THE PRESENCE OF A SHORT REDOX CHAIN IN THE MEMBRANE OF INTACT POTATO-TUBER PEROXISOMES AND THE ASSOCIATION OF MALATE-DEHYDROGENASE WITH THEPEROXISOMAL MEMBRANE

Peroxisomes and mitochondria were purified from potato tubers (Solanum tuberosum L. cv. Bintje) by differential centrifugation followed by s eparation 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]. Contaminatio n by amyloplasts, plasma membrane and endoplasmic reticulum was also m inimal. The peroxisomes were 80% intact as judged by malate dehydrogen ase (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 mumol (mg protein)-1 min-1 in freshly isolated peroxisomes, respe ctively. The active site of the reductase appeared to be on the inner surface of the membrane. The peroxisomes also contained a b-type cytoc hrome. Frozen peroxisomes were subfractionated by osmotic rupture foll owed by centrifugation to separate the soluble proteins from the perox isomal membrane. About half the MDH and 30% of the NADH-ferricyanide r eductase 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 res idual catalase with only a small loss of MDH or NADH-ferricyanide redu ctase activity. MDH appears to be closely associated with the peroxiso mal membrane. When the purified peroxisomal membrane was analyzed by S DS-PAGE followed by silver staining, prominent bands at 22, 40, 41, 48 , 53 and 74 kDa were observed. After immunoblotting the purified perox isomal 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 s hown to be specific for the beta-hydrogen of NADH, the activity could not be due to contamination by endoplasmic reticulum where the reducta se is alpha-specific. We conclude that the peroxisomal membrane contai ns a short redox chain, consisting of a NADH-ferricyanide reductase an d a b-type cytochrome, similar to that of e.g. the plasma membrane. Th e role of this redox chain has yet to be elucidated.