The regulation of alternative oxidase activity by the effector pyruvat
e was investigated in soybean (Glycine max L.) mitochondria using deve
lopmental changes in roots and cotyledons to vary the respiratory capa
city of the mitochondria. Rates of cyanide-insensitive oxygen uptake b
y soybean root mitochondria declined with seedling age. Immunologicall
y detectable protein levels increased slightly with age, and mitochond
ria from younger, more active roots had less of the protein in the red
uced form. Addition of pyruvate stimulated cyanide-insensitive respira
tion in root mitochondria, up to the same rate, regardless of seedling
age. This stimulation was reversed rapidly upon removal of pyruvate,
either by pelleting mitochondria (with succinate as substrate) or by a
dding lactate dehydrogenase with NADH as substrate. In mitochondria fr
om cotyledons of the same seedlings, cyanide-insensitive NADH oxidatio
n was less dependent on added pyruvate, partly due to intramitochondri
al generation of pyruvate from endogenous substrates. Cyanide-insensit
ive oxygen uptake with succinate as substrate was greater than that wi
th NADH, in both root and cotyledon mitochondria, but this difference
became much less when an increase in external pH was used to inhibit i
ntramitochondrial pyruvate production via malic enzyme. Malic enzyme a
ctivity in root mitochondria declined with seedling age. The results i
ndicate that the activity of the alternative oxidase in soybean mitoch
ondria is very dependent on the presence of pyruvate: differences in t
he generation of intramitochondrial pyruvate can explain differences i
n alternative oxidase activity between tissues and substrates, and som
e of the changes that occur during seedling development.