An investigation into the role of subcellular organelles in various species of yeasts

1. The activities of many enzymes in cell extracts of n-hexadecane or glucose-grown Candida stellatoidea and methanol or glucose-grown Candida boidinii were determined. 2. Several enzymes exhibited a latency removable by Triton X-100. 3. Preparation of spheroplasts using zymolyase was necessary to maintain a high degree of organelle integrity in cell extracts. 4. Organelles from n-hexadecane or methanol-grown cells were more fragile than those from glucose-grown cells. 5. The subcellular location of enzymes was investigated by analytical fractionation of cell extracts in sucrose or metrizamide gradients. 6, The mitochondrial zone contained cytochrome c oxidase and all tricarboxylic acid cycle enzymes assayed. 7. The density of microbodies of C. stellatoidea was similar to that of mitochondria on either gradient material. 8. On metrizamide a second catalase peak at p = 1,07 g ml contained organelles up to 1 pm in diameter and activities of many n-hexadecane dissimilatory enzymes. The possibility that cells grown on n-hexadecane contain two populations of microbodies is discussed. 9. Carnitine acetyltransferase and long chain alcohol and aldehyde dehydrogenases were multilocational in these cells. 10. Electron microscopy showed that microbodies of methanol-grown C. boidinii have crystalline cores. 11. These organelles were not separated from mitochondria by rate-zonal centrifugation but in sucrose had a higher density than mitochondria. 12. Whereas glucose-grown cells showed a single population of microbodies, two populations containing catalase and methanol oxidase were distinguished for methanol- grown cells. 13. Formaldehyde and formate dehydrogenases were cytosolic enzymes, whereas some dihydroxyacetone synthase was also associated with mitochondria or vacuoles. 14. Inosine diphosphatase was associated with small membraneous vesicles. 15. Metrizamide was the gradient material of choice for the isolation of cell vacuoles. 16. The role of organelles during respiratory adaptation of anaerobically-grown Saccharomyces uvarum was investigated. 17. Medium containing l6-bromo-9-hexadecenoic acid supported growth under anaerobic glucose-repressed conditions. 18. Fractionations in sucrose gradients revealed a high density population of promitochondria whose density was lowered during glucose-de-repression. 19. Complex density changes in oligomycin-sensitive A T Pase and cytochrome c oxidase distributions on metrizamide gradients were obtained during respiratory adaptation of glucose-de-repressed cells. 20. The significance of these changes are discussed in terms of mitochondrial differentiation. 21, After respiratory adaptation for 3 h, peroxisomes were separated from mitochondria on metrizamide density gradient.