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- Struglics, André, et al.
(författare)
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The presence of a short redox chain in the membrane of intact potato tuber peroxisomes and the association of malate dehydrogenase with the peroxisomal membrane
- 1993
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Ingår i: Physiologia Plantarum. - : Wiley. - 0031-9317 .- 1399-3054. ; 88:1, s. 19-28
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Tidskriftsartikel (refereegranskat)abstract
- Peroxisomes and mitochondria were purified from potato tubers (Solanum tuberosum L. cv. Bintje) by differential centrifugation followed by separation on a continuous Percoll gradient containing 0.3 M sucrose in the lower half and 0.3 M mannitol in the upper half. The peroxisomes band at the bottom and the mitochondria in the middle of this type of gradient. Mitochondrial contamination of the peroxisomes was only 2% [as judged by cytochrome c oxidase (EC 1.3.9.1) activity]. Contamination by amyloplasts, plasma membrane and endoplasmic reticulum was also minimal. The peroxisomes were 80% intact as judged by malate dehydrogenase (MDH, NAD−-dependent; EC 1.1.1.37) latency.The specific activity of NADH-ferricyanide reductase and NADH-Cyt c reductase was 0.22 and 0.051 μmol (mg protein)−1 min−1 in freshly isolated peroxisomes, respectively. The active site of the reductase appeared to be on the inner surface of the membrane. The peroxisomes also contained a b-type cytochrome. Frozen peroxisomes were subfractionated by osmotic rupture followed by centrifugation to separate the soluble proteins from the peroxisomal membrane. About half the MDH and 30% of the NADH-ferricyanide reductase activity was associated with the membrane but only 6% of the catalase (EC 1.11.1.6) activity. A further wash removed 75% of the residual catalase with only a small loss of MDH or NADH-ferricyanide reductase activity. MDH appears to be closely associated with the peroxisomal membrane.When the purified peroxisomal membrane was analyzed by SDS-PAGE followed by silver staining, prominent bands at 22, 40, 41, 48, 53 and 74 kDa were observed. After immunoblotting the purified peroxisomal membrane, a band at 53 kDa showed strong cross-reactivity with antibodies raised against NADH-ferricyanide reductase. Since the NADH-ferricyanide reductase activity in the peroxisomal membrane could be shown to be specific for the β-hydrogen of NADH, the activity could not be due to contamination by endoplasmic reticulum where the reductase is α-specific. We conclude that the peroxisomal membrane contains a short redox chain, consisting of a NADH-ferricyanide reductase and a b-type cytochrome, similar to that of e.g. the plasma membrane. The role of this redox chain has yet to be elucidated.
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