Intracellular Localization of Acid Peptide Hydrolases and Several Other Acid Hydrolases in the Leaf of Pea (Pisum sativum L.)

Abstract

Protoplasts and vacuoles were prepared from pea (P. sativum L.) leaves to determine the acid-hydrolase content of the vacuole. The protoplasts and vacuoles were isolated on sucrose-sorbitol gradients. We conclude that the protoplast preparation was free of enzyme contamination arising from the digestion mixture, after comparing the specific activities of the crude extract and of the isolated protoplasts, and the response to the thiol inhibitor p-chloromercuribenzoate of the crude extract and protoplast acid proteinase with that of the Cellulysin enzyme. N-acetyl-β-D-glucosaminidase, α-mannosidase, acid carboxypeptidase and acid proteinase appeared to be located entirely in the vacuole. Approximately 60% of the acid-phosphatase and 27% of the phosphodiesterase activities were found in the vacuole. We conclude that the acid proteinase and the acid carboxypeptidase are separate enzymes in pea leaves, based on their different sensitivities to the inhibitors phenylmethylsulfonyl fluoride and p-chloromercuribenzoate.