Physical protein regulation of adenylate kinases from muscle, liver, and hepatoma.

hepatoma 3924A have been compared by equilibrium ultracentrifugation, sodium dodecyl sulfate:polyacrylamide gel disc electrophoresis, amino acid analysis, and two-dimensional polypeptide mapping. The hepatoma enzyme is composed of two polypeptide subunits of 13,000 daltons each and has a native molecular weight near 24,000 daltons. Liver adenylate kinase consists of two 13,000 dalton subunits and an 11,000 dalton subunit; the native form exists near 40,000 daltons. The muscle enzyme has four subunits at 13,000 daltons each. Its native form is near 50,000 daltons. A unique polypeptide sequence in the liver adenylate kinase is also indicated from â€oefingerprint†• and amino acid analysis. It is probable that all three forms of adenylate kinase have subunits with similar size, but the liver form also has a unique smaller subunit that may be involved in its regulatabiity by citric acid-cycle metabolites. This unique polypeptide subunit is not present in the hepatoma enzyme. Therefore, if this latter polypeptide is produced from the derepression of a single structural gene, loss of the functioning of this gene in the tumordoesnot necessarily meanlossof enzymatic activity; it probably indicates a loss of metabolite regulatabiity of enzymatic activity. INTRODUCTION AK3 (EC 2.7.4.3) is a central component in the adenylate energy charge system (1†" 2,5†" 7).Several studies (3, 4, 8, 14, 20) observed that it consists of multiple electrophoretic forms in tissues and blood cells. Although it has been assumed that these various electrophoretic forms of AK were isozymes (different combinations ofsimilar subunits with acommon molecular weight), data in this report question this assumption. CA-10906 from the NIH and F73-UF4 from the Florida Division of the American Cancer Society. 2Recipicnt of NIH Research Career Development Award CA-70187. 3The abbreviation used is: AK, adenylate kinase; SDS, sodium dodecyl sulfate. AK was prepared from rat liver, skeletal muscle, and Morris hepatoma 3924A, as previously described (9). Ultracentrifugation analysis was carried out in a Spinco Model E ultracentrifuge with a 3-channel Yphantis cell. Equilibrium analysis was performed by the method of Yphantis (22), and calculations of the molecular weights were made by computer. Each purified AK was analyzed at concentrations of 0.1 and 1.0 mg/ml in 0.05 M phosphate buffer, pH 7.2, containing 1 m@ f3-mercaptoethanol. SDS-polyacrylamide disc gel electrophoresis was accomplished with the use of a rectangular apparatus made in our bioelectronic department. We used glass cylinders (i.d., 0.5 ; length, 8 cm). The enzyme preparations were incubated for 2 hr at …