Addition Of NADP Research Articles

On the Structure and Catalytic Function of Mammary Glucose 6-Phosphate Dehydrogenase

Abstract Experiments were conducted in order to elucidate the relationship between the structure of mammary glucose 6-phosphate dehydrogenase and its catalytic activities with nicotinamide adenine dinucleotide phosphate and nicotinamide adenine dinucleotide. A model is proposed to account for the results which depicts the enzyme as existing in the form of two monomers, X and Y, in rapid, mobile equilibrium with each other, and in which X can be reversibly dissociated into inactive subunits under suitable conditions and Y can be dimerized at high protein concentration. The formation of Y from X is promoted by the addition of NADP+ or NADPH, whereas glycerol or NAD+ promote the formation of X from Y. The NADP-linked activity of Y is greater than that of X, but X possesses greater NAD-linked activity than Y. The experiments here presented in support of the proposed model deal with the interconversion of X and Y. The kinetics of NADPH inhibition of NAD-linked glucose 6-phosphate dehydrogenase is compared in aqueous solution and in 40% glycerol. Sigmoid kinetics is seen in aqueous solution, but normal kinetics obtains in 40% glycerol. Experiments are reported showing that, under the conditions used in the kinetic studies, NADPH does not cause the enzyme to dimerize. It is also shown that the rate and extent of reactivation of glucose 6-phosphate dehydrogenase from inactive subunits are promoted in the presence of NADPH and hindered in the presence of glycerol. Finally, the effects of various reagents upon the NADP-linked and NAD-linked activities of glucose 6-phosphate dehydrogenase are seen to be compatible with their action on the equilibrium between X and Y. Interconversion of X and Y may be of importance in the regulation of mammary glucose 6-phosphate dehydrogenase activity.

Begrenzende Faktoren der Meth�moglobinbildung durch Phenylhydroxylamin in H�molysaten verschiedener Tierarten

1. Addition of ATP, G-6-P, G-6-PDH and NADP to haemolysates of erythroyctes from pigs, cows, and sheep increased the formation of methaemoglobin through phenylhydroxylamine by various degrees, according to the animal source used. The results revealed limiting factors for the formation of methaemoglobin in haemolysates. These factors are compared with the limiting factors found in intact erythrocytes. 2. Phosphorylation of glucose limits NADP reduction and, consequently, the methaemoglobin formation in haemolysates of erythrocytes from the three animals. The individual limiting factors are as follows: in sheep erythrocytes, the activity of hexokinase; in pig erythrocytes, the glucose utilisation; in cow erythrocytes, the ATP concentration. 3. When the hexokinase reaction is circumvented by the addition of G-6-P, the activity of NADPH-diaphorase limits the formation of methaemoglobin in the haemolysates of cow erythrocytes. However, the formation of methaemoglobin in haemolysates of pigs and cows can be increased by the addition of NADP before the activity of diaphorase becomes the limiting factor. 4. After adding a complete NADPH regenerating system, the velocities of methaemoglobin formation in haemolysates from the three animals were found to correlate with the corresponding disphorase activities.

A spectrofluorimetric study of the 7-hydroxylation of coumarin by liver microsomes.

1. The fluorescence characteristics of 3- and 7-hydroxycoumarin, and 7-hydroxy-and 7-methoxy-4-methylcoumarin, have been determined. 7-Hydroxycoumarin shows excited-state ionization from pH1 to 9. 2. A sensitive and specific fluorimetric method for the determination of 7-hydroxycoumarin (umbelliferone), and its application to liver homogenates and other tissue preparations, are described. 3. The enzymic hydroxylation of coumarin to 7-hydroxycoumarin has been studied by this method and the optimum conditions have been determined for rabbit-liver preparations. The enzymic activity was found in the microsomal fraction and required NADPH(2) and oxygen. Activity with NADH(2) was one-third of that with NADPH(2). 4. Addition of NADP was necessary for full activity of 10000g supernatant preparations of liver. Nicotinamide added during preparation preserved coenzymic activity in tissue stored at -12 degrees . Glucose 6-phosphate had no effect on the activity of stored or fresh tissue. 5. Inhibition occurred with p-chloromercuribenzoate, and with the usual inhibitors of the microsomal drug-metabolizing enzymes, SKF acid, SKF 525A, and Lilly 7132, but not with 2,2'-bipyridyl. 6. Liver homogenates from rabbit, guinea pig, coypu, cat and pigeon showed activity, but preparations of rat or mouse liver, and of locust fat bodies, did not hydroxylate coumarin to umbelliferone. The enzyme system was absent from rat-liver homogenates and microsomal preparations. Moreover, rat liver also contained inhibitors of the rabbit-liver coumarin-7-hydroxylase system and of the further metabolism of umbelliferone by guinea-pig liver. Guinea-pig-liver preparations hydroxylated coumarin to umbelliferone and then converted this product into its glucuronide. 7. The coumarin-7-hydroxylase activity of female rabbit liver was two to three times that of male rabbit liver.

ENZYME INHIBITION BY DERIVATIVES OF PHENOTHIAZINE VII. INHIBITION OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE ACTIVITY OF RABBIT ERYTHROCYTES.

The effects of various phenothiazine derivatives were tested, by a NADP+-coupled spectrophotometric assay, on the glucose-6-phosphate dehydrogenase activity of hemolysates of rabbit erythrocytes. Phenothiazine, phenothiazine sulfoxide, thionol, promazine, chlorpromazine, and methylene blue were relatively weak inhibitors. Powerful inhibitors (concentration, for 50% inhibition, I50, in parentheses) were phenothiazone (90 μM) and New Methylene Blue N (75 μM). These two compounds caused a slow direct oxidation of NADPH; however, they appeared to exert an action on the enzyme as well. The enzyme was not inhibited by 2,6-dichlorophenol–indophenol, which is employed in a dye-reduction assay.The enzyme system was readily inactivated by ethanol (I5o = 1.4 M). Prior addition of NADP+afforded some protection.

Inhibition of mitochondrial respiration and phosphorylation by 6-phosphogluconate

In a study of the effect of liver supernatant fractions on isolated mitochondria, it was observed that addition of NADP, but not NAD, caused an inhibition of respiration and phosphorylation. The most obvious explantation for this effect was that 6-phosphogluconate, which could have been formed in the system under study, was the inhibitor. The inhibitory activity of exogenous 6-phosphogluconate has been demonstrated and is reported here.

Conversion of Cholesterol to Progesterone by Corpus Luteum Slices

The conversion of cholesterol-7-3H to progesterone was studied in slices of bovine corpus luteum by measuring both the total amount of progesterone synthesized and the radioactivity in the final product derived from cholesterol-7-3H. The addition of luteinizing hormone to the incubating slices caused an increase in the amount of progesterone synthesized but only a slight increase in the conversion of radioactive cholesterol. The addition of NADP +glucose-6-phosphate, on the other hand, increased both the amount of progesterone and, to a greater extent, the conversion of added cholesterol-7-3H to progesterone. Neither added LH nor added NADP -fglucose-6-P influenced the penetration of the radioactive cholesterol into the slices. It was concluded that NADPH increases progesterone synthesis primarily through its action on the transformation of cholesterol, whereas LH affects the transformation of cholesterol to a slight extent only and acts mainly on precursors prior to cholesterol in the biosynthetic pathwa...