SUMMARY: Spheroplasts of glucose grown and -hexadecane-grown were prepared using snail-enzyme or Zymolyase-5000 and the resultant cell extracts fractionated on sucrose or metrizamide gradients. Organelles from -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 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 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 -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 on metrizamide gradients.


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