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SUMMARY: Spheroplasts of glucose grown and n-hexadecane-grown Candida stellatoidea were prepared using snail-enzyme or Zymolyase-5000 and the resultant cell extracts fractionated on sucrose or metrizamide gradients. Organelles from n-hexadecane-grown cells were more fragile than those from glucose-grown cells and organelle integrity was maintained only after spheroplast formation using Zymolyase-5000. Isopycnic density gradient centrifugation through metrizamide gradients yielded more complex distributions and markedly higher percentage sedimentabilities of marker enzymes than with sucrose gradients. The zone containing cytochrome c oxidase and all tricarboxylic acid cycle enzymes assayed was readily identified. The density of microbodies appears to be similar to that of mitochondria on either gradient material; on metrizamide a second catalase peak at ρ = 1.07 g ml-1 was also observed. This zone was shown by electron microscopy to contain organelles up to 1 μm diameter, and activities of carnitine acetyltransferase and long chain alcohol and aldehyde dehydrogenases. The first enzyme was located mainly in zones containing mitochondria and microbodies; the last two enzymes were multinational and of differing distributions, but were found mainly in mitochondrial and microsomal fractions. The possibility that cells grown on n-hexadecane contain two populations of microbodies is discussed. Most lysosomes were disrupted on sucrose gradients but sedimented to a density of 1.12 g ml-1 on metrizamide gradients.

Subcellular Fractionation of Candida stellatoidea after Growth with Glucose or n-Hexadecane