Localization of Free and Bound Biotin in Cells from Green Pea Leaves

Cytosol and vacuoles from protoplasts, chloroplasts, and mitochondria from green pea ( Pisum sativum) leaves were purified and examined for their biotin content. The bulk of free biotin was shown to be exclusively associated with the cytosolic fraction at a concentration of about 4 pmol/mg protein and no bound biotin was detected. The bulk of bound biotin (biotin-containing carboxylases) was associated with the soluble fraction of chloroplasts and mitochondria at a concentration of about 1.2 and 13 μM, respectively. No free biotin was detected in these organelles. Western blot analysis of total, chloroplastic, and mitochondrial polypeptides, using horseradish peroxidase-labeled streptavidin, revealed three biotin-containing polypeptides with molecular mass of 220,000, 76,000 and 34,000. All were detected in the total pea leaf extract, but the M r 76,000 and the M r 34,000 biotinylated polypeptides were only detected in mitochondria and chloroplasts, respectively. 3-Methylcrotonyl-coenzyme A carboxylase and acetyl-coenzyme A carboxylase activities were measured in these two compartments, respectively. Previously, it has been shown that the M r 76,000 polypeptide was the biotinylated subunit of the mitochondnial 3-methylcrotonyl-coenzyme A carboxylase. In this paper, the origin and putative function of free biotin located in cytosol are discussed.