3-Methylcrotonyl-Coenzyme A Carboxylase Is a Component of the Mitochondrial Leucine Catabolic Pathway in Plants

MCCase activity and the accumulation of the biotin-containing subunit of MCCase in soybean seedlings at 13 DAP. A, Organs of a soybean seedling. B, MCCase activity (mean of five experiments ± se). C, Western blot probed with antiserum to detect the biotin-containing subunit of MCCase. D, Western blot identical to that shown in C, but instead probed with¹²⁵I-streptavidin to detect the biotin prosthetic group on the biotin-containing subunit of MCCase. In C and D protein samples were loaded on the basis of equal MCCase activity (0.1 nmol/min) to detect differences in the catalytic efficiency of the enzyme among organs. The data presented in C and D were gathered from a single experiment; five replicates of this experiment gave similar results.

Effect of cotyledon development on MCCase activity and Leu, protein, and chlorophyll accumulation. A, Chlorophyll and total protein contents. B, MCCase activity and free Leu content. C, Western blot probed with antiserum to detect the biotin-containing subunit of MCCase. D, Western blot identical to that shown in C, but instead probed with ¹²⁵I-streptavidin to detect the biotin prosthetic group on the biotin-containing subunit of MCCase. In C and D protein samples were loaded on the basis of equal MCCase activity (0.1 nmol/min) to detect differences in the catalytic efficiency of the enzyme during cotyledon development. The data presented in A and B are means ± se from three replicates. The data presented in C and D were gathered from a single experiment; three replicates of this experiment gave similar results.

Kinetics of the incorporation of radioactively labeled bicarbonate into acid-stable metabolites by mitochondrial extracts in the presence of MC-CoA and cofactors. Time course of the incorporation of radioactivity into acid-stable products (A), MG-CoA (B), HMG-CoA (C), or acetoacetate (D). Incubations were carried out either without or with 10 μg of avidin (to inhibit MCCase activity), which was added 2.5 min after the start of the reaction (arrows). Each time point is an independent sample and represents two replicates. Because of the rapid incorporation of NaH¹⁴CO₃, the background radioactivity was impossible to obtain. Therefore, the 0-min time point was derived from samples incubated for 1 min in the presence of 10 μg of avidin.

Steady-state levels of the mRNA coding for the biotin-containing subunit of MCCase in organs of soybean seedlings at 13 DAP and during cotyledon development. Ethidium bromide-stained agarose gel containing approximately equal amounts (20 μg) of RNA, isolated from various organs (A) or from cotyledons at different stages of development (B). Northern blot depicting the accumulation of the mRNA coding for the biotin-containing subunit of MCCase in various organs (C) or in cotyledons at different stages of development (D). The data presented in this figure were gathered from a single experiment. Five replicates (A and C) and three replicates (B and D) of these experiments gave similar results.