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SUMMARY: Homogenates from Polytomella caeca were fractionated by differential centrifugation. All the mitochondria (located by measurement of cytochrome c oxidase) and most of the peroxisomes (located by measurement of catalase) sedimented at 105g min. Most of the NADPH-cytochrome c oxidoreductase, IDPase and acid p-nitrophenyl phosphatase were still present in the supernatant. This supernatant also contained a cytochrome b, cytochromes P-450 and P-416. Zonal centrifugation indicated that catalase and cytochrome oxidase were entirely sedimentable at 6 × 106g min and that NADPH- and antimycin A-insensitive NADH-cytochrome c oxidoreductases were not mitochondrial. Peroxisomes (ρ = 1.25) were separated from mitochondria (ρ = 1.20) by both rate and isopycnic-zonal centrifugation. Two zones of acid p-nitrophenyl phosphatase activity were detected, one at ρ = 1.15 and the other at ρ = 1.21, but no other acid hydrolases were found. Separation, of polysomes and various microsomal enzymes (IDPase, acid p-nitrophenyl phosphatase, NADH- and NADPH-cytochrome c oxidoreductases) was also achieved.

Subcellular Fractionation of the Colourless Alga Polytomella caeca by Differential and Zonal Centrifugation