COVALENT AND NONCOVALENT DIMERS OF THE CYANIDE-RESISTANT ALTERNATIVE OXIDASE PROTEIN IN HIGHER-PLANT MITOCHONDRIA AND THEIR RELATIONSHIP TOENZYME-ACTIVITY
Al. Umbach et Jn. Siedow, COVALENT AND NONCOVALENT DIMERS OF THE CYANIDE-RESISTANT ALTERNATIVE OXIDASE PROTEIN IN HIGHER-PLANT MITOCHONDRIA AND THEIR RELATIONSHIP TOENZYME-ACTIVITY, Plant physiology, 103(3), 1993, pp. 845-854
Evidence for a mixed population of covalently and noncovalently associ
ated dimers of the cyanide-resistant alternative oxidase protein in pl
ant mitochondria is presented. High molecular mass (oxidized) species
of the alternative oxidase protein, having masses predicted for homodi
mers, appeared on immunoblots when the sulfhydryl reductant, dithiothr
eitol (DTT), was omitted from sodium dodecyl sulfate-polyacrylamide ge
l sample buffer. These oxidized species were observed in mitochondria
from soybean (Glycine max [L.] Merr. cv Ransom), Sauromatum guttatum S
chott, and mung bean (Vigna radiata [L.] R. Wilcz). Reduced species of
the alternative oxidase were also present in the same mitochondrial s
amples. The reduced and oxidized species in isolated soybean cotyledon
mitochondria could be interconverted by incubation with the sulfhydry
l reagents DTT and azodicarboxylic acid bis(dimethylamide) (diamide).
Treatment with chemical cross-linkers resulted in cross-linking of the
reduced species, indicating a noncovalent dimeric association among t
he reduced alternative oxidase molecules. Alternative pathway activity
of soybean mitochondria increased following reduction of the alternat
ive oxidase protein with DTT and decreased following oxidation with di
amide, indicating that electron flow through the alternative pathway i
s sensitive to the sulfhydryl/disulfide redox poise. In mitochondria f
rom S. guttatum floral appendix tissue, the proportion of the reduced
species increased as development progressed through thermogenesis.