Histone-specific protein-arginine methyltransferase from wheat germ.

Abstract

Protein methylase I (S-adenosyl-L-methionine:protein (arginine) N-methyltransferase; EC 2.1.1.23)has been purified from wheat germ approximately 90-fold with a yield of 160%.This unusually high yield of the enzyme activity suggested the presence of an inhibitor.Subsequently, this inhibitor was purified to homogeneity and was found to be identical with adenosine; W absorption spectrum, retention on a high performance liquid chromatography column, migration upon isoelectric focusing (PI value of 6.54), and type of inhibition pattern (competitive) with Ki of 1.58 X M.Among various protein methylases from different sources, wheat germ protein methylase I was found to be the most sensitive toward this inhibitor.When the 90-fold purified protein methylase I was subjected to molecular sieve chromatography on a Sephadex G-100 column, the enzyme activity could not be detected in any of the fractions.However, when the fractions eluted in the void volume and in the inclusion region were combined by ammonium sulfate precipitation, the enzyme activity reappeared.This indicates a presence of cofactor for the enzyme activity.This cofactor was further characterized to be heat-labile, dialyzable, small molecular weight, and peptide in nature.Protein methylase I of wheat germ has several distinctive properties from those of the calf brain protein methylase I: optimum pH, the end product analysis, and high specificity toward histone.Furthermore, the endogenous substrate for the wheat germ enzyme has been identified as histone H4 based on the analysis of the radiolabeled endogenous substrate proteins on acid-urea and sodium dodecyl sulfate gel electrophoresis.K,,, values for histone and S-adenosyl-L-methionine were 5.5 X M and 5.7 X lo-" M, respectively.Among various derivatives of S-adenosyl-L-homocysteine, S-adenosyl-L-homocysteine, A9145C, S-inosyl-~-(2-hydroxy-4-methylthio)butyrate, and sinefungin were the most powerful inhibitors for the enzyme with Ki values of to lo-' M. 3-Deazaadenosine, thioethanoladenosine, n-butylthioadenosine, and 5"methylthioadenosine were moderate inhibitors.ATP, ADP, AMP, and various nucleosides and bases were inactive as inhibitors for the enzyme. Methylated arginines have been found to occur in many