained by proteolytic enzymes trypsin, chymotrypsin and thermolysin: NH(2)-Ser Asp Glu Lys Ala Ser Pro Asp Lys His His Arg Phe Ser Leu Ser Arg Tyr Ala Lys Leu Ala Asn Arg Leu Ser Lys Trp Ile Gly Asn Arg Gly Asn Arg Leu Ala Asn Pro Lys Leu Leu Glu Thr Phe Lys Ser Val-COOH. The number of amino acids according to the sequence were 48, the molecular mass 6385. As predicted from the sequence, seminalplasmin very likely contains two alpha-helical domains in which residues 8-17 and 40-48 are involved. No evidence for the existence of beta-sheet structures was obtained. Treatment of seminalplasmin with the above proteases as well as with amino peptidase M and carboxypeptidase Y completely eliminated biological activity.

Journal Article↗
PubMed1983

Purification and spectral characterization of seminalplasmin, an antimicrobial protein from bull semen.

A new method for the purification of seminalplasmin, an antimicrobial protein from bull semen, was developed. The last step of the procedure involved preparative high performance liquid chromatography on a reversed phase column. Highly purified seminalplasmin was characterized by CD, absorption, fluorescence spectroscopy, double immunodiffusion and biological activity. Analytical ultracentrifugation revealed a molecular mass of 6300 Da. Amino-acid analysis of the protein preparation indicated the absence of sulfur-containing amino acids cysteine and methionine.

Amino Acids↗
PubMed1982

Synthesis and physicochemical properties of two analogs of poly(dA): poly(2-aminopurine-9-beta-D-deoxyribonucleotide) and poly 2-amino-deoxyadenylic acid.

Polymerization of chemically synthesized dn2h6ATP and dn2ATP by deoxynucleotidyl transferase from calf thymus furnished poly(dn2h6A) and poly(dn2A) respectively. The synthetic polynucleotides were characterized by spectroscopic, ultracentrifugation and enzymatic methods. In polynucleotide-polynucleotide interaction, poly(dn2h6A) and poly(dn2A) behaved like analogs of poly(dnA).

Animals↗
PubMed1982

Substrate specificity of CTP synthetase from Escherichia coli.

The stoichiometry of the enzymatic reaction catalyzed by CTP synthetase from Escherichia coli was analyzed by high-performance liquid chromatography. The results revealed that for every mole of UTP transformed to CTP, one mole of ATP was converted to ADP. The substrate specificity of CTP synthetase from E. coli was investigated by means of UTP analogs. Chemical modification of UTP involved either the uracil, ribose or 5'-triphosphate part. None of the UTP analogs studied proved to be a substrate. The capacity of the UTP analogs to inhibit CTP synthetase was investigated. From the UTP derivatives employed only 2-thiouridine 5'-triphosphate was found to inhibit the enzyme competitively with reasonable affinity: Ki/Km(UTP) = 1. This study indicated that the three main structural elements of the UTP molecule: uracil, ribose and 5'-triphosphate moiety, contribute to substrate specificity. The behaviour of a limited number of CTP analogs as product-like inhibitors supported this view.

Carbon-Nitrogen Ligases↗
PubMed1981

A minimal mechanism for abortive initiation of transcription of T7 DNA.

A steady state kinetic analysis of product inhibition in abortive initiation of transcription at promoters A1 and A3 of T7 DNA was carried out. The obtained kinetic data are in agreement with a mechanism of ordered product release with pppApU being released first from the central complex. A minimal general mechanism for abortive initiation satisfying the kinetic results is given. The stimulation of transcription of T7 DNA by pppApU at low substrate concentrations was investigated.

DNA, Viral↗
PubMed1981

Properties of P3 esters of nucleoside triphosphates as substrates for RNA polymerase from Escherichia coli.

P3-[(2,4-Dinitrophenyl)amino]ethyl (DNPNHEt) and P3-methyl phosphate esters of nucleoside 5'-triphosphates have been synthesized. Their properties as substrates in the initiation and elongation steps of transcription have been examined by using RNA polymerase from Escherichia coli and poly[d(A-T)] or T7 DNA as templates. It is shown that transcription can be initiated by ATP-EtNHDNP and that 2,4-dinitrophenyl residues are incorporated at the 5' end of the RNA molecules. Steady-state kinetic experiments of abortive initation on promoters A1 and A3 of T7 DNA revealed that ATP-EtNHDNP, ADP-EtNHDNP, and ATP-OCH3 have lower Km values and markedly reduced Vmax values compared to those of ATP. The two classes of esters, NTR-EtNHDNP and NTP-OCH3, were found to differ regarding their utilization as substrates for elongation. Both ATP-OCH3 and UTP-OCH3 are substrates for transcription. However, only the pyrimidine derivatives of NTP-EtNHDNP are elongation substrates which release DNPNHEt-PP upon utilization. This dramatic difference between the purine and pyrimidine derivatives of NTP-EtNHDNP reflects a selective process in the transcriptional complex for purines and pyrimidines.

DNA-Directed RNA Polymerases↗