Carl F. Cori

The isolation of pyridoxal-5-phosphate from crystalline muscle phosphorylase

Abstract Dialyzed and Norit-treated muscle phosphorylase a, after recrystallization from versene-glycerophosphate, contains 8 organic phosphate groups per mole or 2 phosphate groups per subunit of molecular weight of 125,000. Four of these phosphate groups are extracted by precipitation of the enzyme with trichloroacetic or perchloric acid. The extracted phosphate compound was isolated as the barium salt and identified as pyridoxal-5-phosphate by its spectrum and by specific enzymic tests. Column chromatography of the trichloroacetic acid extract and paper electrophoresis did not reveal the presence of other phosphorylated compounds. In particular, pyridoxamine-5-phosphate, adenylic acid and other nucleotides could not be detected. Free pyridoxal, pyridoxamine or pyridoxine were also absent.

Regulation of glycolysis in muscle

Abstract The question was raised, what causes the sudden increase in the rate of glycolysis when muscle contracts and the equally rapid decrease in rate when the muscle relaxes. For the purpose of this study, isolated frog sartorii were stimulated electrically while immersed in anaerobic Ringers solution without glucose. The rate was varied between 3 and 48 shocks per min and the duration was 30 min. Under these conditions, the conversion of glycogen to lactate is the main energy producing reaction. The results indicated that two reactions are of prime importance in the regulation of glycolysis in the working muscle: the formation of glucose-1-P from glycogen and inorganic P through the action of the phosphorylase system and the removal of fructose-6-P through the phosphofructokinase reaction. From the relation of the concentration of hexosemonophosphates to the flow rate over the glycolytic system it was concluded that during stimulation these two enzyme systems increase their activity synchronously and proportionately. This is in contrast to the action of epinephrine which causes a much greater increase in phosphorylase activity than in phosphofructokinase activity and thereby leads to a large accumulation of hexosemonophosphates. From measurements of tissue concentration and rate of efflux of lactate from muscle immediately after cessation of stimulation it was concluded that lactate formation returned to the resting rate within 5 min. On the basis of measurements of the tissue concentrations of ATP, ADP, AMP, and Pi it was concluded that changes in the concentrations of these compounds in stimulated muscle were too small to permit the increase in enzymatic rates actually observed in vivo. This applies particularly to the phosphorylase reaction for which in vitro data are available which permit predictions of rates at different concentrations of the above reactants. The concentrations of glucose-6-P, fructose-6-P and fructose-1,6-di-P were likewise not well correlated to the rate increase. The only two compounds that increased proportionately with the frequency of stimulation were end products of anaerobic glycolysis in muscle, namely lactate and α- l -glycero-P. A detailed investigation of the phosphorylase b [rlhar2] a interconversion in stimulated muscle suggested that the phosphorylase b kinase underwent an activation-inactivation cycle closely connected with the contraction and relaxation phases of muscle. An analysis of results obtained with a strain of mice incapable of forming phosphorylase a during stimulation indicated that this interconversion, while not essential for lactate formation per se, was of importance for the time of onset and the final speed of lactate formation. A hypothesis based on localization and structural organization of the glycolytic chain in muscle is proposed which could explain the results summarized above. A mechanism is suggested which is similar in nature to the activation and inactivation of actomyosin (and perhaps of phosphorylase b kinase) by Ca++ ions. It contains the idea of access of reactants to the catalyst during contraction and separation from reactants during relaxation.

Mechanism of Formation of Hexosemonophosphate in Muscle and Isolation of a New Phosphate Ester

Summary In minced and washed frog muscle incubated in phosphate buffer, added adenylic acid transfers inorganic phosphate to carbohydrate resulting in the formation of hexosemonophosphate. The first phosphorylation product proved to be a new ester which was isolated as the crystalline brucin salt and had the properties of glucose-1-phosphoric acid; when added to frog muscle extract it was converted in a few minutes to the Embden ester.

The metabolism of fructose in liver. Isolation of fructose-1-phosphate and inorganic pyrophosphate

Fructose is converted to glucose in a dialyzed liver homogenate fortified with an oxidizable substrate, Mg++ and a catalytic amount of ATP. In the presence of fluoride, the conversion of fructose to glucose is inhibited, and an acid-labile phosphate ester accumulates. Isolation and characterization by its strong negative specific rotation, hydrolysis constant in acid, and liberation of inorganic phosphate on forming the osazone, identified this ester as funcoose-1-phosphate. Another acid-labile phosphate ester which accumulated even in the absence of a phosphate acceptor was isolated as the crystalline sodium salt and identified as inorganic pyrophosphate. A protein fraction precipitated from liver extract between 0.45 and 0.55 saturation with ammonium sulfate catalyzed the conversion of fructose-1-phosphate to hexose-6-phosphate. This mutase reaction was accelerated by Mg++ ions and inhibited by fluoride. In liver homogenate, in the absence of fluoride, the mutase reaction is followed by dephosphorylation of hexose-6-phosphate to free glucose and inorganic phosphate. These three enzymatic reactions explain the mechanism of conversion of fructose to glucose in the liver. The formation of fructose-1-phosphate by liver fructokinase is not inhibited by glucose, in contrast to the strong inhibition glucose exerts on the phosphorylation of fructose by yeast and brain hexokinase. Fructose utilization in the liver is thus independent of glucose utilization, the latter being catalyzed by a separate enzyme. The liver fructokinase is much more active in an aerobic system with catalytic amounts of ATP than in an anaerobic system with an excess of ATP. A summary of the enzymatic reactions concerned with the metabolism of fructose in various tissues is presented.

The combination of diphosphopyridine nucleotide with glyceraldehyde phosphate dehydrogenase

Abstract The theory has been examined that glyceraldehyde phosphate dehydrogenase from rabbit muscle contains two catalytic sites, having dissociation constants with DPN which differ by a factor of 100 or more. The facts in favour of a very slightly dissociated site are that the enzyme retains on recrystallization or dialysis a stoichiometric amount of DPN. From observations made in kinetic measurements this DPN does not measurably dissociate on five fold dilution of the enzyme. Furthermore, evidence is presented that DPNH is also bound to the enzyme and that it can be displaced by added DPN to an extent which indicates relative affinities of the protein for the oxidized and reduced forms of at least the same order of magnitude. The fact that bound DPN can be removed from the enzyme by adsorption on charcoal and that it exchanges rapidly with DPN labelled with P32 allows the conclusion (a) that the binding is not of the covalent type and (b) that bound DPN has a measurable dissociation. Other approaches to the problem did not reveal differences between the reaction with enzyme-DPN and the reaction with a catalytic amount of enzyme plus added DPN. In both cases, in the presence of an excess of substrate, the reaction was first order with respect to the total DPN concentration, and the pH optimum was the same. The equilibrium constants with bound and with added DPN were also the same. Iodoacetate inhibited the reaction at the bound site. Kinetic studies involving simultaneous reaction of bound and added DPN showed that with increasing concentrations of the latter a saturation value was approached, but the data could not be resolved to give an unequivocal answer in terms of two catalytic sites. Enzyme DPNH was shown to react rapidly with lactic dehydrogenase plus pyruvate, or in the reverse reaction, bound DPN was found to react with lactic dehydrogenase plus lactate. On the basis of the assumption that bound DPNH has a very low dissociation, the observed rate of reaction with lactic dehydrogenase would have to be attributed to collisions between protein molecules. In the light of available evidence the hypothesis that glyceraldehyde phosphate dehydrogenase has two catalytic sites which differ in their affinity for DPN requires further examination.

Multiple biochemical effects of a series of x-ray induced mutations at the albino locus in the mouse.

Further studies of the four radiation-induced lethal albino mutations causing glucose 6-phosphatase deficiency in the mouse have shown that there is also a deficiency of tyrosine aminotransferase in the newborn albino mutants. These two enzymes can be induced in fetal and newborn heterozygous or homozygous normal littermate controls by injection of glucagon or dibutyryl cyclic AMP, but not in the albino mutants. Microsomal NADH-cytochrome c reductase was found to be increased in the albino mutants. The multiple biochemical abnormalities in the albino mutants, in addition to the lack of gene dosage effect in the heterozygotes, suggest the involvement of genes other than the structural genes for particular enzymes. When a new radiation-induced lethal albino mutation was tested against the four original alleles, complementation resulted, and double heterozygotes were found to be viable, with normal enzyme levels.

The rate of absorption of a mixture of glucose and galactose.

Summary When glucose and galactose are absorbed from a mixture of equal parts of these two sugars, the rate of absorption of both sugars is reduced to such an extent, that the total amount of sugar absorbed is not greater than if glucose alone or galactose alone were being absorbed.

The interaction of phosphorylase with protamine.

Abstract Protamine at 0°C forms a highly insoluble complex with phosphorylase a which contains one or two moles of protamine per mole of phosphorylase, depending on whether or not protamine is present in excess. Precipitates formed at 30° contain up to 7 moles of protamine per mole of enzyme, and the precipitation curves are diphasic owing to a solubilizing effect of an excess of protamine on the complex. The pH dependence of the precipitation shows that phosphorylase and protamine must be oppositely charged in order to form a precipitate. The protein which protamine precipitates directly from crude muscle extract contains at least 50% phosphorylase a. Protamine may be used to separate phosphorylase a and b, since the latter forms a much more soluble complex than the former. A crystalline protamine-phosphorylase b complex has been described containing equimolar ratios of the proteins. The enzymic activity of the precipitated phosphorylase a complex becomes progressively lower as the molar ratio of protamine in the complex is increased. The diphasic activity curves of phosphorylase a in the presence of increasing amounts of salmine as reported by Krebs in the preceding paper1, may be explained by similar diphasic precipitition curves of phosphorylase and salmine, as well as by the varying enzymic activities of the different complexes. From the temperature dependence of the phosphorylase a reaction in the absence and presence of adenylic acid it was calculated that the energy of activation was 24,600 calories in the former and 20,800 calories in the latter case. Thus adenylic acid increases the rate of the phosphorylase a reaction by lowering the energy of activation. In the phosphorylase b reaction, which does not occur unless adenylic acid is added, the energy of activation was 21,200 calories, or approximately the same as for phosphorylase a in the presence of adenylic acid.

The Absorption of Glycine and d, 1-Alanine.:

The rate of intestinal absorption of glycine and alanine has been studied with the method described in a previous publication. 1 Groups of 10 rats were killed 1, 2 and 3 hours after feeding a 15 per cent solution of the amino acid by stomach tube. The average absorption coefficients (i. e., the amount absorbed per 100 gm. of body weight per hr.) for glycine were as follows: 1 hr., 0.048 gm.; 2 hours, 0.050 gm.; 3 hours, 0.046 gm. The values obtained for d, 1-alanine were: 1 hour, 0.044 gm.; 2 hours, 0.044 gm.; 3 hours, 0.047 gm. If these values are plotted against time, it will be found that they fall on a straight line. Since the rate of absorption of different hexoses and pentoses was also found to follow a straight line, 1 the same considerations that were made with respect to the sugars, apply to the amino acids. The rate of absorption is, within wide limits, independent of the absolute amount and the concentration of amino acid present in the intestine. If this were not the case, the rate of absorption would be represented by a curve rather than a straight line. A mixture of equal parts of glycine and alanine was absorbed at a rate of 0.061 gm. per 100 gm. of body weight per hour. One would expect a rate of absorption of 0.09 gm., if each amino acid was absorbed independently from the mixture. It has been found previously, 2 that when glucose and galactose are absorbed from a mixture, the rate of absorption of both sugars is reduced to such an extent that the total amount of sugar absorbed is not much greater than if glucose alone or galactose alone were being absorbed.

Glucose 6-phosphatase deficiency, mechanisms of genetic control and biochemistry.

Mechanisms are discussed by which several radiation-induced lethal mutations in the mouse control enzyme activity and morphogenesis. These mutations behave as alleles at the albino locus in chromosome I; furthermore, all of them suppress glucose 6-phosphatase activity, resulting in perinatal death of albino homozygotes. Additional pleiotropic effects, and the absence of a dosage effect in heterozygotes, are not easily explained by a mutational change in the structural gene for glucose 6-phosphatase. The mutations might affect membrane proteins and thus render membrane structure abnormal; such membranes may lack the ability to bind tyrosinase as well as glucose 6-phosphatase and may also be responsible for morphogenetic abnormalities.