Abstract
Abstract Adenylosuccinate synthetase has been purified more than 250-fold from extracts of rabbit muscle acetone powder by heating to 60°, ammonium sulfate fractionation, phosphocellulose and hydroxylapatite chromatography, and Sephadex G-150 gel filtration. Crystals have been obtained by dialysis against 60 to 67% ammonium sulfate. Enzyme activity is stable for about 2 weeks in phosphate buffer containing 1 mm dithiothreitol; for longer periods, dialysis against saturated ammonium sulfate is preferable. The enzyme is quite basic, has a molecular weight of approximately 54,000, and does not appear to dissociate into subunits upon treatment with sodium dodecyl sulfate or mercaptoethanol. A broad maximum in enzyme activity occurs at pH ∼ 6.6, and the kinetic parameters of the enzyme differ significantly from those of the Escherichia coli or Ehrlich ascites-tumor cell enzymes. Km values for IMP, GTP, and l-aspartate are 2 x 10-4 m, 1 x 10-5 m, and 3 x 10-4 m, respectively, for the muscle enzyme. GDP, AMP, adenylosuccinate, argininosuccinate, phosphate, arsenate, and sulfate inhibit enzyme activity. Enzyme activity is unaffected by incubation for 24 hours with EDTA, azide, and phenyl-methanesulfonyl fluoride, but is completely destroyed by freezing or by 18-hours incubation with 5,5'-dithiobis(2-nitrobenzoic acid). Adenylosuccinate synthetase activity has also been found in crude extracts of acetone powders of rabbit heart, liver, kidney, brain, and lung.
Highlights
This paper describes the preparation of nearly homogeneous adenylosuccinate synthetase from rabbit muscle
The rabbit muscle enzyme appears to be more basic than the E. coli enzyme since it will not stick to DEAE-cellulose, sticks quite well to phosphocellulose and carboxymethylcellulose, and migrates very slowly in the high pH buffers used for Ornstein-Davis gel electrophoresis
Since K, for IMP and Ki for AMP for the rabbit muscle enzyme are on the order of concentrations usually found in the cell, enzyme activity may be quite dependent on changes in cellular concentrations of these two compounds, affording a very sensitive mechanism for the control of AMP synthesis
Summary
Dithiothreitol (Cleland’s reagent), u- and L-aspartic acid, Lglutamic acid, L-glutamine, L-malic acid, cellulose phosphate, imidazole, inosine, hypoxanthine, and the sodium salts of XMP, IMP, IDP, ITP, GTP, dGTP, GDP, GMP, CTP, UTP, and. AhlP were supplied by Sigma Chemical Co. N-2-Hydroxycthylpiperazine-N’Zethanesulfonic acid (HEPES buffer) and the sodium salt of UMP were purchased from Calbiochem (Los Angeles, Calif .). Enzyme grade ammonium sulfate and phenylmethanesulfonyl fluoride were purchased from Schwarz-Mann (Orangeburg, N.J.). DEAE-cellulose (gel and paper) and carboxymethylcellulose were supplied by Reeve Angel (Clifton, N.J.), DEAE
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