Gerty T. Cori

The effect of epinephrine and other glycogenolytic agents on the phosphorylase A content of muscle

Abstract 1. Injection of epinephrine in rats caused a marked rise in the phosphorylase a content of muscle. Nor-epinephrine had the same effect but required much larger doses. 2. Epinephrine prevented the complete disappearance of phosphorylase a in a fatigued muscle and accelerated the resynthesis of phosphorylase a during recovery from fatigue. 3. The concentration of active phosphorylase a in thin frog muscles, incubated in a solution containing epinephrine, aerobically or anaerobically rose at the expense of inactive phosphorylase b . 4. Versene inhibited the above rise. 5. The glycogenolytic agents dinitrophenol and caffeine reduced the phosphorylase a content when it was high and caused no change when it was low.

Enzymic conversion of phosphorylase a to phosphorylase b

Abstract Phosphorylase b, the product of the action of a muscle enzyme (PR) on crystalline rabbit muscle phosphorylase a, has one half the molecular weight of the a form. The molecular weights are 242, 000 and 495, 000 respectively.

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.

Enzymatic Breakdown of Glycogen in Liver Extracts

It has been shown in previous papers 1 that dialyzed liver extracts contain an enzyme which forms glucose-1-phosphoric acid (1-ester) from glycogen and inorganic phosphate and that this enzyme is activated by adenylic acid. When 1-ester is added to liver extract, inorganic phosphate is split off due to the presence of a phosphatase. It is shown in this paper that the combined action of these two enzymes converts glycogen to glucose, a reaction which has hitherto been ascribed exclusively to a diastatic enzyme. A typical experiment is recorded in Table I. The liver of a fasted rabbit was cooled, ground in a mortar and extracted twice with ice-cold distilled water. The extract was dialyzed for 4 hours in thin collodion sacs against running tap water of 10° it was then centrifuged at high speed for 10 minutes and used at once. Additions were made to the extract as shown in Table I, and analyses were performed before and after incubation. The glycogen, after digestion in 30% NaOH, was precipitated from boiling alcohol, centrifuged, redissolved in water and again precipitated. The fermentable sugar was determined in HgSO4-BaCO3 filtrates, inorganic phosphate in trichloracetic acid filtrates. The formation of hexosemonophosphate was calculated from the amount of inorganic P which was esterified during incubation. Table I shows that addition of phosphate to the reaction mixture increases very markedly the disappearance of glycogen and that addition of adenylic acid causes a further increase. In the latter case the rate of disappearance of glycogen corresponds to 1.4 g per 100 g liver per hour which would be sufficiently rapid to meet physiological needs of blood sugar formation in that organ. Phlorhizin, which is known to inhibit the disruptive phosphorylation of glycogen in muscle hash or extract, 2 also has an inhibitory effect in liver extracts.

Formation of Glucose-1-Phosphoric Acid in Muscle Extract

In recent experiments 1 on minced and washed frog muscle, adenylic acid, in amounts corresponding to 4 mg. P per 100 gm. muscle, increased very markedly the formation of hexosemono-phosphate. The small amount of phosphorylation which occurred without addition of adenylic acid was attributed to the presence of nucleotides which had not been removed by washing, since the washed muscle contained 2 to 4 mg. % of organic P. The first phosphorylation product was found to be glucose-1-phosphoric acid which was isolated as the crystalline brucine salt and which has since been synthesized and identified as an α-compound. This is of significance in view of the fact that glycogen, the carbohydrate from which this ester is formed, consists of α-glucosidic linkages. Glucose-1-phosphoric acid, when added to fresh muscle extract, is converted in a few minutes to hexose-6-phosphoric acid by a wandering of the phosphate group. The same change occurs in minced and washed muscle but at a slower rate, so that after short periods of incubation and with phosphorylation accelerated by the addition of adenylic acid, the rate of formation of the 1-ester exceeds its rate of conversion to the 6-ester, these being the conditions which made possible the isolation of the new ester. The experiments in Table I show that a dialyzed rabbit muscle extract behaves in the same way as minced and washed frog muscle. Without addition of adenylic acid phosphorylation was slow and glucose-1-phosphoric acid did not accumulate in significant amounts. With adenylic acid added phosphorylation was increased about 4 times, resulting in an accumulation of glucose-1-phosphoric acid. Between 60 and 90 minutes phosphorylation no longer occurred and the gain in 6-ester (+3.5 mg.) was balanced by the decrease in 1-ester (—3.4 mg.).

Relation Between Absorption and Utilization of Galactose.

Previous experiments 1 have shown that when galactose and glucose are absorbed together, the rate of absorption of galactose is greatly reduced. Folin and Berglund 2 had reported previously that in men the sugar excretion is less than one-tenth as great when a mixture of glucose and galactose (100 gm. each) is ingested as the excretion obtained from 100 gm. of galactose when taken alone. They suggested that the extent to which galactose is utilized in the human organism depends on the quantity of available glucose. Corley 3 administered glucose and galactose intravenously and obtained no evidence that the presence of an excess of glucose in the blood increased the ability of the rabbit to utilize circulating galactose. However, when Corley 4 administered glucose and galactose by mouth, the urinary excretion of galactose decreased, in confirmation of the results of Folin and Berglund. These observations made it desirable to establish a more definite relationship between the rate of absorption and the extent of utilization of galactose in the body. Such experiments were made 2 years ago and are now here reported. Experimental. Each group, consisting of 4 to 6 rats, fasted previously for 48 hours, was fed a different amount of galactose by stomach tube, the amount introduced being known in each case. In one series of experiments galactose alone was fed, in a second series a galactose-glucose mixture of equal parts was given, and in a third series, galactose was presented to the rats in the form of lactose. The collection of urine was extended beyond the period of absorption in order to make allowance for any lag in sugar excretion. Figure 1 is a graphic illustration of the average values obtained in these experiments.

Fate of Glucose and Other Sugars in the Eviscerated Animal.

Rats fasted previously for 24 hours were operated upon under amytal anesthesia. The whole intestinal tract, including spleen and pancreas, and occasionally kidneys and adrenals, was removed. The blood sugar falls in such a preparation owing to the fact that the liver is out of circulation. The sugar content of the whole rat was determined by passing the heat coagulated tissues through a meat grinder and extracting them with hot water. Separation of sugar from non-sugar reducing substances was attempted. Sugar (100 mg. per 100 gm. rat) was injected intravenously after evisceration and groups of rats were killed and analyzed, (a) 10 minutes after the sugar injection, (b) after 1 hour of sugar injection at a constant rate, and (c) after 1 hour of sugar injection plus insulin. The average percentages of sugar recovered in the 3 cases are shown in the following summary: The specificity of the insulin action is a very striking phenomenon. In experiments with injection of larger amounts of sugar and extending over 2 hours, glucose, in the absence of insulin injection, was found to disappear to the same extent as the other sugars. The glycogen content of the muscles increased under these conditions.

Comparative study of the blood sugar concentration in the liver vein, the femoral artery and the femoral vein during insulin action.

A new method has been developed for the collection of blood from the liver vein, whereby admixture of blood from the vena cava is avoided. The puncture of the liver vein requires no narcosis of the animal and can be repeated several times. The normal sugar values for different blood vessels obtained on rabbits which were previously starved for 24 hours were as follows: 1. For the difference in blood sugar concentration of the liver vein and the neck vein: 28 mg. (average of 9 experiments). 2. For the difference in the blood sugar concentration of the femoral artery and femoral vein: 8 mg. (average of 20 experiments). 3. For the difference in blood sugar concentration of the liver vein and the femoral artery: 23 mg. (average of 9 experiments). The following two questions were studied: 1. Does the liver show a diminished output of sugar into the bloodstream during insulin action? 2. Are the muscles as well as the liver influenced by insulin? Simultaneous analysis of the blood of the liver vein, the femoral artery and the femoral vein showed that the factors causing the fall of the blood sugar during insulin action are: 1. A diminished output of sugar by the liver into the blood stream. 2. A larger intake of sugar than normal by the muscle from the blood stream. Insulin may cause a fall of blood sugar in three ways: a. By its action on the liver (diminished output of sugar). b. By its action on the muscle (increased intake of sugar). c. By its combined action on both the liver and the muscle, the former showing diminished output and the latter simultaneously increased intake of sugar.