Subcellular Distribution of Enzymes in Ochromonas malhamensis*

Abstract

SYNOPSIS. The activity and distribution of 7 enzymes in Ochromonas malhamensis were studied. Subcellular organelles were separated by centrifugation at 648,000 g min to precipitate the larger particles; the resulting supernatant was centrifuged at 5,560,000 g min to separate the microsomal fraction from the supernatant. Sixty‐four percent of the cytochrome oxidase (1.9.3.1 ferrocytochrome c:oxygen oxidoreductase, 81% of the catalase (1.11.1.6 hydrogen‐peroxide: hydrogen‐peroxide oxidoreductase) and 70% of the urate oxidase (1.7.3.3 urate:oxygen oxidoreductase) activity was associated with the larger particles, altho only 20% of the total protein was found in this fraction. Three acid hydrolases, cathepsin (3.4.4.9 cathepsin C, acid phosphatase (3.1.3.2 orthophosphoric monoesterphosphohydrolase) and acid ribonuclease (2.7.7.17 ribonucleate nucleotido‐2′‐transferase) were found mostly in the supernate (50‐60%, yet their latency and their similar subcellular distribution indicated the presence of lysosomes. After 2.5 hr centrifugation in a sucrose density gradient (ρ= 1.08–1.25, the acid hydrolases showed a broad distribution which differed greatly from cytochrome oxidase associated with mitochondria. Catalase, which could not be separated from cytochrome oxidase by centrifuging on this gradient, had a different distribution after centrifugation on a kinetic gradient. Urate oxidase had a similar distribution to catalase and both these enzymes were latent, indicating the presence of peroxisomes.