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H Tschesche

Publications and source records attributed to H Tschesche.

At least 163 records · Page 9Linked to original sources
PubMed1983

Characterisation of beta 1-anticollagenase from human plasma and its reaction with polymorphonuclear leukocyte collagenase by disulfide/thiol interchange.

A beta 1-serum component, beta 1-anticollagenase, capable of inhibiting various mammalian tissue collagenases, was isolated from human plasma by gel filtration, affinity chromatography and ion-exchange chromatography. The inhibitor contains 1-2 free sulfhydryl groups, which are a prerequiste for inhibitory activity and for binding to the thiol-Sepharose affinity support. Alkylation of beta 1-anticollagenase by iodoacetamide blocks inhibitory activity. The inhibitor was purified to apparent homogeneity and exhibited a Mr = 30500 determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The amino acid and carbohydrate composition was determined. According to its composition and the isoelectric focussing beta 1-anticollagenase is an acidic protein with an isoelectric point of 5.6. Inhibition of human leukocyte collagenase proceeds in a strong 1 : 1 stoichiometric reaction. The mechanism of this association takes place by a disulfide/thiol interchange reaction as has been previously indicated for human leukocyte collagenases in forming the latent enzyme [Macartney, H. W. and Tschesche, H. (1980) FEBS Lett. 119, 327-332]. The beta 1-anticollagenase--leukocyte-collagenase complex (latent enzyme) is activatable by disulfide-containing compounds such as cystine, oxidised glutathione, insulin, relaxin, trypsinogen and others, but not by 179,203-di(S-carboxymethyl)trypsinogen, or its trypsin derivative. Compounds containing inaccessible disulfide bonds, e.g. chymotrypsin, or sulfhydryl groups, e.g. D-penicillamine, do not activate the complex. Activation is, however, easily obtained with the oxidised-glutathione-generating system myeloperoxidase/H2O2/glutathione as was previously demonstrated for the human leukocyte latent collagenase activatable in a phagocytosis-simulated respiratory burst [Tschesche, H. and Macartney, H. W. (1981) Eur. J. Biochem. 120, 183-190].

Amino Acids↗
PubMed1982

A new acrosin inhibitor from boar spermatozoa.

A new proteinase inhibitor (Mr 7500) was isolated to apparent homogeneity from boar spermatozoa by repeated gel filtration on Sephadex G-50 and affinity chromatography on concanavalin-A--Sepharose 4B. The inhibitor strongly inhibits boar acrosin in a competitive 1:1 stoichiometric reaction with Kass = 7 x 10(10)1 mol-1. The inhibitor is a glycoprotein and represents a first member of a new class of proteinase inhibitor with a rather short polypeptide backbone of only 42 amino acid residues and a low cystine content. The basic protein (isoelectric point 9.4) contains a single disulfide loop, which is easily reducible by sodium borohydride. Upon reduction the inhibitory activity is lost, but rapidly regained after air reoxidation of the corresponding half-cystine residues. The reactive site residue was established to be arginine by inhibition with 2,3-butanedione. The inhibitor is rather specific for acrosin and inhibits bovine trypsin only to a limited extent. However, incubation with catalytic amounts of trypsin (or acrosin) at acid pH (pH2-3) rapidly leads to a limited proteolysis at the reactive site with formation of 67% modified (reactive site hydrolysed), but still active inhibitor. The equilibrium constant was established to be Khyd = 2.0.

Acrosin↗
PubMed1982

Separation of the human leucocyte enzymes alanine aminopeptidase, cathepsin G, collagenase, elastase and myeloperoxidase.

A simple and rapid procedure is described for the separation of the human leucocyte enzymes alanine aminopeptidase, cathepsin G, collagenase, elastase and myeloperoxidase. The enzymes are prepared from leucocytes, obtained from buffy coat, by repeated extraction with buffer A(1 M salt concentration). The pooled extracts are successively subjected to batch adsorption on concanavalin A-Sepharose, gel filtration on Sephacryl S-300, affinity chromatography on collagen-Sepharose 4-B, batch adsorption on CM-Sephadex C-50 and adsorption chromatography on hydroxyapatite. The yields of the isolated enzymes of a typical preparation are 47% alanine aminopeptidase, 9% cathepsin G, 90% latent and active collagenase, 23% elastase and approximately 100% myeloperoxidase with respect to the pooled extracts. The cathepsin G, collagenase and elastase preparations are essentially free from other proteolytic enzymes and may be used without further purifications.

Aminopeptidases↗
PubMed1981

The metal ion requirement for activation of latent collagenase from human polymorphonuclear leucocytes.

Latent human PMN leucocyte collagenase (enzyme-inhibitor complex) was shown to require zinc for the property of being activatable by various disulfides [see Macartney, H.W. and Tschesche, H. (1980) FEBS Lett. 119, 327--332]. The active enzyme also requires zinc for activity, indicating a possible participation in the enzyme's reaction mechanism and/or stabilization of the active site. The zinc in the latent enzyme may be removed by dialysis against EDTA, or cysteine. This produces a zinc-free latent enzyme which cannot be activated by any of the disulfide-containing activators. Readdition of zinc to the EDTA-inhibited latent enzyme, at the same concentration as the EDTA, produces an activatable latent enzyme once again. However, excessive zinc concentrations (more than three times the concentration of EDTA) exhibited an inhibitory effect on the activation process. Thereafter the inhibitor cannot be removed by disulfides from the enzyme-inhibitor complex of the latent enzyme. The zinc in the latent enzyme may be replaced by other double-positive metal ions such as cobalt, manganese, magnesium and copper.

Cations, Divalent↗
PubMed1981

N-Acetylalanine aminopeptidase, a new enzyme from human erythrocytes.

A new enzyme liberating N-acetylalanine from N-acetylalanine peptides with high specificity has been isolated from the cytosol of human erythrocytes. The N-acetylalanine aminopeptidase was purified by ammonium sulfate precipitation at 60% saturation, followed by chromatography on columns of Sephadex G-200, SP-Sephadex C-50, and DEAE-Sephadex A-50. About 2 000-fold enrichment was achieved from hemolyzed erythrocytes. The enzyme was homogeneous according to polyacrylamide disc electrophoresis and had a specific activity of 18.1 U/A280 unit. An apparent molecular weight of 300 000 +/- 15 000 was obtained from gel filtrations and was confirmed in the ultracentrifuge in "an active enzyme centrifugation" giving a corrected sedimentation value, s20w of 12 S. The pH optimum in triethanolamine/HCl buffer was around pH 8.3 with N-acetylalanine-4-nitroanilide as substrate, the Km was 0.616 mmol/l. The enzyme was stable between pH 6.0 to 8.0, but lost enzymic activity rapidly below pH 5 and with organic solvents. It is stabilized in a 0.1 M solution of ammonium sulfate. The activity was destroyed by high concentrations of chloromercuribenzoate and di(2-pyridyl)disulfide in an unspecific manner and could not be restored by cysteine. Various protein endoproteinase inhibitors are without influence on the enzymic activity. The enzyme exhibits an aminopeptidase-like activity with release of N-acetylalanine in order of decreasing activity from N-acetylalanine-4-nitroanilide, N-acetylalanyl-alanylalanine, N-acetylalanyl-tyrosyl-isoleucine, N-acetylalanylalanine, N-acetylalanyl-alanyl-alanylalanine, and N-acetylalanine ethyl ester. Several unacetylated peptides and alanine-4-nitroanilide as well as protein substrates were not hydrolyzed. The enzymic activity has not been found in the cytosolic compartment of highly purified human leucocytes. Its physiological function in erythrocytes is still unknown.

Aminopeptidases↗
PubMed1980

Carbon-13 nuclear magnetic resonance studies of the selectively isotope-labeled reactive site peptide bond of the basic pancreatic trypsin inhibitor in the complexes with trypsin, trypsinogen, and anhydrotrypsin.

A previously characterized modification of the basic pancreatic trypsin inhibitor (BPTI), with the carbonyl carbon atom of Lys-15 selectively enriched in 13C, the peptide bond Arg-39--Ala-40 cleaved, and Arg-39 removed, was used for 13C NMR studies of the reactive site peptide bond Lys-15--Ala-16 in the complexes with trypsin, trypsinogen, and anhydrotrypsin. The chemical shift of [1-13C]Lys-15 was 175.7 ppm in the free inhibitor, 176.4 ppm in the complexes with trypsin and anhydrotrypsin and the ternary complex with trypsinogen and H-Ile-Val-OH, and 175.7 ppm in a neutral solution containing the inhibitor and trypsinogen. These data show that the trypsin--BPTI complex does not contain a covalent tetrahedral carbon atom in the position of the reactive site peptide carbonyl of the inhibitor. They would be consistent with the formation of a noncovalent complex but cannot at present be used to further characterize the degree of a possible pyramidalization of the carbonyl carbon of Lys-15 in such a complex. The identical chemical shifts in the complexes with trypsin and anhydrotrypsin indicate that the gamma-hydroxyl group of Ser-195 of trypsin does not have an important role in the binding of the inhibitor. The previously described [Perkins, S. J. & Wüthrich, K. (1980) J. Mol. Biol. 138, 43--64] stepwise transition from the trypsinogen conformation to an intermediate conformational state in the trypsinogen--BPTI complex and a trypsin-like conformation in the ternary complex trypsinogen--BPTI--H-Ile-Val-OH appears to be manifested also in the chemical shift of [1-13C]Lys-15 of labeled BPTI.

Aprotinin↗
PubMed1980

The effect of nisin on murein synthesis.

Nisin inhibits murein synthesis with concomitant accumulation of undecaprenyl-pyrophospho-MurHAc(pentapeptide) (lipid intermediate I). This inhibition is caused by the formation of a complex between the antibiotic and lipid intermediate I. Undecaprenyl-pyrophospho-MurNAc(pentapeptide)-GlcNAc (lipid intermediate II) also forms a complex with nisin. However, when murein synthesis is inhibited by nisin, this latter complex is not formed since lipid intermediate II is not longer synthesized.

Hydrogen-Ion Concentration↗
PubMed1980

Synthesis and analytical use of 3-carboxypropionyl-alanyl-alanyl-valine-4-nitroanilide: a specific substrate for human leukocyte elastase.

A simple synthesis is described for 3-carboxypropionyl-Ala-Ala-Val-4-nitroanilide, a convenient and very specific substrate for human leukocyte elastase (Km = 1.0mM, kcat = 8.7 s-1). The substrate does not undergo appreciable spontaneous hydrolysis. It is not cleaved by trypsin or chymotrypsin and only rather slowly by porcine pancreatic elastase (Km = 9.1mM, kcat = 1.4 s-1).

Humans↗
PubMed1979

Structural characterization by nuclear magnetic resonance of a reactive-site 13carbon-labelled basic pancreatic trypsin inhibitor with the peptide bond Arg-39--Ala-40 cleaved and Arg-39 removed.

With the use of an enzymatic replacement method, 90%-enriched [1-13C]lysine was introduced into the reactive site of the basic pancreatic trypsin inhibitor. Characterization of the labelled inhibitor with 13C nuclear magnetic resonance (NMR), 1H NMR and chemical methods showed that while the reactive-site peptide bond Lys-15--Ala-16 was properly resynthesized, the polypeptide chain was cleaved at the peptide bond Arg-39--Ala-40 and Arg-39 was removed. Detailed 1H NMR studies showed further that, with the exception of the immediate environment of the modification site, the average spatial structure of the native inhibitor was preserved in the modified protein. Compared to the native inhibitor, the thermal stability of the globular conformation was found to be reduced, interior amide protons exchanged at a faster rate and the internal mobility of aromatic rings located outside the immediate environment of the cleaved peptide bond was essentially unchanged. These observations coincide closely with previous reports on different modifications of the inhibitor and can be explained by a recently proposed dynamic multi-state model for globular proteins. Since the fundamental structural properties of the native inhibitor and full inhibitory activity are preserved after resynthesis, the [1-13C]lys-15-labelled inhibitor with the peptide bond Arg-39--Ala-40 cleaved and Arg-39 removed should be suitable for 13C NMR studies of mechanistic aspects of proteinase-inhibitor interactions.

Alanine↗