Arginine Decarboxylase from Escherichia coli

Abstract

Abstract Several lines of evidence are presented which, together with previous data that arginine decarboxylase (mol wt = 820,000; s020,w = 23.3 S) contains an integral multiple of five subunits, indicate that the enzyme is composed of 10 identical or very nearly identical polypeptide chains. (a) Acrylamide gel electrophoresis in sodium dodecyl sulfate and mercaptoethanol gives a single band containing a material having a molecular weight of approximately 96,000. (b) In denaturing solvents, 6.7 moles of sulfhydryl groups react per 82,000 g of protein with 5,5'-dithiobis(2-nitrobenzoic acid). Since 7.0 residues of cysteic acid were found after performic acid oxidation, the molecule contains no disulfide bonds. In the native (23 S) enzyme, none of the sulfhydryl groups are reactive, while in the dissociated (mol wt = 160,000, s020,w = 8.0 S) form of the enzyme, 0.9 mole of sulfhydryl groups reacts per 82,000 g. Similar results were obtained for the dissociated species by reaction with both p-chloromercuribenzoate and N-ethylmaleimide. Thus, the dissociated (8 S) species has 1 mole of uniquely reactive sulfhydryl residue per 82,000 g. (c) Amino-terminal analysis by the isocyanate procedure gives 6 to 8 moles of methionine residues per 820,000 g; this result was confirmed by dinitrophenylation. Digestion with carboxypeptidase A releases 10.3 moles of alanine per 820,000 g and hydrazinolysis gives 5 to 6 moles of alanine. (d) Peptide maps of tryptic hydrolysates have 54 to 58 ninhydrin-positive spots; the expected number, for 10 identical polypeptide chains, is 61. (e) Isoelectric focusing of arginine decarboxylase which had been reassociated from the 8 S to the 23 S species gave a single peak with a pI of 4.44. A revised purification procedure, suitable for preparing large quantities of enzyme, is also presented.