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Biomedical subjects

H Tschesche

Publications and source records attributed to H Tschesche.

At least 199 records · Page 11Linked to original sources
PubMed1976

Occurrence of multiple forms of bull and ram acrosin during proenzyme activation and inhibition of activation by p-nitrophenyl p'-guanidinobenzoate.

Proacrosin was extracted from freshly ejaculated bull and ram spermatozoa with acidic buffer solution pH 3.0, and was partially purified by gel filtration to remove acrosin inhibitors. The occurrence of multiple active forms during proacrosin activation at pH 7.8 was monitored by active enzyme staining of samples after polyacrylamide gel electrophoresis at pH 3.8. For comparison, the protein pattern of such activation samples was also determined after sodium dodecylsulfate-polyacrylamide gel electrophoresis. Proacrosin activation was completely prevented in the presence of 10(-4) M p-nitrophenyl p'-guanidinobenzoate.

Acrosin↗
PubMed1976

Hydrolysis-resynthesis equilibrium of the lysine-15--alanine-16 peptide bond in bovine trypsin inhibitor (Kunitz).

Catalytic amounts of bovine beta-trypsin, bovine alpha-chymotrypsin and porcine plasmin establish a true thermodynamic equilibrium between virgin (I) (reactive site Lys15-Ala16 peptide bond intact) and modified (I) (this bond hydrolyzed) bovine trypsin/kallikrein inhibitor (Kunitz). The very slow reaction rates for attaining equilibrium are pH-dependent and differ for different enzymes. Optimal rates are for beta-trypsin at pH 3.75, for alpha-chymotrypsin at pH 5.5, and for plasmin at pH 5.0. Under conditions of optimum pH the equilibrium is reached with the highest rate by plasmin. In 10(-5)M inhibitor solutions the equilibrium concentrations of virgin and modified inhibitor are established by plasmin after almost 300 days starting from either pure virgin or pure modified inhibitor. Thus, the hydrolysis constant KHyd = [I]/[I] is determined to be 0.33 at pH 5.0. In spite of many unsuccessful attempts, this demonstrates that the reactive site peptide bond Lys15-Ala16 in the bovine trypsin inhibitor (Kunitz) can be hydrolyzed by catalytic amounts of endopeptidase. It further confirms that the hydrolyzed Lys15-Ala16 peptide bond in modified inhibitor is subject to thermodynamic control resynthesis.

Alanine↗
PubMed1976

Activation of chymotrypsinogen by boar acrosin and its prevention by antiboar acrosin rabbit gamma-globulins.

Activation of chymotrypsinogen by bovine trypsin or boar sperm acrosin was followed up using Nalpha-acetyl-L-tyrosine ethyl ester in a highly sensitive test system. Inhibition studies employing antiboar acrosin rabbit gamma-globulins showed the following results. 1) Whereas the acrosin-induced activation velocity was significantly depressed in the presence of the antibodies, the trypsin-catalyzed activation rate was not diminished. 2) The antibodies enhanced the acrosin-catalyzed cleavage rate of BzArgOEt significantly, but not the trypsin-catalyzed cleavage rate of this substrate. 3) Autodigestion of acrosin was considerably reduced in the presence of the antibodies. The enzymatic test system used is especially suitable to study the specificity of acrosin antibodies or their affinity to related enzymes if only small amounts of these substances are available.

Acrosin↗
PubMed1976

Influence of boar acrosin antibodies produced in rabbit and sheep on chymotrypsinogen activation catalyzed by acrosin from boar, bull, ram, rabbkt and human.

Activation of bovine chymotrypsinogen is catalyzed with increasing velocity by human, rabbit, boar, bull and ram acrosin. Antiboar-acrosin rabbit gamma-globulins cause a significant reduction in the proenzyme activation rate induced by boar and bull acrosin, but only a weak reduction or none if ram or rabbit acrosin is the activating agent. The antiboar-acrosin gamma-globulins from sheep strongly inhibit chymotrypsinogen activation by ram, bull and boar acrosin, and significantly inhibit the human acrosin-catalyzed reaction.

Acrosin↗
PubMed1976

Quantitative mass spectral identification of p-bromophenylthiohydantoins from Edman degradations by tetradeuterated internal standards.

p-Bromophenylisothiocyanate is a reagent well suited for the Edman degradation of peptide chains[1-5]. The relative rate of the coupling reaction at the N-terminus is slightly more rapid than with phenyl- or methylisothiocyanate, but less rapid than with pentafluorophenylisothiocyanate. The latter reagent, however, is rapidly hydrolyzed under the conditions of the coupling reaction. The p-bromo substituent facilitates mass spectral identification of the p-bromophenylthiohydantoins obtained, due to its characteristic double peak[1-4]. Quantitation of the amount of p-bromophenylthiohydantoin obtained can easily be achieved in the parent mass spectrum by addition of a known amount of tetradeutero-p-bromophenylthiohydantoin as an internal standard. The tetradeuterated standards give rise to molecular ion peaks four mass units higher than the corresponding undeuterated probes. Standard and probe volatilize almost equally well. The composition in the vapor indicated by the H/D ratio reflects the molar concentrations over the entire range of probe evaporation. A procedure for the preparation of the tetradeutero-p-bromophenyl-isothiocyanate and the respective tetradeuterated amino acid p-bromophenylthiohydantoins is given.

Acetanilides↗
PubMed1976

[The disulfide bridges of the trypsin-kallikrein inhibitor K from snails (Helix pomatia). Thermal inactivation and proteolysis by thermolysin (author's transl)].

Isoinhibitor K is the main component of the complex mixture of isoinhibitors of broad specificity secreted into the mucus by the Roman snail (Helix pomatia). The disulfide pairing was determined after the amino acid sequence had been elucidated. Two cystine-containing peptides with the disulfide bridges Cys32-Cys53 and Cys32-Cys53 plus Cys7-Cys57 were obtained after thermolytic hydrolysis of the native inhibitor at 80 degrees C and chromatographic separation of the peptides using SE-Sephadex. The Cys16-Cys40 disulfide bridge could be reduced selectively by sodium borohydride with no loss in biological activity. This property and the covalent structure correspond to that of the intracellular inhibitor from bovine organs, which is largely homologous in its amino acid sequence to the secretory inhibitor from the snail. The complete covalent structure of isoinhibitor K will be presented. The snail inhibitor is less stable against proteolytic inactivation by thermolysin and against thermal denaturation at pH 8.0 than the inhibitor from bovine organs (Kunitz inhibitor).

Amino Acid Sequence↗
PubMed1975

The amino-acid sequence of isoinhibitor K form snails (Helix pomatia). A sequence determination by automated Edman degradation and mass-spectral identification of the phenylthiohydantoins.

Performic-acid-oxidized isoinhibitor K of snails (Helix pomatia) was subjected to arginine-directed tryptic proteolysis. Six peptide fragments including one overlap peptide from limited cleavage of the Arg-3-Pro-4 bond were purified to homogeneity. Four arginine peptides and the C-terminal peptide were sequenced by automatic Edman degradation using a special peptide program. The phenylthiohydantoins were all identified by chemical ionization mass spectrometry, except four cysteic acid residues that were identified on an amino acid analyzer after acid hydrolysis. Quantitative evaluation of the phenylthiohydantoins by chemical ionization mass spectrometry using total molecular-ion beam integration greatly facilitated sequencing. The mass spectrum of the dipeptide less than Glu-Gly revealed that the N-terminus was blocked by pyroglutamic acid. The complete amino acid sequence of isoinhibitor K was determined. An almost 50% homology between the sequences of the snail inhibitor and the bovine trypsin-kallikrein inhibitor (Kunitz) became obvious. A comparison of all homologous sequences of this particular class of proteins known to date is presented.

Amino Acid Sequence↗
PubMed1975

Trypsin-kallikrein isoinhibitor K (type Kunitz) from snails (Helix pomatia). Purification and characterization.

A basic proteinase inhibitor, isoinhibitor K, was purified by SE-Sephadex C-25 column chromatography from the mixture of acid-stable and heat-stable isoinhibitors of the snail (Helix pomatia). Isoinhibitor K is homogeneous in polyacrylamide gel, cellulose acetate and polyacrylamide-dodecylsulfate electrophoresis. From the electrophoretic mobility in dodecylsulfate-polyacrylamide gel and apparent molecular weight of 6500 +/- 200 was estimated. From the amino acid composition the inhibitor consists of 58 amino acid residues. It contains three disulfide bridges, a C-terminal valine and a lysine residue at the reactive site. Isoinhibitor K inhibits the enzymes: bovine trypsin and chymotrypsin, porcine plasmin and pancreatic kallikrein, the trypsin-like component of Streptomyces griseus proteinase-pronase E, and fungi proteinase K from Tritirachium album Limber, which is only inhibited very slightly in contrast to the effect of the mixture of isoinhibitors. The inhibitory effect of isoinhibitor K against these enzymes is compared to that of the mixture or of other isoinhibitors. The following enzymes are not inhibited by isoinhibitor K: Aspergillus proteinase P and alkaline bacillus proteinase 2231 (Röhm), which both are inhibited by the mixture of isoinhibitors. Porcine elastase, bacterial proteinase N (M) (Röhm), and a trypsin-like proteinase from wheat are not inhibited, porcine acrosin and porcine serum kallikrein only to a very minor extent by the mixture of isoinhibitors. Reactive-site peptide-bond cleavage during inhibition could not be detected. Thus, the inhibitory behaviour is just as broad in specificity and as unusual as that of the trypsin-kallikrein inhibitor (Kunitz) from bovine organs. The N-terminus is blocked by pyroglutamic acid. Isoinhibitor K is the main component of the isoinhibitors secreted into the mucus and amounts to 35-40% of the mixture.

Amino Acids↗
PubMed1975

Kinetics of binding of bovine trypsin-killikrein inhibitor (K unitz) in which the reactive-site peptide bond Lys-15--Ala-16 is cleaved, to alpha-chymotrypsin and beta-trypsin.

Equilibrium measurements of the binding of reactive-site-cleaved (modified) bovine trypsin-kallikrein inhibitor (Kunitz) to alpha-chymotrypsin and beta-trypsin show a stoichiometric 1:1 association with high binding constants. At least in the case of chymotrypsin much evidence is presented that the reaction with modified inhibitor leads to the same complex as the reaction with virgin inhibitor does. The association rate constant of modified inhibitor with chymotrypsin at pH 7, 22.5 degrees C is 15.8 M-1 S-1. This is about 2 x 10(4) times slower than the binding of virgin inhibitor to that enzyme. In the analogous reaction of modified inhibitor with beta-trypsin, however, the association rate constant (1.2 x 10(4) M-1 s-1 at pH 6.9, 22.5 degrees C) is of about the same order of magnitude as it is in the reaction of virgin inhibitor and trypsin. These and analogous phenomena observed in the reactions of virgin and modified soybean trypsin inhibitor (Kunitz) with alpha-chymotrypsin and beta-trypsin suggest that the specificity of both inhibitors to trypsin is strongly reflected in the association rate constants of the modified forms. The dissociation rate constants of the complexes of trypsin-kallikrein inhibitor with chymotrypsin or with trypsin towards the modified inhibitor are estimated to be unmeasurably slow (half-life times of 45 or 1.5 x 10(4) years, respectively).

Alanine↗
PubMed1975

The effect of cleaving the reactive-site peptide bond Lys-15--Ala-16 on the conformation of bovine trypsin-kallikrein inhibitor (K unitz) as revealed by solvent-perturbation spectra, circular dichroism and fluorescence.

Spectroscopic measurements of virgin bovine trypsin-kallikrein inhibitor and its modified species (in which the reactive-site peptide bond Lys-15--Ala-16 is split) indicate a conformational difference between both proteins. The inhibitor contains four tyrosines but no tryptophan. In the modified inhibitor a tyrosyl blue shift is seen in the difference absorption spectrum of modified against virgin inhibitor. The solvent perturbation spectra show an increase of the fraction of exposed tyrosyls from 0.45 in the virgin inhibitor to 0.59 in the modified form. Comparison of the circular dichroism spectra of the modified and virgin inhibitors reveals a decrease of the mean residue ellipticity in the tyrosine and peptide bond region of the modified inhibitor. In the fluorescence spectra a 50% increase in the quantum yield of the tyrosine fluorescence is observed in the modified inhibitor. All these spectroscopic data support the idea, which is also evidenced by the X-ray crystallographic model, that in the modified inhibitor up to five residues from Ala-16 to Arg-20 gain rotational freedom.

Alanine↗
PubMed1975

On the carbohydrate composition of bovine colostrum trypsin inhibitor.

The trypsin inhibitor from bovine colostrum has been separated into several forms by CM-Sephadex and DEAE-cellulose chromatography. These forms differ in the amount and composition of the carbohydrate they contain, which has been quantitated for four components by gas-liquid chromatography and standrad colorimetric procedures. The monosaccharides fucose, mannose, galactose, galactosamine, glucosamine and sialic acids have been determined. A microheterogeneity was establish ed in the carbohydrate moiety, which amounts to about 40% of the total molecular weight (Mr 11 000 - 14 000) of bovine colostrum inhibitor.

Animals↗