title: Characterisation of a highly hydrophobically modified lactate dehydrogenase.
abstract: 1. Lysine residues of porcine H4 lactate dehydrogenase (L-lactate:NAD+ oxidoreductase EC 1.1.1.27) were modified with methyl-epsilon-(N-2,4-dinitrophenyl)aminocaproimidate - HCl. With increasing incorporation of the reagent a linear decrease of enzymatic activity was noticed. No essential lysyl group with an extraordinary reactivity was modified. 2. The active forms of the modified enzyme with different incorporation values were separated from denatured material by fractional precipitation and gel chromatography. An epsilon-(N-2,4-dinitrophenyl)aminocaproamidinate lactate dehydrogenase was obtained with an average incorporation of 38 groups per tetramer and a residual activity of 42%. This material proved to be homogenous in cellulose electrophoresis. 3. The epsilon-(N-2,4-dinitrophenyl)aminocaproamidinate lactate dehydrogenase is soluble only in glycine buffer at pH 8 and can be stabilized as ternary complex with NAD+ and sodium sulfite. Gel chromatography and ORD measurements show no strong conformational change. 4. epsilon-(N-2,4-dinitrophenyl)aminocaproamidinate lactate dehydrogenase has similar Km values for pyruvate, NADH, lactate and NAD+ as the native enzyme, and shows a lower thermostability due to a diminished stabilization by the hydrate layer on the surface.
mesh_terms: D000614:Aminocaproates / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D004355:Drug Stability; D006358:Hot Temperature; D007202:Indicators and Reagents; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008239:Lysine / Q000032:analysis; D009206:Myocardium / Q000201:enzymology; D009578:Nitrobenzenes / Q000494:pharmacology; D011485:Protein Binding; D011489:Protein Denaturation; D013552:Swine
chemical_list: D000614:Aminocaproates; D007202:Indicators and Reagents; D009578:Nitrobenzenes; D007770:L-Lactate Dehydrogenase; D008239:Lysine