Acetyl‐Coenzyme‐A Carboxylase in Cultured Hepatocytes

Abstract

Studies were made on the content, synthesis and degradation of acetyl-coenzyme-A carboxylase in JTC-25 . P3 cells, hepatocytes which can be maintained in a protein-free and lipid-free chemically defined medium. The addition of corn oil or fatty acid to the medium resulted in a decrease in the activity level of the enzyme without impairing the viability of cells. All the fatty acids tested exhibited this effect, although linoleic acid and oleic acid were more effective than palmitic acid, stearic acid and arachidonic acid. Immunochemical titration and Ouchterlony double-diffusion analysis indicated that the decrease in the activity level of the enzyme observed in cells incubated in medium supplemented with fatty acid can be ascribed to a reduction of the quantity of the enzyme. Isotopic leucine incorporation studies with the use of immunochemical techniques demonstrated that this reduction of the enzyme content is due to a decrease in the rate of synthesis of the enzyme. The rate of degradation of the enzyme was essentially unaffected, the half-life being 25 and 28 h, respectively, in cells incubated in the presence and absence of fatty acid. It was shown that most of the isotopic fatty acid added to the medium was incorporated into cellular phospholipids, while a very small portion of it was recovered in triglyceride and nonesterified fatty acid.Acetyl-coenzyme-A carboxylase catalyzes the first step leading specifically to long-chain fatty acid synthesis and plays a critical role in the regulation of this synthetic process (see [l]). The activity level of this enzyme in mammalian tissues varies in accord with the rate of fatty acid synthesis under various dietary, hormonal, developmental and genetic conditions of the animal (see [2,3]). Particular emphasis has recently been placed on the questions, whether the variations in the level of acetyl-CoA carboxylase activity in tissue extracts observed under the various conditions are actually determined by changes in the quantity of the enzyme, and if so, whether these changes are due to alterations in the rate of synthesis or in that of degradation of the enzyme. Immunochemical studies with rats subjected to different Enzymes. Acetyl-CoA carboxylase or acetyl-CoA : carbondioxide ligase (ADP-forming) (EC 6.4.1.2) ; lactate dehydrogenase or L-lactate : NAD' oxidoreductase (EC 1.1.1.27); ATP-citrate lyase or ATP : citrate oxaloacetate-lyase (pro-3S-CH2COO-+ acetyl-CoA; ATP-dephosphorylating) (EC 4.1.3.8); malic enzyme or L-malate : NADP' oxidoreductase (oxaloacetate-decarboxylating) (EC 1.1.1.40).dietary manipulations [4-61 and alloxan-diabetes [6] as well as with genetically obese mice [7] demonstrated that the fluctuations of the level of acetyl-CoA carboxylase activity in the liver of these animals reflect changes in the quantity of the enzyme. Furthermore, it was shown with the use of combined immunochemical and isotopic techniques that the increase in the acetyl-CoA carboxylase content of the liver of rats fasted and subsequently refed a fat-free diet can be ascr...

Glucose-dependent induction of acetyl-CoA carboxylase in rat hepatocyte cultures

“…, (Nakanishi & Numa, 1971). The half-life of acetyl-CoA carboxylase as determined in primary hepatocyte cultures in the present study (39 h) was higher than that observed in an established hepatocyte cell line [28h (Kitajima et al, 1975)]. However, it was smaller than the half-life in intact rats [48 h (Majerus & Kilburn, 1969); 59h (Nakanishi & Numa, 1970)].…”

Acetyl-Coenzyme-A Carboxylase from Rat Liver. Subunit Structure and Proteolytic Modification

“…The procedures used were ammonium sulfate fractionation of the soluble supernatant fraction, calcium phosphate gel adsorption and elution, a second ammonium sulfate fractionation, DEAE-cellulose chromatography, and Sepharose 2 B chromatography. Thc final enzyme preparations exhibited a specific activity of 5-7 U/mg of protein at 37 C. Their homogeneity was ascertained as described previously [18] Beckman SW-65L Ti rotor at 19 "C and 40000 rev./ min for 100 min. In order to confirm that the radioactivity found in the purified enzyme was actually located in its biotinyl prosthetic group, 150 pg of the labelled enzyme (25 500 counts/min) was first heated at 60 "C for 5 min and then digested with 50 pg proteinase K at 37 "C for 24 h in 60 pl0.1 M Tris-C1 buffer pH 7.0.…”

Evidence that acyl coenzyme A synthetase activity is required for repression of yeast acetyl coenzyme A carboxylase by exogenous fatty acids.

“…Furthermore, isotopic leucine incorporation studies with the use of immunochemical techniques have revealed that the reduction of the enzyme content upon addition of fatty acid to the culture medium can be ascribed to a decrease in the rate of synthesis of the enzyme, while the rate of degradation of the enzyme is not affected by exogenous fatty acid (5). On the basis of these findings, it appears reasonable to assume that the observed decrease in the acetyl-CoA carboxylase content of S. cerevisiae likewise represents a repression of the enzyme.…”