Kevin L. Schalinske

Carnitine palmitoyltransferase. Activation by palmitoyl-CoA and inactivation by malonyl-CoA

Extraction of rat liver mitochondria twice with 0.5% Triton X-100 in a salt-free medium leaves less than 10% of the carnitine palmitoyltransferase membrane bound. The remaining membrane-bound enzyme is inhibited virtually completely by 10 microM malonyl-CoA. Preincubation of the extracted membranes with palmitoyl-CoA and salts (KCI) for several minutes activates the enzyme and makes it increasingly insensitive to malonyl-CoA. Addition of malonyl-CoA to the preincubation reverses this desensitization. In albumin-containing media salts also decrease the binding of palmitoyl-CoA to albumin and stimulate carnitine palmitoyltransferase by increasing substrate availability in free solution. The reverse reaction shows accelerated desensitization by palmitoylcarnitine and resensitization by malonyl-CoA.

Partial purification and functional reconstitution of GDP sensitive brown adipose tissue mitochondria uncoupling protein using octyl glucoside.

A partial purification of the uncoupling protein of brown adipose tissue mitochondria (BATM) was achieved by extraction of BATM with 40 mM octyl glucoside, followed by affinity chromatography on ATP-agarose. The isolated protein was functionally reconstituted into liposomes using octyl glucoside dialysis. Proteoliposomes containing the uncoupling protein had an increased proton or chloride conductance when subjected to a valinomycin-induced potassium diffusion potential. The increased ion conductance was consistently found to be inhibited by 200 microM GDP.

Quantitation of carbon flow through the hepatic folate-dependent one-carbon pool in rats.

The quantitation of carbon flow through the folate-dependent one-carbon pool in regard to the synthesis of methionine from the amino acid precursor, serine, was determined in rat liver. Utilizing duodenal cannulated rats and in vivo tracer kinetic techniques where [3-14C]serine was continuously infused at a rate of 3.32 microCi/h, a steady-state (plateau) specific radioactivity was achieved within 200 min from the onset of the infusion period. This resulted in an irreversible loss rate of 431 +/- 12 mumol/h for hepatic serine. In conjunction with the specific radioactivity measurements of hepatic methionine, the percentage of the total entry into the hepatic methionine methyl carbon pool that came from serine (i.e., transfer quotient) was calculated to be 51.7 +/- 5.2%. Similar experiments utilizing [methyl-3H]methionine as the infusate resulted in a value of 112 +/- 6 mumol/h for the irreversible loss rate of hepatic methionine. Using the irreversible loss rate of methionine and the transfer quotient to methionine from serine, the flux of the beta-carbon of serine to remethylate homocysteine and generate methionine was calculated to be 57.9 mumol/h. These results not only present a methodology for the determination of folate-dependent carbon flow in vivo, but also demonstrate the high degree to which the homocysteine moiety of methionine is conserved in vivo to meet the methylation requirements in the rat.

13-cis-retinoic acid and hepatic steatosis in rats

The effect of administration of 13-cis-retinoic acid (100 mg/kg diet) on lipid metabolism was examined in male rats fed either a 20% casein + 0.3% methionine diet, a 20% casein diet, a 10% casein + 0.3% methionine diet, or a 10% casein + 0.6% methionine diet for 10 days. Hepatic triglyceride concentrations of rats fed either 10% casein diet were 3-fold greater than animals receiving diets containing 20% casein. The addition of 13-cis-retinoic acid to the diet further increased the total hepatic lipid (43-56%) and triglyceride (approximately 2-fold) concentrations in rats fed the 10% casein diets. 13-cis-Retinoic acid supplementation did not alter the total liver lipid or triglyceride concentrations in rats fed either of the 20% casein diets. Thus, under specific dietary conditions, the administration of 13-cis-retinoic acid resulted in a marked accumulation of hepatic lipids which did not appear to be related to the total methionine content of the diet nor to the hepatic concentrations of S-adenosylmethionine and glutathione. In addition, all four groups of 13-cis-retinoic acid-fed rats exhibited elevations in the concentration of serum triglycerides, and 10-20% reductions in serum cholesterol concentrations.

Brown adipose tissue from fetal rhesus monkey (Macaca mulatta): morphological and biochemical aspects

Brown adipose tissue (BAT) from fetal rhesus monkeys microscopically resembled adult rodent BAT containing multiocular fat cells with numerous mitochondria. Mitochondrial carnitine palmitoyl transferase activity was lower than that in adult rodents and adenine nucleotide translocase activity was similar to that reported for rats. Rhesus monkey BAT mitochondria (BATM) possess an uncoupling protein that is characteristic of BAT as evidenced by the binding of [3H]GDP, the inhibition by GDP of the high Cl- permeability or rapid alpha-glycerol-3-phosphate oxidation. Electrophoretic analysis of BATM showed the presence of a 32,000 mol.wt protein which was enriched by procedures established for the isolation of BATM uncoupling protein.