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G B Kresze

Publications and source records attributed to G B Kresze.

9 recordsLinked to original sources
PubMed1983

Lipoamide dehydrogenase from baker's yeast. Improved purification and some molecular, kinetic, and immunochemical properties.

The flavoprotein lipoamide dehydrogenase was purified, by an improved method, from commercial baker's yeast about 700-fold to apparent homogeneity with 50-80% yield. The enzyme had a specific activity of 730-900 U/mg (about twice the value of preparations described previously). The holoenzyme, but not the apoenzyme, possessed very high stability against proteolysis, heat, and urea treatment and could be reassociated, with fair yield, with the other components of yeast pyruvate dehydrogenase complex to give the active multienzyme complex. The apoenzyme was reactivated when incubated with FAD but not FMN. As other lipoamide dehydrogenases, the yeast enzyme was found to possess diaphorase activity catalysing the oxidation of NADH with various artificial electron acceptors. Km values were 0.48 mM for dihydrolipoamide and 0.15 mM for NAD. NADH was a competitive inhibitor with respect to NAD (Ki 31 microM). The native enzyme (Mr 117000) was composed of two apparently identical subunits (Mr 56000), each containing 0.96 FAD residues and one cystine bridge. The amino acid composition differed from bacterial and mammalian lipoamide dehydrogenases with respect to the content of Asx, Glx, Gly, Val, and Cys. The lipoamide dehydrogenases of baker's and brewer's yeast were immunologically identical but no cross-reaction with mammalian lipoamide dehydrogenases was found.

Amino Acids↗
PubMed1979

Inactivation and disassembly of the pyruvate dehydrogenase multienzyme complex from bovine kidney by limited proteolysis with an enzyme from rat liver.

Mammalian pyruvate dehydrogenase multienzyme complex is inactivated when treated with a leupeptin-sensitive enzyme (termed 'inactivase') obtained from rat liver lysosomes. However, the inactivation of the overall reaction does not affect any of the component activities of the enzyme complex. By several methods it is demonstrated that treatment with the inactivase provokes the disassembly of the complex into its constituent enzyme components which, though being enzymatically active when assayed separately, are unable to catalyze the coordinated reaction sequence of pyruvate oxidation. The dissociation occurs as a consequence of limited proteolysis of the lipoate acetyltransferase core of the multienzyme complex. Isolated nicked acetyltransferase retains its complete enzymatic activity and behaves as a high-molecular-weight aggregate. The lipoamide dehydrogenase and pyruvate dehydrogenase components, however, are not cleaved by the inactivase.

Animals↗
PubMed1977

Reaction of yeast fatty acid synthetase with iodoacetamide. 3. Malonyl-coenzyme A decarboxylase as product of the reaction of fatty acid synthetase with iodoacetamide.

Yeast fatty acid synthetase possesses very low malonyl-CoA decarboxylase activity. Treatment with iodoacetamide, while abolishing synthetase activity, induces a strong malonyl decarboxylase activity which, in turn, can be inhibited by N-ethylmaleimide. Kinetic analysis shows that the emergence of the decarboxylase activity is synchronized to the disappearance of the fatty-acid-synthesizing activity and thus, is due to carboxamidomethylation of the peripheral SH-groups of the multienzyme complex. Strong decarboxylase activity was also found after treatment of the synthetase with methylmalonyl-CoA. A hypothetical scheme is proposed which explains the origination of the decarboxylase activity as a consequence of conformational changes of the condensing enzyme component which happen when the peripheral SH-group is acylated or alkylated.

Binding Sites↗
PubMedDate not supplied

Removal of endoproteinases from biological fluids by "sandwich affinity chromatography" with alpha 2-macroglobulin bound to zinc chelate-Sepharose.

A new method for removing nearly all active endoproteinases from fluids called "sandwich affinity chromatography" is described. It is based on strong chelate binding of alpha 2-macroglobulin (alpha 2M) and its proteinase complexes to Zn2+-bis-carboxymethylamino-Sepharose (Zn chelate-Sepharose) and its ability to complex most active endoproteinases. The preferred performance minimizing unspecific protein adsorption is binding first alpha 2M to Zn chelate-Sepharose and then adsorbing the proteinase to the alpha 2M-Zn chelate-Sepharose using elevated salt concentrations. A suitable standard buffer, in which most proteases and alpha 2M are active and the protease-alpha 2M complex remains bound to Zn chelate-Sepharose, is 0.02 mol/liter sodium phosphate, pH 6.5, containing 0.15 mol/liter NaCl. As an example, the reaction of trypsin with alpha 2M-Zn chelate-Sepharose was studied. After saturating Zn chelate-Sepharose first with alpha 2M and then with trypsin under standard conditions, the bound alpha 2M equals the bound trypsin activity (measured with Chromozym TRY). The specific binding capacity of alpha 2M-Zn chelate-Sepharose for proteases was determined in this way to be 30-40 U trypsin, i.e., 0.40-0.54 mg/ml of gel. The balance and the fact that the bound trypsin is inaccessible to soybean trypsin inhibitor indicate that at these conditions no unspecific trypsin binding occurs. Chymotrypsin, thermolysin, elastase, bromelain, ficin, and papain are also bound at standard conditions but not exoproteases like carboxypeptidases A and Y. Advantages of the sandwich affinity chromatography are the simple loading procedure by adsorption, the high capacity of the gel material, and the possibility to reuse the Zn chelate-Sepharose after eluting reacted alpha 2M and reloading with new alpha 2M.

Animals↗