On the Subunit Structure of Yeast Inorganic Pyrophosphatase

Abstract

Abstract Evidence based upon a variety of experimental approaches is in accord with the conclusion that native yeast inorganic pyrophosphatase (EC 3.6.1.1, pyrophosphate phosphohydrolase) comprises two identical subunits approximately 32,000 in molecular weight with threonine and valine the NH2- and COOH-terminal residues, respectively. Gel filtration of reduced and carboxymethylated pyrophosphatase on a column of 6% agarose in 6 m guanidine hydrochloride revealed a single protein component of molecular weight 33,000; a value of 35,500 was obtained after exhaustive succinylation of the alkylated enzyme. The molecular weight thus derived for S-carboxymethylated pyrophosphatase was in close agreement with the values calculated from polyacrylamide gel electrophoresis in sodium dodecyl sulfate and amino acid compositional data which were, respectively, 33,000 and 30,250. Threonine was identified as the sole NH2-terminal residue by the dimethylaminonaphthalene-5-sulfonyl chloride end group procedure. Hydrolysis of alkylated pyrophosphatase by carboxypeptidase A was attended by the rapid release of COOH-terminal valine in quantities stoichiometric with the amount of monomeric protein present. Peptide maps of trypsin digests from S-carboxymethylated enzyme revealed a total of 30 peptides, five of which gave a positive test for arginine and three of which stained positively for tryptophan. These results are in accord with what would be expected on the basis of the lysine, arginine, and tryptophan content of the enzyme subunit. Finally, the partial sequence of NH2-terminal amino acid residues presented below was elucidated by Edman degradation with the aid of automated sequencing equipment. [see PDF for sequence] Yields of the phenylthiohydantoin amino acid derivatives obtained after each cycle of the degradative procedure were consistent with the quantity of monomeric protein subjected to analysis.