THE PRESENCE OF A SHORT REDOX CHAIN IN THE MEMBRANE OF INTACT POTATO-TUBER PEROXISOMES AND THE ASSOCIATION OF MALATE-DEHYDROGENASE WITH THEPEROXISOMAL MEMBRANE
A. Struglics et al., THE PRESENCE OF A SHORT REDOX CHAIN IN THE MEMBRANE OF INTACT POTATO-TUBER PEROXISOMES AND THE ASSOCIATION OF MALATE-DEHYDROGENASE WITH THEPEROXISOMAL MEMBRANE, Physiologia Plantarum, 88(1), 1993, pp. 19-28
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.