Intracellular distribution of fumarase in rat skeletal muscle.

The distribution of fumarase activity between the mitochondrial and cytoplasmic compartments of rat skeletal muscle was studied using the method of Fatania and Dalziel (Biochim. Biophys. Acta 631 (1980) 11-19), fractional extraction technique and a method based on the calculation of mitochondrial protein content in the tissue and on the determination of fumarase activity both in the tissue homogenate and in the isolated mitochondria. We found 10%, 5% and 0% of the total fumarase activity in the cytoplasm using these methods, respectively. The results suggest that no more than 10% of the total fumarase activity is present in the cytosolic fraction of rat skeletal muscle. The metabolic consequences of such distribution of fumarase in skeletal muscle are discussed.

Acetate-induced changes of adenine nucleotide levels in rat liver.

The changes in adenine nucleotide concentration induced by acetate were investigated in rat liver in situ and in isolated rat hepatocytes. Adenosine monophosphate (AMP) concentration increased approximately threefold within 15 minutes after intraperitoneal injection of sodium acetate. A small but significant decrease in adenosine triphosphate (ATP) concentration also occurred. Consequently, the ATP/AMP ratio decreased from approximately 14 (the value found in control or sodium chloride-injected rats) to approximately 3 (the value found in sodium acetate-injected rats). Adenosine diphosphate (ADP) concentration increased slightly, but this was statistically nonsignificant. Total adenine nucleotide concentrations after acetate injection remained essentially the same as those in control rats. Adenylate energy charge decreased after acetate administration. No significant changes in nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) concentrations were found after sodium acetate injection. Similar patterns of changes in adenine nucleotide concentrations were found in isolated rat hepatocytes incubated in the presence of acetate. These data indicate that acetate, which appears in human blood either during hemodialysis with acetate-containing solution or after ethanol consumption, may alter energy equilibrium of adenine nucleotides in the liver. This is due to the conversion of ATP to AMP in the course of acetate to acetyl-coenzyme A (CoA) activation. It is therefore possible that accelerated ATP turnover in the liver may contribute both to the "intolerance to acetate" in patients subjected to dialysis with the sodium acetate-containing solution and to the pathogenesis of gout associated with excessive ethanol consumption.

Inhibition by alanine of AMP-deaminase from rabbit skeletal muscle.

1. Alanine inhibits rabbit muscle AMP-deaminase while aspartate, histidine and glutamate are ineffective. 2. The degree and type of inhibition of AMP-deaminase by alanine depend on pH; at pH 6.5 alanine behaves like an allosteric effector exerting a negative heterotropic effect. At pH 7.0 the inhibition is non-competitive, Ki being as high as 19 mM. 3. The probable significance of the effect of alanine on AMP-deaminase in muscle metabolism is discussed.

Two forms of AMP deaminase in bovine heart.

Two forms of bovine heart AMP deaminase were separated by phosphocellulose column chromatography. Form A with lesser affinity to phosphocellulose exhibited a hyperbolic type of substrate curve and was relatively insensitive to ATP. Form B was strongly activated by 1 mM ATP and its substrate saturation kinetics (without ATP) indicated a cooperative effect. The alteration of adenylate energy charge affected forms A and B differently but within the physiological range, the activity of both forms decreased with the increase in energy charge. The Mr of the two forms was identical as estimated by gel filtration. These activities are compared to those of AMP deaminases from the hearts of other species.

Inhibition of pyruvate oxidation by skeletal muscle mitochondria by phenylpyruvate.

1. Phenylpyruvate inhibits pyruvate plus malate oxidation in human and rat skeletal muscle mitochondria in state 3 and in the uncoupled state, it has, however, no effect in state 4. 2. Inhibition by phenylpyruvate of pyruvate oxidation by intact uncoupled rat muscle mitochondria was competitive, with the Ki value about 0.18 mM. 3. It is suggested that the inhibition of pyruvate oxidation is due to the action of phenylpyruvate on muscle pyruvate dehydrogenase, and is the principal cause of the elevated concentration of pyruvate and lactate in blood plasma of phenylketonuric patients.

AMP deaminase from the rat heart injured by high doses of vitamin D.

Adenylate deaminase (EC 3.5.4.6) has been isolated from the hearts of healthy rats and from the rats with cardionecrosis induced by administration of vitamin D in high doses. Crude extract from the necrotic hearts catalysed adenylate deamination with a higher maximum velocity and a higher apparent Km value than the extract from healthy hearts. The enzyme from healthy animals was activated by ATP (Km-type of activation); the maximum velocity of the ATP-activated enzyme could be increased in about 50% by preincubation with phosphatidylcholine-containing liposomes. Liposomes had no effect on the enzyme isolated from the hearts of vitamin D-intoxicated animals, whereas other kinetic and regulatory properties were similar to those of the enzyme from healthy hearts. Kinetic and regulatory parameters of adenylate deamination in the hearts of rats which survived cardionecrosis returned to normal values 6 weeks after vitamin D intoxication.

Diversity of the effect of phosphatidylcholine and sphingomyelin on adenylate deaminase from pig brain.

Liposomes made of sphingomyelin were found to inhibit both ATP-activated and non-activated AMP deaminase from pig brain, in contrast to liposomes made of egg yolk phosphatidylcholine which exhibited an activating effect on the ATP-activated enzyme, being without effect on AMP deaminase in the absence of ATP. Dioleoylphosphatidylcholine exerted a similar effect as egg yolk phosphatidylcholine but dipalmitoylphosphatidylcholine was without effect.

Purification and regulatory properties of pig kidney AMP deaminase.

AMP deaminase (EC 3.5.4.6) from pig kidney was purified about 1200-fold by chromatography on cellulose phosphate. The enzyme showed a sigmoid-shaped substrate saturation curve which was converted to hyperbolic by addition of ATP. The ATP-activated enzyme was sensitive to phosphatidylcholine-containing liposomes which caused a further increase of activity by lowering the S0.5 value and increasing V max. In the absence of ATP, the enzyme was not sensitive to phosphatidylcholine-containing liposomes. Phosphatidate-containing liposomes exerted an inhibitory effect both in the presence and absence of ATP. In the presence of ATP phosphatidate was a non-competitive inhibitor. Orthophosphate was found to be a competitive inhibitor of AMP deaminase from pig kidney. When the phosphatidylcholine/phosphatidic acid ratio in liposomes was 2.7, AMP deaminase was activated, whereas when this ratio dropped below 2.1, liposomes exerted a non-competitive inhibitory effect.

Changes of nucleotide content in human and rat heart during cardiac surgery and ischemia.

The influence of ischemia on purine nucleotide and their catabolite concentration in human myocardium was investigated during surgery of acquired and congenital heart defects. This was compared with the influence of ischemia on rat heart. Concentrations of adenine and guanine nucleotides and their catabolites were measured in the extracts of heart biopsies taken at the onset of ischemia and at the time of reperfusion. The content of myocardial ATP in human heart decreased from the initial value of 22.3 +/- 1.1 to 14.6 +/- 1.5 nmol/mg protein and total adenine nucleotide pool decreased from 34.2 +/- 1.8 to 27.6 +/- 1.5 nmol/mg protein during the operation. Significant increases in myocardial concentrations of purine catabolites were also observed with the most prominent rise in inosine from below 0.5 at the onset of the ischemia to 3.0 +/- 0.5 nmol/mg protein at the time of reperfusion. A positive correlation was demonstrated between the concentration of purine catabolites in the heart at the end of ischemia with the decrease of both ATP and the total nucleotide pool. An interesting metabolic specificity of the ischemic human heart appeared to be only a small accumulation of inosine monophosphate (IMP). The increase of IMP in the rat heart after ischemia was several-fold higher. Thus, cardiac surgery of congenital and acquired heart defects was associated with a significant decrease in myocardial adenylate pool and a single biopsy collected at the end of ischemia seems to be sufficient to evaluate the extent of this metabolic and possibly functional impairment of the heart.

Adenylate degradation products release from the human myocardium during open heart surgery.

Purine degradation products were determined in the human heart coronary sinus effluent collected from patients undergoing cardiac surgery, during infusion of a cardioplegic solution. At the onset of cardiopulmonary bypass the mean concentrations of adenosine, inosine and hypoxanthine were 0.1, 0.5 and 0.3 mumol/l, respectively. Ischemic arrest leads to a progressive increase of the respective levels to 1.4 17.8 and 9.6 mumol/l after 60-80 min of ischemia. Xanthine concentration was undetectable (less than 0.2 mumol/l) throughout. A substantial urate release (20 mumol/l) was observed which decreased with the duration of ischemia. Xanthine oxidoreductase activity in human myocardium was found to be below the detection limit (0.1 mU/g wet weight). Thus, urate release represented wash out of urate which had accumulated in the tissue.

Purines, lactate and phosphate release from child and adult heart during cardioplegic arrest.

The release of lactate, phosphate and purine catabolites from the heart in adult and children undergoing cardiac surgery was recorded. The compounds were determined in the coronary effluent collected during subsequent infusions of cardioplegic solution into the coronary root. As compared to the infusion just after onset of ischemia, both in adults and children manifold increase of the release was observed during subsequent infusions. The rates of release of lactate, phosphate and purines (adenosine + inosine + hypoxanthine) were 1.5 to 2.5 times higher in children than in adult hearts during the second cardioplegic infusion and 3 to 7 times higher during the third cardioplegic infusion in spite of a more frequent infusion of cardioplegic solution in children. A much greater increase of the release of lactate, phosphate and purines provides evidence for more severe metabolic injury during cardioplegic arrest to the heart in children than in adults.

Metabolic characterization of three hamster melanoma variants.

Some enzyme activities and metabolic features of the black Ma melanotic, brown MI melanotic and Ab amelanotic melanomas of hamster were investigated. The activities of hexokinase and phosphofructokinase were similar in all three melanomas, the activity of NAD-dependent glycerol-3-phosphate dehydrogenase was higher in the amelanotic melanoma and that of pyruvate kinase and lactate dehydrogenase were slightly lower in MI than in the other tumors. The activities of citrate synthase, succinate dehydrogenase and malate dehydrogenase were higher in the Ma and MI melanotic melanomas than in the Ab amelanotic melanoma. The rate of labeled CO2 production from 6-14C-glucose, 1,5-14C-citric acid and U-14C-glutamine was about 2 times higher in melanotic melanomas than in amelanotic one, while no significant differences among the three melanomas were found in respect to 1-14C-glucose and U-14C-glycerol-3-phosphate. The production of 14CO2 was much higher from 1-14C-glucose than from 6-14C-glucose in all the melanomas studied. L-DOPA stimulated the production of 14CO2 from 1-14C-glucose much stronger in the Ma and MI melanomas than in the Ab melanoma. In none of the tumors the incorporation from 6-14C-glucose to CO2 was affected by L-DOPA. It is postulated that oxidation of glucose via the pentose phosphate cycle is involved in melanogenesis.