Hyperanodic forms of human glucose-6-phosphate dehydrogenase.

Pure glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) is transformed into 'hyperanodic forms' when incubated at acidic pH and in the presence of NADP+ with excess of glucose-6-phosphate or with some 'NADP+ modifying proteins' purified from the same cells. The enzyme hyperanodic forms exhibit low isoelectric point, altered kinetic properties and high lability to heat, urea, and proteolysis. Differences between hyperanodic and native forms of glucose-6-phosphate dehydrogenase are also noted by microcomplement fixation analysis, ultraviolet absorbance difference spectrum and fluorescence emission spectrum. Drastic denaturation of the enzyme by urea and acid treatment did not suppress the difference of isoelectric point between native and hyperanodic forms of glucose-6-phosphate dehydrogenase. From our data we suggest that the conversion into hyperanodic forms could be due to the covalent binding on the enzyme of a degradation product of the pyridine nucleotide coenzyme. This modification could constitute a physiological transient step toward the definitive degradation of the enzyme.     

Characterization of glucose-6-phosphate dehydrogenase in Thailand

Summary Characterization of partially purified eryrhrocyte G-6-PD from 50 enzymedeficient males in 45 unrelated Thai families revealed 6 enzyme variants. Thirty-five subjects in 31 families had G-6-PD variant with normal electrophoretic mobility, slightly low Km G-6-P, normal substrate-analog utilization, normal pH-optimum curve, and slightly increased heat stability. This enzyme variant is called G-6-PD Mahidol.Six subjects had enzyme with fast electrophoretic mobility (106–108% of normal), low Km G-6-P, slightly increased substrate-analog utilization, biphasic pH-optimum curve, and slightly low to normal heat stability. This variant was identical to G-6-PD Canton.Five subjects had G-6-PD with fast electrophoretic mobility (103–106% of normal), low Km G-6-P, very high substrate-analog utilization except for DPN which it did not use as cofactor, markedly biphasic pH-optimum curve and very low heat stability. This variant is called G-6-PD Union (Thai).Two brothers had G-6-PD with normal electrophoretic mobility, low Km G-6-P, slightly increased substrate-analog utilization, biphasic pH-optimum curve and low heat stability. This variant is designated G-6-PD Siriraj.G-6-PD from one patient had slightly fast electrophoretic mobility, increased substrateanalog utilization, especially of DPN, and very low thermal stability. It is called G-6-PD Kan.One subject had G-6-PD with normal electrophoretic mobility, Km G-6-P, pH-optimum curve and heat stability, and increased substrate-analog utilization. This G-6-PD variant is named G-6-PD Anant.G-6-PD Mahidol is far more common than any other known variants in Thailand.

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Zusammenfassung Eine Charakterisierung von teilweise gereinigtem Erythrocyten-G-6-PD von 50 Männern mit Enzym-Defekt aus 45 nicht miteinander verwandten Thai-Familien ergab 6 Enzym-Varianten. 35 Personen in 31 Familien hatten eine G-6-PD-Variante mit normaler elektrophoretischer Wanderungsgeschwindigkeit, einen leicht verminderten G-6-P-Km-Wert, einer normalen Substratanalog-Verwertung, einer normalen pH-Optimum-Kurve und einer leicht erhöhten Hitze-Stabilität. Diese Enzym-Variante wurde G-6-PD Mahidol genannt.Sechs Personen hatten ein Enzym mit rascher elektrophoretischer Wanderung (106–108% der Norm), niedrigem Km für G-6-P, leicht erhöhter Substrat-Verwertung, einer biphasischen pH-Optimum-Kurve und normaler bis leicht erniedrigter Hitzestabilität. Diese Variante ist identisch mit G-6-PD Canton.Fünt Personen hatten G-6-PD mit rascher elektrophoretischer Wanderung (103–106%), niedrigem Km G-6-P, sehr hoher Substratanalog-Verwertung—mit Ausnahme von DPN, das nicht als Cofactor wirkte—, einer stark biphasischen pH-Optimum-Kurve und sehr geringer Hitze-Stabilität. Diese Variante wurde als G-6-PD Union (Thai) bezeichnet.Zwei Brüder hatten ein G-6-PD mit normaler elektrophoretischer Wanderung, niedrigem Km G-6-P, leicht erhöhter Substratanalog-Verwertung, einer biphasischen pH-Optimum-Kurve und geringer Hitze-Stabilität. Diese Variante erhielt den Namen G-6-PD Siriraj.G-6-PD eines Patienten hatte eine leicht erhöhte elektrophoretische Wanderungsgeschwindigkeit, eine erhöhte Substratanalog-Verwertung, besonders für DPN, und eine sehr geringe Hitze-Stabilität (G-6-PD Kan).Eine Person zeigte ein G-6-PD mit normaler elektrophoretischer Wanderungsgeschwindigkeit, Km G-6-P pH-Optimum-Kurve und Hitze-Stabilität. Nur die Substratanalog-Verwertung war erhöht. Diese Variante wurde G-6-PD Anant gennant.G-6-PD Mahidol ist die bei weitem häufigste Variante in Thailand.

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This investigation received financial support from the World Health Organization.     

Characterization of glucose-6-phosphate dehydrogenase and 6-phosphogluconic acid dehydrogenase from hazel cotyledons

NADP reduction was shown to occur in a crude cytosolic extract from the cotyledonary material of hazel seed prior to the addition of erogenous dehydrogenase substrate. This activity interfered with the assay of glucose-6-phosphate dehydrogenase and 6-phosphogluconic acid dehydrogenase activities. The inherent NADP reduction was removed by ammonium sulphate fractionation. Subsequent de-salting of the resulting partially-purified fraction permitted assay of G6PDH and 6PGDH. Both enzymes were shown to be NADP specific. Typical Michaelis-Menten kinetics were shown for each enzyme, towards NADP and their respective substrate.     

Characterization of an inhibitor of glucose-6-phosphate dehydrogenase

A strong inhibitor of G6PDH has been detected in rat liver homogenates. The inhibitor, isolated by ultrafiltration methods, proved to be very stable under incubation with trypsin and high temperatures. Gel-filtration through Sephadex G-75 showed it to have a molecular weight of 3,500 daltons, though perchloric acid treatment produced a light form of 900 daltons. Both forms of inhibitor act as competitive inhibitor with respect to G6P and exhibit non-competitive inhibition pattern with respect to NADP+. Physical and kinetic properties, and the increase of G6PDH activity at low NADP+ concentrations, in the presence of NADPH and inhibitor or palmitoyl-CoA, in relation to the G6PDH activity in presence of NADPH, lead to the identification of the low-molecular weight inhibitor as palmitoyl-CoA.     

Isoenzymes of glucose-6-phosphate dehydrogenase from tobacco cells

Two anodic isoenzymes of glucose-6-phosphate dehydrogenase (G6PDH) were isolated from tobacco suspension culture WR-132, utilizing fractional ammonium sulfate precipitation and DEAE-cellulose chromatography. The pH optimum was 9.0 for isoenzyme G6PDH I and 8.0–8.3 for G6PDH IV. Isoenzyme G6PDH I exhibited Michaelis-Menten kinetics for both substrates, G6P and NADP⁺, with K_m's of 0.22 mM and 0.06 mM, respectively. G6PDH IV exhibited Michaelis-Menten kinetics for G6P with a K_m of 0.31 mM. The NADP⁺ double reciprocal plot showed an abrupt transition between two linear sections. This transition corresponds to an abrupt increase in the apparent K_m and V_max values with increasing NADP⁺, denoting negative cooperativity. The two K_m's for high and low NADP⁺ concentrations were 0.06 mM and 0.015 mM, respectively. MWs of the isoenzymes as determined by SDS disc gel electrophoresis were 85 000–91 000 for G6PDH I and 54 000–59 000 for G6PDH IV. Gel filtration chromatography on Sephadex G-150 showed MW's of 91 000 for G6PDH I and 115 000 for G6PDH IV. A probable dimeric structure for IV is suggested, with two NADP⁺ binding sites.     

Specific activities of 6-phosphogluconate dehydrogenase,glucose-6-phosphate dehydrogenase and glucose-6-phosphate isomerase during Bufo bufo development

It has been suggested by some authors that during amphibian development, due to the higher glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activity compared to that of 6-phosphogluconate dehydrogenase (EC 1.1.1.43), 6-phosphogluconate could accumulate in the embryo tissues and regulate the channelling of glucose-6-phosphate into glycolysis. Here, on the base of the specific activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glucose-6-phosphate isomerase (EC 5.3.1.9) found in the embryos of Bufo bufo during development, it is discussed whether 6-phosphogluconate can accumulate and play a regulative role on glucose-6-phosphate metabolism in the anuran embryo.     

Rapid purification of glucose-6-phosphate dehydrogenase from mammal's erythrocytes

A procedure for rapid purification to homogeneity of glucose-6-phosphate dehydrogenase (G6PD) is herein presented. Our method is not new, but represents a simplification of the method of De Flora et al. (Arch. Biochem. Biophys. 169, 362-3, 1975) which consisted of three steps: DEAE-Sephadex, phosphocellulose (P11) and affinity chromatography on 2'5' ADP-Sepharose. These authors eluted the enzyme from the P11 with phosphate and from 2'5' ADP-Sepharose with KC1 and NADP. By our method, the DEAE-Sephadex step is omitted, the G6PD is eluted from P11 with citrate and NADP, and from 2'5' ADP-Sepharose with KC1, NADP and EDTA. The elution of the enzyme from the phosphocellulose was studied in detail and the temperature effect has been described. We report here an application of this method to a rapid microscale purification starting from 3.5-4 ml of rabbit blood, which can be performed in about 8 hours and a macroscale purification starting from 180-200 ml of human blood, which takes a day and a half.