Charles J. Stewart

Carboxylation of acetonyldethio-coenzyme a by acetyl coenzyme a carboxylase

Acetonyldethio-coenzyme A has been synthesized [l] together with its 2’-phosphate isomer as a unique analogue of acetyl-CoA; in this analogue, hereafter referred to as acetone-CoA, the sulfur is replaced by methylene. Acetone-CoA was shown to exhibit strong competitive inhibition of citrate synthase, which mediates the formation of citrate from acetyl-CoA and oxaloacetate [ 11. The present investigation was designed to examine the effect of acetone-CoA on acetyl-CoA carboxylase (acetyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1.2), which catalyzes the ATP-dependent carboxylation of acetyl-CoA to form malonyl-CoA, ADP and orthophosphate [2] and plays a critical role in the regulation of fatty acid biosynthesis [3]. The results obtained have demonstrated that acetone-CoA serves as a substrate of acetyl-CoA carboxylase from rat liver; the apparent K, value for acetone-CoA is approximately livetimes as high as that for acetyl-CoA, while the V max value for acetone-CoA is approximately one-

Gas chromatographic determination of β-phenethylbiguanide and its metabolite p-hydroxy-β-phenethylbiguanide in serum and urine

A gas chromatographic method is presented for the detection of β-phenethylbiguanide (PEBG) and its metabolite, p-hydroxy-β-phenethylbiguanide (p-OH PEBG). The procedure is applicable for the determination of the drug and its metabolite in the serum and urine of rats. The detection limit is 0.2 μg PEBG and 0.5 μg p-OH PEBG per ml of serum or urine. A time course study of blood concentration and elimination rate following intraperitoneal injection of 100 mg/kg of PEBG to normal rats was performed. β-PEBG was found to be present in the blood and the urine, p-OH PEBG was only detected in the urine. Twenty-four hours following intraperitoneal injection, the urine contained 32% of the administered dose, 20% as unaltered PEBG and 12% as p-OH PEBG.

Inhibition of Glucose Oxidation by 6-Deoxy-D-Glucose.

Summary 6-Deoxy-D-glucose has been shown to be an inhibitor of glucose oxidation in rat kidney slices, mouse adipose tissue, and rat diaphragm. The raio of 6-DOG to glucose which gave a 50% inhibition of glucose oxidation corresponded to 12:1 in rat kidney slices and rat diaphragm and 4:1 in mouse adipose tissue. The inhibition of glucose oxidation in mouse adipose tissue was shown to be reversible. Experiments using rat diaphragm tissue showed that 6-DOG inhibited uptake of glucose in proportion to decrease in glucose oxidation. Incubation of mouse adipose tissue with 6-DOG uniformly labeled with C14 did not result in formation of measurable amounts of C14O2. It is concluded that this compound is incapable of complete oxidation in this tissue. It is believed that the site of competitive inhibition is either at the cell entry mechanism or at the hexokinase reaction. Thus, the metabolic block produced by 6-DOG should be less complex than that produced by the 2-position modified sugars. The use of 6-DOG may be very useful for studying cell permeation mechanisms.

Enzymatic Synthesis of Acetono-CoA, A Competitive Inhibitor of Acetyl-CoA with Citrate Synthase

The synthetic acetyl-CoA analog, acetono-CoA (acetonyldethiocoenzyme A), in which the sulfur atom has been replaced by a methylene group, has been previously synthesized and found to be a competitive inhibitor (KI= 13.2 µM) of acetyl-CoA with citrate synthase. To determine whether the acetono-group would interfere with CoA biosynthesis, a crude preparation of the bifunctional enzyme complex which converts ATP and pantetheine 4’-phosphate to CoA was investigated. Incubation mixtures of the crude complex, ATP, and acetonyldethio-pantetheine 4’-phosphate were prepared. Products and reactants were isolated by DEAE-cellulose column chromatography and peaks were identified by TLC. Enzymatic synthesis of acetono-CoA was verified by establishing its KI (25 µM) in the citrate synthase reaction.