Immunological analysis of acetyl-CoA carboxylase mass, tissue distribution and subunit composition.
Changes in the mass and subunit structure of liver acetyl-CoA carboxylase (ACC) accompany altered nutrition in vivo. Enzyme activity in different tissues and cell lines is also, in part, determined by variations in both total mass and ACC isoenzyme composition. ACC isoenzyme mass and hetero/homo-isoenzyme association were quantified by three sandwich e.l.i.s.a. assays, i.e. an avidin-based assay that measured total isoenzyme mass and two antibody-sandwich assays which measure polypeptide association. Results from the avidin-based assay reveal that the two major isoenzymes, of molecular mass 265 kDa (ACC 265) and 280 kDa (ACC 280), are present in markedly variable concentration in several rat and mouse tissues and in cell lines of rat and mouse origin. Hepatic ACC mass has been reported to be distributed between mitochondrial and cytosolic fractions and to undergo only a change in subcellular distribution without alteration in total mass on induction/repression of activity in vivo [Roman-Lopez, Shriver, Joseph & Alfred (1989) Biochem. J. 260, 927-930]. However, in the present study, immunoblotting and e.l.i.s.a. analysis reveals that, in rat liver, the mass of both isoenzymes is predominantly cytosolic in distribution, is markedly diminished on fasting and rises 6-8-fold on refeeding of a high-carbohydrate diet. These data support the results of several other investigations of hepatic ACC mass, and are consistent with known nutritionally altered changes in ACC mRNA content. By the two antibody-sandwich e.l.i.s.a. assays, isoenzyme complexes either composed of both ACC 280 and 265 or with multiple copies of ACC 265 are detectable in rat liver enzyme; their concentration varies independently of total ACC mass with the nutritional state of the rat, being lowest in fasting and highest on fasting/refeeding. E.l.i.s.a. analysis, applicable to crude tissue/cell extracts, provides a simple, sensitive and quantitative measurement of ACC mass and subunit composition. Its use may permit needed quantitative insight into the role of variable total ACC and isoenzyme mass and of alterations in ACC subunit composition that occur in vivo or in isolated cells in response to a variety of hormonal and nutritional influences.
Iverson
A J
AJ
Department of Medicine, Dartmouth Medical School, Hanover, NH 03756.
Bianchi
A
A
Nordlund
A C
AC
Witters
L A
LA
D000103:Acetyl-CoA Carboxylase / Q000032:analysis*; D000273:Adipose Tissue / Q000201:enzymology; D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D005215:Fasting; D005260:Female; D005502:Food; D015151:Immunoblotting; D007527:Isoenzymes / Q000032:analysis*; D008025:Ligases / Q000032:analysis*; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009132:Muscles / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D051381:Rats; D011919:Rats, Inbred Strains; D014018:Tissue Distribution
D007527:Isoenzymes; D046911:Macromolecular Substances; D008025:Ligases; D000103:Acetyl-CoA Carboxylase