Biotin carboxylases in mitochondria and the cytosol from skeletal and cardiac muscle as detected by avidin binding.
Biotin carboxylases in mammalian cells are regulatory enzymes in lipogenesis and gluconeogenesis. In this study, endogenous biotin in skeletal and cardiac muscle was detected using avidin conjugated with alkaline phosphatase and applied in high concentrations to muscle sections. The avidin binding was subsequently visualized by histochemical demonstration of the alkaline phosphatase activity. All cardiac muscle cells showed high affinity for avidin with only the nuclei and the intercalated discs remaining unstained. In skeletal muscle a diffuse reaction could be detected in the sarcoplasm of the muscle fibres. A granular reaction was noted in the same fibres that showed activity for succinic dehydrogenase. The specificity of the coloured reaction product in the muscle sections was investigated and is suggested to be caused by avidin binding to biotin moieties in mitochondria and the cytosol. Mitochondrial and cytosolic preparations of skeletal muscle were electrophoresed in sodium dodecyl sulphate gels. After blotting and incubation with 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 biotin-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 cytosolic band might be a cleavage product of acetyl-CoA carboxylase. We propose that using alkaline phosphatase-conjugated avidin it is possible to detect the mitochondrial and cytosolic biotin-dependent carboxylases in striated muscle.
Kirkeby
S
S
Department of Oral Function, Faculty of Health Sciences, University of Copenhagen, Denmark.
Moe
D
D
Bøg-Hansen
T C
TC
van Noorden
C J
CJ
D000328:Adult; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001360:Avidin / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D019731:Carbon-Nitrogen Ligases; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D066298:In Vitro Techniques; D037102:Lectins / Q000378:metabolism; D008025:Ligases / Q000737:chemistry / Q000378:metabolism*; D008297:Male; D008929:Mitochondria, Heart / Q000201:enzymology; D008931:Mitochondria, Muscle / Q000201:enzymology; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology*; D011485:Protein Binding; D051381:Rats
D037102:Lectins; D001360:Avidin; D001710:Biotin; D000469:Alkaline Phosphatase; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C019791:biotin carboxylase