BIOTIN CARBOXYLASES IN MITOCHONDRIA AND THE CYTOSOL FROM SKELETAL ANDCARDIAC-MUSCLE AS DETECTED BY AVIDIN BINDING

Biotin carboxylases in mammalian cells are regulatory enzymes in lipog enesis and gluconeogenesis. In this study, endogenous biotin in skelet al and cardiac muscle was detected using avidin conjugated with alkali ne phosphatase and applied in high concentrations to muscle sections. The avidin binding was subsequently visualized by histochemical demons tration of the alkaline phosphatase activity. All cardiac muscle cells showed high affinity for avidin with only the nuclei and the intercal ated discs remaining unstained. In skeletal muscle a diffuse reaction could be detected in the sarcoplasm of the muscle fibres. A granular r eaction was noted in the same fibres that showed activity for succinic dehydrogenase. The specificity of the coloured reaction product in th e muscle sections was investigated and is suggested to be caused by av idin binding to biotin moieties in mitochondria and the cytosol. Mitoc hondrial and cytosolic preparations of skeletal muscle were electropho resed in sodium dodecyl sulphate gels. After blotting and incubation w ith conjugated avidin, two bands with molecular weights of 75 kDa and 130 kDa respectively were evident in the mitochondrial preparation. It is suggested that the 75-kDa band represents comigration of the bioti n-containing subunits of propionyl-CoA carboxylase and methylcrotonyl- CoA carboxylase. The 130-kDa band may represent the biotin-containing pyruvate carboxylase. In the cytosolic preparation a 270-kDa band was stained in blots that had been incubated with conjugated avidin; this band is suggested to represent acetyl-CoA carboxylase. A 190-kDa cytos olic band might be a cleavage product of acetyl-CoA carboxylase. We pr opose that using alkaline phosphatase-conjugated avidin it is possible to detect the mitochondrial and cytosolic biotin-dependent carboxylas es in striated muscle.