PubMed Health⌕ Search

Biomedical subjects

H R Wenzel

Publications and source records attributed to H R Wenzel.

31 records · Page 2Linked to original sources
PubMed1987

Kinetics of inhibition of human plasma kallikrein by a site-specific modified inhibitor Arg15-aprotinin: evaluation using a microplate system and comparison with other proteases.

Human plasma kallikrein, a product of contact-activated plasma proteolysis, is moderately inhibited by aprotinin, a small polypeptide from bovine lung that has been used as an experimental drug in human disease states. Aprotinin has a Lys residue in the P1 (reactive center) position occupying residue 15. Since kallikrein is an arginine-directed serine protease, we hypothesized that an altered form of aprotinin, Arg15-aprotinin, might be a better inhibitor. Kinetic evaluations were performed in 96-well microplates. We found that the KL (loose or Michaelis-Menten complex) was unchanged by the modification. However, the association rate constant was increased from 1.14 X 10(4) (mol/L)-1s-1 to 1.5 X 10(5) (mol/L)-1s1, thus indicating that the inhibition rate was increased 14-fold for the modified protein. The Ki (at equilibrium) was decreased from 3.2 X 10(-7) mol/L to 1.5 X 10(-8) mol/L after substituting Arg for Lys in the P1 position. Therefore, the modified inhibitor binds to plasma kallikrein more tightly than the natural protein. We also investigated the effect of Arg15-aprotinin on tissue kallikrein, plasmin, factor XIIa, factor XIa, and thrombin and found that the Ki slightly decreased from 5.1 X 10(-7) mol/L to 1.2 X 10(-7) mol/L for tissue kallikrein and slightly decreased from 2 X 10(-8) mol/L to 1 X 10(-8) mol/L for plasmin. Arg15-aprotinin did not inhibit thrombin or factor XIIa, even though both enzymes are arginine-directed serine proteases. However, factor XIa, although it was not inhibited by aprotinin, had a Ki of 3.4 X 10(-8) mol/L for Arg15-aprotinin. Therefore, Arg15-aprotinin is a more effective inhibitor of plasma kallikrein as well as factor XIa but shows minimal preference for plasmin and tissue kallikrein. This study also indicates that it is possible and practical to perform kinetic analyses directly in microplates.

Aprotinin↗
PubMed1984

The refined 2.2-A (0.22-nm) X-ray crystal structure of the ternary complex formed by bovine trypsinogen, valine-valine and the Arg15 analogue of bovine pancreatic trypsin inhibitor.

Large orthorhombic crystals of the complex formed by bovine trypsinogen and a semisynthetic homologous bovine pancreatic trypsin inhibitor with the reactive-site lysine residue replaced by an arginine residue [( Arg15]PTI) have been obtained which are isomorphous with the crystals of PTI-trypsinogen [Bode, W., Schwager, P. and Huber, R. (1978) J. Mol. Biol. 118, 99-112]. The X-ray crystal structure of the ternary complex of trypsinogen-[Arg15]PTI with the dipeptide Val-Val has been determined by X-ray data to 2.2-A (0.22-nm) resolution by means of difference Fourier methods and has been crystallographically refined to a final R-value of 0.17. Replacement of the reactive-site Lys15 by an arginine residue is accompanied in the complex by small movements of polar side groups of trypsin and enclosed solvent molecules within the specificity pocket. Only solvent molecule 414 OH which mediates the hydrogen bond interactions between Lys15 NZ and Asp189 carboxylate is expelled, thus allowing the bulkier guanidyl group to approach this carboxylate. The dipeptide Val-Val binds in the pocket accepting the Ile-Val N-terminus in trypsin. The cavity left by the CD-methyl group of Ile16 upon replacement by a valine residue is only partially filled by slight rearrangements of neighbouring peptide side chains. Part of the positive free energy change observed upon replacement of Ile-Val may allow for the maintenance of this cavity.

Animals↗
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↗
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

Ternary complex formation of pig heart lactate dehydrogenase with spin-labelled coenzyme and inhibitors as studied by electron spin resonance.

The formation of ternary inhibitor and 'dead end' complexes of pig heart lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) was studied by means of two NAD derivatives, spin-labelled at N6 and C-8 of the adenine ring. Dissociation constants calculated for the inhibitors oxamate and oxalate from their corresponding ternary complexes are in excellent agreement with data from literature derived from sedimentation experiments. However, the recently postulated enzyme-NADH-sulfite complex was not observed. The mobility of the spin-label, i.e. the protein conformation near the adenine binding pocket in various ternary complexes depends on the type of inhibition or substrate employed.

Animals↗
PubMed1979

The nature of the substrate inhibition in lactate dehydrogenases as studied by a spin-labeled derivative of NAD.

The formation of the ternary complex of lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) from pig heart and skeletal muscle with the adduct of pyruvate to NAD", spin-labeled at N6 was studied by ultraviolet spectroscopy and ESR techniques. According to ultraviolet measurements we found identical binding characteristics for the natural coenzyme and its spin-labeled analog. The rate by which the ESR signal of free spin-labeled NAD+ decreased upon addition of pyruvate to the binary complexes was substantially different in the two isozymes. With the heart type an initial drop followed by a further linear decrease, zero order in the enzyme and coenzyme concentration was observed. In case of the skeletal muscle isozyme no immediate reaction and a first order process occurred. The initial reaction can be attributed to a non-covalent enzyme/spin-labeled NAD+/pyruvate complex with a dissociation constant for pyruvate of 11 +/- 1 mM, thus explaining the well-known substrate inhibition in the heart isozyme above 2 mM pyruvate. The further reaction is then determined by the buffer dependent enolization of pyruvate. In the muscle isozyme formation of the covalent adduct is not assisted by prior binding of pyruvate in a non-covalent ternary complex and therefore the rate depends on the binary complex concentration.

Animals↗
PubMed1976

The synthesis of spin-label derivatives of NAD+ and its structural components and their binding to lactate dehydrogenase.

Spin-labelled derivatives of NAD+ and its structural components (i.e. adenosine, adenine, AMP, ADP and ADPR) have been synthesized. Their binding to pig heart lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) has been studied and dissociation constants have been determined. The spin-labelled derivatives of ADP and ADPR exhibit a tighter binding than the corresponding NAD+ derivative. This may be attributed to the repulsion of the positively charged nicotinamide ring by an histidine side chain in the active center of the enzyme.

Animals↗
PubMedDate not supplied

Pressure induced structural fluctuations in hemoglobin, studied by EPR-spectroscopy.

A quartz based cavity for pressure dependent EPR measurements on liquid samples allowing pressures up to 0.6 GPa was constructed. First investigations with this setup were done on spin labeled horse hemoglobin derivatives both in ferric and ferrous state of oxidation. The second derivative EPR spectra show changes of the label's mobility, which are not correlated with spin state changes of the Fe-porphyrin complex, but which point out structural fluctuations inside the globin protein matrices.

Cyclic N-Oxides↗
PubMedDate not supplied

Thermodynamic and magnetic resonance studies on the hydration of polymers: II. Protein-water interactions in powered ribonuclease.

ESR studies on spin-labeled amorphous RNase A as a function of varying concentrations of sorbed H2O and D2O will be presented. A relaxation analysis of saturation transfer (ST-)ESR spectra of 14N(1H) nitroxide spin-label molecules essentially fixed at amino acid residue His-105 will be given. A characteristic correlation has been observed between the microdynamic behavior--expressed by the rotational correlation times of the paramagnetic label--and the macroscopic thermodynamic entropy for the sorption process of H2O and D2O at RNase. This correlation is particularly pronounced at low water concentrations, vis., nH2O/nprotein less than or equal to 100. A significant difference in this concentration range exists between the two systems "RNase-H2O" and "RNase-D2O", which is manifested not only by the thermodynamic data but also by the microdynamic behavior extracted from the corresponding non-linear ESR absorption line shapes.

Computer Simulation↗