Carboxypeptidase from Yeast

Abstract

Abstract In order to render the carboxypeptidase from yeast more readily available for use in sequence studies in protein chemistry, the procedure of Hata, Hayashi, and associates for the preparation of the enzyme has been adapted to a larger scale through cooperation with the New England Enzyme Center. From 100 pounds of bakers' yeast, the yield was about 1.4 g of the chromatographically purified, electrophoretically homogeneous enzyme, free of other proteinase activity. The carboxypeptidase from the yeast of different manufacture has properties corresponding very closely to those observed initially by Hayashi and Hata; the glycoprotein has a molecular weight of about 61,000, has a nitrogen content of 12.74%, yields 15.1 g of hexose per 100 g, and consists of a single polypeptide chain of 442 residues with 16 residues of glucosamine in the carbohydrate moiety. Lysine is at the NH2 terminus and -Asp-Ser-Thr-Leu is the COOH-terminal sequence. The broad carboxypeptidase activity at pH 5, which includes the liberation of proline, has been tested with the present preparation on glucagon, the B chain of insulin, and reduced and carboxymethylated pancreatic ribonuclease. The amino acids in these sequences released most slowly were glycine and aspartic acid. The carboxypeptidase retains activity in 6 m urea and can be used for the study of proteins that may have inaccessible COOH termini in the usual aqueous solution; native pancreatic ribonuclease is one example.