Age-related changes of glucose-6-phosphate dehydrogenase activity in mouse oocytes

Summary Glucose-6-phosphate dehydrogenase (G6PDH) activity was measured in follicular oocytes and in ovulated eggs of prepubertal, adult and aged mice. G6PDH activity in ovulated eggs was 60% of the activity in follicular oocytes in all age groups. The mean G6PDH activity was significantly higher in follicular oocytes of adult mice than in oocytes of both prepubertal and aged mice. In aged mice, the decreased mean activity in follicular oocytes as well as in ovulated eggs was mainly due to a high percentage of cells with extremely low activity (25 and 18%, respectively). The percentage of preovulatory oocytes with low activity in prepubertal mice was 9% and in adult mice 0.3%. For ovulated eggs these percentages were 0% for both prepubertal and adult mice. In every age group, all ovulated eggs showed a normal morphology. When ovulated eggs with extremely low G6PDH activity can still be fertilized, it can be questioned whether this loss of activity could cause disturbances in development of (preimplantation) embryos. Our findings emphasize the potentialities of investigating intact single oocytes for changes in enzyme activities, which could be applied as parameters for quality control of these cells.     

In vivo uptake of mitochondrial malate dehydrogenase from watermelon by mitochondria of Xenopus laevis oocytes

The heterologous in vivo translation system of Xenopus laevis oocytes was used to translate messenger RNA isolated from water-melon cotyledons. Immunocytochemistry was used to localize the translation products in situ within the oocyte. In addition, the translation products were immunoprecipitated from homogenized oocytes, separated on SDS-polyacrylamide electrophoresis and visualized by fluorography. A variety of watermelon proteins encoded in the injected mRNA were translated within the oocytes. Among them was the mitochondrial isoenzyme of malate dehydrogenase (mtMDH). The mtMDH was correctly imported into the mitochondria of the oocytes, as detected by immunocytochemistry.     

A simple screening procedure for adenylate kinase, hexokinase and glucose-6-phosphate dehydrogenase deficiencies

A simple screening procedure for the detection of adenilate kinase (AK), hexokinase (Hx) or glucose-6-phosphate dehydrogenase (G6PD) deficiencies in blood, is described. It consists of two assays : in the first, the ATP formed by blood AK is coupled to Hx and G6PD, and in the second, the glucose-6-phosphate formed by blood Hx is coupled to G6PD. The enzyme activities are visually estimated by the reduction of NADP+ (non-fluorescent) to NADH (fluorescent). The appearance of fluorescence in the first assay indicates that the three enzyme activities are present. The absence of fluorescence could be due to the deficiency of any one of the three enzymes; in this case the second assay used in combination with the Beutler's screening test for G6PD permits the detection of the specific enzymatic deficiency.     

Changes in hexokinase and glucose-6-phosphate dehydrogenase in red cells during hypo and hyperthyroidism

Activities of hexokinase and glucose-6-phosphate dehydrogenase have been measured in red blood cells from thyroidectomized, triiodothyronine treated and hyperthyroid rats. After thyroidectomy, significant decrease in the activities of hexokinase and glucose-6-phosphate dehydrogenase was observed as compared to controls. The effects were reversed with triiodothyronine administration to the thyroidectomized rats. Hyperthyroidism increased both enzymes. The observations further confirm the hypermetabolic effects of thyroid hormones on the cellular metabolism.     

Adenosine triphosphate-linked control of Pseudomonas aeruginosa glucose-6-phosphate dehydrogenase

Extracts of Pseudomonas aeruginosa (ATCC 7700) cells grown on glucose, gluconate, or glycerol had enzyme activities related to the Entner-Doudoroff pathway. These activities were present in no more than trace amounts when the bacteria were grown on succinate. Fructose-1,6-diphosphate aldolase could not be detected in extracts of the bacteria grown on any of the above carbon sources. Therefore, it appears that P. aeruginosa degrades glucose via an inducible Entner-Doudoroff pathway. The apparent absence of fructose-1,6-diphosphate aldolase in cells growing on succinate suggests that the bacteria can form hexose and pentose phosphates from succinate by an alternate route. d-Glucose-6-phosphate dehydrogenase, a branch-point enzyme of the Entner-Doudoroff pathway, was purified 50-fold from glucose-grown cells. Its molecular weight, estimated by sucrose density gradient centrifugation, was found to be approximately 190,000. The enzyme was strongly inhibited by adenosine triphosphate, guanosine triphosphate, and deoxyguanosine triphosphate, which decreased the apparent binding of glucose-6-phosphate to the enzyme. It is suggested that adenine nucleotide-linked control of glucose-6-phosphate dehydrogenase may regulate the overall catabolism of hexose phosphates and prevent their wasteful degradation under certain conditions requiring gluconeogenesis.     

Frequency of glucose-6-phosphate dehydrogenase, pyruvate kinase and hexokinase deficiency in the Saudi population

The frequencies of glucose-6-phosphate dehydrogenase (G-6-PD), pyruvate kinase (PK) and hexokinase (HK) deficiency were determined in different regions of Saudi Arabia. G-6-PD deficiency was found to range from 0.045 to 0.220 for the male and 0.020 to 0.125 for the female population. The highest frequencies were found to exist in the regions which are endemic to malarial parasite and have high frequencies of sickle cell and thalassaemia genes. Partial deficiencies of PK and HK were encountered in each region, however, no case of complete deficiency of these enzymes was identified. Further investigations are in progress to determine the clinical manifestations of enzyme deficiencies in the Saudi population.     

Kinetics and control of bovine adrenal glucose-6-phosphate dehydrogenase.

Michaelis-Menten kinetics are observed in studies of highly purified bovine adrenal glucose-6-phosphate dehydrogenase at pH8.0 in 0.1 M bicine. The Km for NADP+ is 3.8 muM and for glucose-6-phosphate, 61 muM. At pH 6.9 Km for NADP+ increases to 6.5 muM. The enzyme is inhibited by NADPH both at pH 6.8 and at 8.0 with a Kip of 2.36 muM at pH 8.0. Inhibition is competitive with respect to both substrates implying that addition of substrates is random ordered. The data are also interpreted in terms of "reducing charge", the mole fraction of coenzyme in the reduced form. This appears to be the major mechanism for regulation of the pentose shunt. D-glucose, oxidized by the enzyme at a very slow rate, is also a competitive inhibitor for the natural substrate with a Ki of 0.29 M. Phosphate is a competitive inhibitor for glucose-6-phosphate oxidation but both phosphate and sulfate accelerate glucose oxidation suggesting a common binding site for the two anions and the phosphate of the natural substrate. While binding of ACTH to our enzyme preparations has been observed, we have not been able, in spite of repeated attempts, to demonstrate augmentation of the activity of the enzyme by the addition of ACTH.     

In vivo synthesis and turnover of cytoplasmic ribosomal RNA by stage 6 oocytes of Xenopus laevis.

Cytoplasmic ribosomal RNA (rRNA) synthesis was detected in white-banded stage 6 oocytes taken from female Xenopus laevis which were injected with [³H]guanosine 7 days previously. The specific radioactivity of the rRNA in oocytes collected from injected females by weekly laparotomies displays first-order exponential decay. Calculated values for the half-life of rRNA ranged from 9.1–30.9 days in experiments on four animals. The concept of ribosomes in large ovarian oocytes of amphibians as an absolutely stable, long-term storage product appears incorrect.     

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.     

Effect of diabetes and insulin-induced hypoglycemia on hexokinase and glucose-6-phosphate dehydrogenase in red blood cells

Activities of hexokinase and glucose-6-phosphate dehydrogenase have been measured in red blood cells from control, diabetic and insulin treated rats. After an initial decrease, the enzyme activities increased, but remained lower than control levels. A reversal of the diabetes effect was seen with insulin administration. Insulin induced hypoglycemia increased both enzymes. An overall control of glucose metabolism by insulin in red blood cells was observed.     

Fructose-6-phosphate is not a substrate for glucose-6-phosphate dehydrogenase

D-Fructose-6-phosphate was shown not to be a substrate for glucose-6-phosphate dehydrogenases (EC. 1.1.1.49) from human erythrocytes, bovine adrenal, rat liver, three yeasts (brewer's yeast, baker's yeast, and Candida utilis), and Leuconostoc mesenteroides. These findings contrast with those of G.M. Kidder (J. Exp. Zool., 226:385-390, '83).     

Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase

Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase has found many applications in biomedical research. However, up to several years ago, the methods used often appeared to be unreliable because many artefacts occurred during processing and staining of tissue sections or cells. The development of histochemical methods preventing loss or redistribution of the enzyme by using either polyvinyl alcohol as a stabilizer or a semipermeable membrane interposed between tissue section and incubation medium, has lead to progress in the topochemical localization of glucose-6-phosphate dehydrogenase. Optimization of incubation conditions has further increased the precision of histochemical methods. Precise cytochemical methods have been developed either by the use of a polyacrylamide carrier in which individual cells have been incorporated before staining or by including polyvinyl alcohol in the incubation medium. In the present text, these methods for the histochemical and cytochemical localization of glucose-6-phosphate dehydrogenase for light microscopical and electron microscopical purposes are extensively discussed along with immunocytochemical techniques. Moreover, the validity of the staining methods is considered both for the localization of glucose-6-phosphate dehydrogenase activity in cells and tissues and for cytophotometric analysis. Finally, many applications of the methods are reviewed in the fields of functional heterogeneity of tissues, early diagnosis of carcinoma, effects of xenobiotics on cellular metabolism, diagnosis of inherited glucose-6-phosphate dehydrogenase deficiency, analysis of steroid-production in reproductive organs, and quality control of oocytes of mammals. It is concluded that the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase is of highly significant value in the study of diseased tissues. In many cases, the first pathological change is an increase in glucose-6-phosphate dehydrogenase activity and detection of these early changes in a few cells by histochemical means only, enables prediction of other subsequent abnormal metabolic events. Analysis of glucose-6-phosphate dehydrogenase deficiency in erythrocytes has been improved as well by the development of cytochemical tools. Heterozygous deficiency can now be detected in a reliable way. Cell biological studies of development or maturation of various tissues or cells have profited from the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.