Joseph Mendicino

Reversible inactivation of d-fructose 1,6-diphosphatase by adenosine triphosphate and cyclic 3′, 5′-adenosine monophosphate☆

Abstract An analytical method for the quantitative recovery and purification of fructose 1,6-diphosphatase from incubation mixtures containing crude kidney extracts has been described. Utilizing this procedure it has been demonstrated that renal fructose 1,6-diphosphatase was inactivated on incubation with ATP and cyclic-3′,5′-AMP. Fructose 1,6-diphosphatase present in crude kidney extracts was inactivated by ATP and Mg ++ alone, and the rate of this inactivation was increased in the presence of cyclic 3′,5′-AMP. Purified enzyme was inactivated by ATP only in the presence of crude kidney extracts. Fructose 1,6-diphosphatase was also inactivated when kidney slices were incubated in the presence of epinephrine. When fructose 1,6-diphosphatase was incubated in the presence of an AT 32 P generating system in crude extracts, 32 P was incorporated into the enzyme protein with a concomitant decrease in enzyme activity. Very little or no 32 P i was incorporated in the absence of adenosine nucleotide. The inactivated enzyme was reactivated on incubation with crude kidney extracts. The possible relationship of these effects to those obtained with three other enzymes involved in the synthesis and degradation of glycogen and the mechanism of action of epinephrine is discussed.

URIDINE DIPHOSPHATE-D-GLUCOSE-GLYCOGEN GLUCOSYLTRANSFERASE FROM MAMMARY GLAND.

Abstract Uridine diphosphate- d -glucose-glycogen glucosyltransferase (UDPglucose: α-1,4-glucan α-4-glucosyltransferase, EC 2.4.1.11), the enzyme which catalyzes the synthesis of α-1,4-glucosyl residues of glycogen from UDP- d -glucose, was purified approx. 20-fold from lactating mammary gland extracts by ammonium sulfate fractionation, protamine precipitation and chromatography on TEAE-cellulose. The purified enzyme required d -glucose -6-P for maximal activity and the extent of activation, 2 to 3-fold, was found to be dependent on the concentration of UDP- d -glucose and glycogen. The pH optimum of the reaction, in the presence or absence of d -glucose -6-P , was approx. 6.5. The apparent Michaelis constant for UDP- d -glucose was 5·10 −4 M in the presence of d -glucose -6-P and 2·10 −3 M in the absence of this phosphate ester. The glucosyltransferase activity was found to be located almost exclusively in the soluble subcellular fraction in this tissue.

[68] Phosphoprotein phosphatase from swine kidney

Publisher Summary This chapter describes an assay method and the purification procedure for the enzyme phosphoprotein phosphatase from swine kidney. The enzyme hydrolyzes phosphoryl groups attached to specific serine residue in polypeptide substrates and shows little or no activity with other low-molecular-weight monophosphate esters. The enzyme activity can be measured by the rate of 32 P i release from 32 P-labeled enzymes or by changes in the activity of the phosphorylated forms of phosphorylase or glycogen synthase. One enzymic assay is based on the ability of phosphoprotein phosphatase to catalyze the conversion of glycogen phosphorylase to a dephosphorylated form that is not dependent on the presence of adenosine monophosphate (AMP) for activity. The other assay is based on the ability of the phosphatase to convert glycogen synthase to a dephosphorylated form dependent on the presence of glucose 6-phosphate for activity. The purification procedure involves homogenization, acid precipitation, precipitation with ammonium sulfate, chromatography on diethylaminoethyl (DEAE)-Sephadex A-50, gel filtration on Sephacryl S-200, chromatography on Blue Dextran–Sepharose 4B and hexanediamine-Sepahrose 4B, chromatography on polylysine-Sepharose 4B, and gel filtration on Sephadex G-100. The molecular weight of the enzyme estimated by gel filtration on Sephadex G-100 is 70,000. The pH optimum of the purified enzyme is 7.0; 10 mM MgCl 2 is required for maximum activity.

[31] cAMP-dependent protein kinase, soluble and particulate, from swine kidney

Publisher Summary This chapter describes an assay method, purification, and properties of cAMP-dependent protein kinase—soluble and particulate—from swine kidney. The homogeneous preparations of protein kinase isolated from swine kidney catalyze the phosphorylation of purified homologous glycogen synthase, phosphorylase kinase, phosphofructokinase, pyruvate kinase, and fructose-l,6-bisphosphatase. The regulation of these enzymes through the action of the cyclic AMP-dependent forms of protein kinase may mediate the intracellular effects of hormones on the adenylate cyclase system in kidney. The enzymic assay is based on the ability of protein kinase to catalyze conversion of glycogen synthase to a form that is dependent on the presence of glucose 6-phosphate for activity. The rate of decrease in the activity of glycogen synthase is measured in the absence of glucose 6-phosphate. The purification process of the cytosolic catalytic subunit of protein kinase involves homogenization, chromatography on cellulose phosphate, pH 5.5 treatment, chromatography on diethylaminoethyl (DEAE)-cellulose, chromatography on Blue Dextran–Sepharose 4B, chromatography on cellulose phosphate, and gel filtration on Sephadex G-100.

[19] Isomers of α-d-apiofuranosyl 1-phosphate and α-d-apiose 1,2-cyclic phosphate

Publisher Summary The study of the biosynthetic reactions involving the intermediates of D-apiose is impeded by the lack of chemically pure phosphate esters of the branched-chain sugar. Four monophosphate esters are formed when β -D-apiose tetraacetate is treated with anhydrous phosphoric acid. Two isomers of D-apiose, α-D-apio-L-furanosyl 1-phosphate and α-D-apio-D-furanosyl 1-phosphate, are obtained in the highest yield. Several cyclic phosphate esters of D-apiose are also formed when β-D-apiose tetraacetate is treated with crystalline phosphoric acid. The α-D-1,2-cyclic D- and L-furanosylapiose phosphodiesters are formed from the corresponding α-D-apiose 1-phosphate esters when they are treated with alkali to remove the O -acetyl groups. The diisopropylidene derivative is prepared from D-apiose. One of the cyclic phosphodiester derivatives has properties identical with those of a compound formed during the enzymic conversion of UDP-D-glucuronic acid to D-apiose derivatives in the extracts of parsley and Litaneutria minor . The analytical and chromatographic data shows that the isolated products are isomeric monophosphate esters of D-apiose.

[17] Aldose 1,6-diphosphates

Publisher Summary This chapter describes the method for the preparation of aldose 1,6-diphosphates. A microanalytical procedure is developed to measure the effect of time of incubation and temperature on the rate and extent of the phosphorylation reaction. In this assay, the sugar diphosphate product is partially purified and the rate of formation of acid-labile phosphate is measured. The fully acetylated derivative of D-glucose-6-P is prepared by a modification of the procedure used for the synthesis of completely acetylated derivatives of free hexoses with acetic anhydride in pyridine at 3%. Characterization of a-D-glucose 1,6-diphosphate is discussed. The procedure described in the chapter, with slight modifications has been used to prepare α -D-ribose 1,5-diphosphate, α -D-galactose 1,6-diphosphate, α -D-mannose 1,6-diphosphate, and N-acetyl- α -D-glucosamine 1,6-diphosphate. All the sugar diphosphates prepared by this procedure were active when they were assayed for enzymic activity with rabbit muscle phosphoglucomutase. Some precautions must be taken when preparing sugar diphosphates other than α -D-glucose-l,6-P 2 . Several sugar monophosphate samples contain varing amounts of D-glucose-6-P. To remove this impurity, 5-g samples of D-galactose-6-P and D-mannose-6-P were treated with D-glucose- 6-P dehydrogenase and NADP + to convert traces of D-glucose 6-P to 6-P-gluconic acid. One other precaution that must be taken in the preparation of α -D-mannose-l,6-P 2 and α -D-ribose-l,5-P 2 is to reduce the concentration of HC1 from 10 to 5 m M, during the removal of the residual sugar monophosphate. These sugar diphosphates are considerably more labile to acid hydrolysis than α -D-glucose-l,6-P 2 .