RT Journal Article
SR Electronic
T1 Characterization of Biotin and 3-Methylcrotonyl-Coenzyme A Carboxylase in Higher Plant Mitochondria
JF Plant Physiology
JO Plant Physiol.
FD American Society of Plant Biologists
SP 450
OP 455
DO 10.1104/pp.99.2.450
VO 99
IS 2
A1 Baldet, Pierre
A1 Alban, Claude
A1 Axiotis, Stella
A1 Douce, Roland
YR 1992
UL http://www.plantphysiol.org/content/99/2/450.abstract
AB Mitochondria from green pea (Pisum sativum) leaves were purified free of peroxisomes and chlorophyll contamination and examined for their biotin content. The bulk of the bound biotin detected in plant mitochondria was shown to be associated with the matrix space to a concentration of about 13 micromolar, and no free biotin was detected. Western blot analysis of mitochondrial polypeptides using horseradish peroxidase-labeled streptavidin revealed a unique biotin-containing polypeptide with a molecular weight of 76,000. This polypeptide was implicated as being the biotinylated subunit of 3-methylcrotonyl-coenzyme A (CoA) carboxylase. Fractionation of pea leaf protoplasts demonstrated that this enzyme activity was located largely in mitochondria. The 3-methylcrotonyl-CoA carboxylase activity was latent when assayed in isotonic media. The majority of the enzyme activity was found in the soluble matrix of mitochondria. Maximal 3-methylcrotonyl-CoA carboxylase activity was found at pH 8.3 in the presence of Mg2+. Kinetic constants (apparent Km values) for the enzyme substrates were: 3-methylcrotonyl-CoA, 0.05 millimolar; ATP, 0.16 millimolar; HCO3−, 2.2 millimolar. The involvement of 3-methylcrotonyl-CoA carboxylase in the leucine degradation pathway in plant mitochondria is proposed.