Detection of endogenous avidin-binding proteins in rat liver cells by transblot and electron microscopy.
Peroxidase-conjugated avidin was used to detect the endogenous avidin-binding proteins in rat tissues. By a transblot method, avidin-peroxidase interacted with proteins of mitochondrial fractions of rat liver with estimated molecular weights of 120,000 and 74,000. The proteins were identified as pyruvate carboxylase (120 kDa, pI 6.4) and methylcrotonyl-CoA carboxylase (74 kDa, pI 7.2) by two-dimensional gel electrophoresis and transblot method. The estimated molecular weight of an additional band (220,000) detected in the cytosolic fraction of rat liver was consistent with acetyl-CoA carboxylase. Intense staining also occurred with kidney, heart, ovary and adipose tissue, moderate with large and small intestine, cerebrum and cerebellum, and very faint with testis, lung and spleen. The sections of rat liver embedded in LR white were incubated with avidin-colloidal gold conjugate and examined under an electron microscope. The glutaraldehyde-perfused rat liver blocks were also incubated with streptavidin-ferritin conjugate and the ultrathin sections were cut and examined. The majority of gold and ferritin particles were found in the mitochondria of liver cells. No other cellular compartment was labeled except the cytosol which accounted for approximately 20% of the total labeling of the hepatocytes. The present procedure is a simple, rapid and inexpensive method for detecting the intracellular localization of endogenous avidin-binding proteins in the cells.
Haneji
T
T
Department of Anatomy, School of Medicine, Chiba University, Japan.
Toyama
Y
Y
Nagano
T
T
D000273:Adipose Tissue / Q000378:metabolism; D000818:Animals; D001360:Avidin / Q000378:metabolism*; D019735:Carbon-Carbon Ligases; D003600:Cytosol / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D015151:Immunoblotting; D007668:Kidney / Q000378:metabolism; D008025:Ligases / Q000096:biosynthesis* / Q000737:chemistry; D008099:Liver / Q000378:metabolism*; D008297:Male; D008854:Microscopy, Electron; D008861:Microsomes / Q000378:metabolism; D008930:Mitochondria, Liver / Q000378:metabolism; D008970:Molecular Weight; D009206:Myocardium / Q000378:metabolism; D010053:Ovary / Q000378:metabolism; D011766:Pyruvate Carboxylase / Q000096:biosynthesis* / Q000737:chemistry; D051381:Rats; D017208:Rats, Wistar
D001360:Avidin; D008025:Ligases; D019735:Carbon-Carbon Ligases; D011766:Pyruvate Carboxylase; C021573:methylcrotonoyl-CoA carboxylase