Search

Glucose-6-phosphate dehydrogenase has been purified 192-fold from Methylomonas M15, grown on methanol as sole source for carbon and energy. The purification procedure involved N-cetyl- N,N,N,-trimethylammonium bromide precipitation, ion-exchange chromatography and affinity chromatography on blue-dextran-Sepharose. The final enzyme preparations were homogeneous as judged by acrylamide gel electrophoresis and by sedimentation in an ultracentrifuge. The molecular weight of the enzyme was calculated to be 108000 ± 5000 from sedimentation equilibrium experiments. Electrophoresis in sodium dodecylsulfate gels indicates that the enzyme is a dimer composed of two probably identical subunits each having a molecular weight of 55000. The enzyme exhibits activity with either NAD or NADP as electron acceptor. Mg2+ or an other bivalent cation is not essential for enzyme activity. The Km values were found to be 0.2 mM for NAD, 0.014 mM for NADP, 0.29 mM for glucose 6-phosphate with NAD and 0.11 mM for glucose 6-phosphate with NADP as electron acceptor. The reduced coenzymes NADPH and NADH as well as ATP are powerful inhibitors of the enzyme. The intracellular levels of glucose 6-phosphate, ATP, NAD and NADP were measured and found to be approximately 2.5 mM, 1.5 mM, 1 mM and 2 mM respectively, during exponential growth. From 3 consideration of the substrate pool sizes and types of inhibitors, it is concluded th3.t this enzyme may function in two roles m the cell: NADH production for energetics and NADPH production for reductive biosynthesis. [ABSTRACT FROM AUTHOR]

An improved procedure was developed for the isolation of pyruvate decarboxylase from wheat germ. Its final step, an electrophoresis of the native apoenzyme in concave pore gradient polyacrylamide gels, followed by superficial activity-staining, produced two bands of different molecular masses and chain compositions. The highmolecular-mass band occurred in Iow quantity and consisted of, probably eight, apparently identical chains of Mr = 33 000, as judged from sodium dodecyl sulfate electrophoreses. The low-molecular-mass band contained two types of chains with Mrα = 63 000-65 000 and Mrβ = 61 000-62 000. The N termini of both chains were threonine, whereas their C-terminal sequences were different: α, -(Val)-(Ser)-(Ala)-Leu; β, -(His)-(Asp)-(Ala)-Ser. Their amino acid composition was too different to be compatible with our original concept of one chain being produced from the other by proteolytic shortening. Limited proteolysis by Staphylococcus aureus V8 proteinase yielded peptides partly identical size and partly quite different. In all properties investigated, the low-molecular-mass enzyme largely resembled yeast pyruvate decarboxylase; the holoenzyme appeared to possess (αβ)2 structure, the apoenzyme αβ. SH reagents inactivated the enzyme. Binding and fluorescence of 2-p-toluidinonaphthalene-6-sulfonate indicated a similar lipophilicity of the active site as found earlier for the yeast enzyme. 2-Hydroxy-5-nitrobenzyl modification of exposed tryptophan residues left the holoenzyme intact, but in the apoenzyme it destroyed most of the cofactor-binding ability and hence the activity. The strength of cofactor binding and the maximal specific activity were found somewhat lower than in yeast pyruvate decarboxylase. [ABSTRACT FROM AUTHOR]

A simple procedure for the extraction and purification of monoamine oxidase from pig brain mitochondria is described. The enzyme purified in this way appears to be homogeneous by cellulose acetate electrophoresis and the molecular weight was estimated to be approximately 102,000 by gel-filtration. The purified enzyme is inhibited by iproniazid, chelating agents and a sulphydryl reagent. The Km value for tyramine has been determined as has its Ki value for high substrate inhibition. [ABSTRACT FROM AUTHOR]

L-6-Hydroxynicotine oxidase, an inducible enzyme of nicotine catabolism in Arthrobacter oxidans, was obtained as a homogeneous protein, as judged by electrophoresis and ultracentrifuge studies; it forms hexagonal crystals in ammonium sulfate solution. Its molecular weight was determined to be 93,000. It contains two moles of FAD per mole of enzyme. In the presence of guanidine hydrochloride, enzymatically inactive components of molecular weight 47,000 were formed which appear to represent the subunits of the protein. No evidence for a participation of metal ions in the electron transport of the enzyme was obtained. Although Hg2+ and p-chloromercuriphenylsulfonate strongly affect activity, N-ethylmaleimide and 5,5′-dithiobis-(2-nitrobenzoate) do not inhibit the enzyme. The latter compound reacts with one thiol group only after exposure of the enzyme to 7.5 M urea. [ABSTRACT FROM AUTHOR]