Abstract
AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) has been purified to apparent homogeneity from rat muscle. The preparation exhibits a single polypeptide band with a molecular weight of 60,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme has a sedimentation coefficient of 11.3 S. Analysis by sedimentation equilibrium techniques showed the nat-ive enzyme to have a molecular weight of 238,000, whereas the enzyme, when analyzed in 6 M guanidine hydrochloride and 10 mM 2-mercaptoethanol, had a molecular weight of only 59,500. The amino acid composition of the enzyme was determined and peptide mapping was performed on a tryptic digest of S-carboxymethylated enzyme. NH2-terminal analysis by both the dansylation and cyanate procedures failed to identify a free NH2 terminus. Treatment of the enzyme with carboxypeptidase A resulted in the release of approximately 0.5 mol each of valine and leucine per 60,000 g of enzyme. The data presented indicate that hte native enzyme has a tetrameric structure consisting of four polypeptide chains each having a molecular weight of 60,000. The COOH-terminal analysis can be interpreted either as an indication of subunit heterogeneity or as a result of incomplete digestion of a -X-Leu-Val sequence at the end of a single type of polypeptide chain. Tryptic peptide maps strongly support the latter interpretation and suggest that the subunits are essentially identical.
Highlights
The kinetic properties of AMP deaminase from a number of tissues have been examined in several laboratories, and there is general agreement that the enzyme is subject to very stringent regulation
This paper reports a purification procedure and a detailed analysis of the subunit structure of AMP deaminase from rat muscle
Since the enzyme obtained by this method was not homogeneous, either by the criterion of sedimentation velocity or Na dodecyl-S04-gel electrophoresis, it was necessary to introduce new steps into the purification procedure
Summary
AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) has been purified to apparent homogeneity from rat muscle. The physiological role of AMP deaminase remains obscure, the relatively high concentration of the enzyme in muscle tissue, together with the fact that it is found tightly complexed to myosin [2, 3], implies that it may be significant in. Directing the energy flow required for musclecontraction It has recently been proposed by Chapman and Atkinson [4] that the role of AMP deaminase in liver is to stabilize the adenylate energy charge. The kinetic properties of AMP deaminase from a number of tissues have been examined in several laboratories, and there is general agreement that the enzyme is subject to very stringent regulation. This paper reports a purification procedure and a detailed analysis of the subunit structure of AMP deaminase from rat muscle. This, we feel, is an initial effort in the direction of correlating the catalytic, regulatory, and molecular properties of the enzyme
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