细胞质雄性不育水稻幼穗和花药的呼吸酶活性研究

研究珍汕97A和珍汕97B的雌雄蕊原基形成期、花粉母细胞形成期和花粉母细胞减数分裂期的幼穗及单核期、二核期和三核期的花药中呼吸代谢三羧酸循环（TCA）的苹果酸脱氢酶（MDH）和异柠檬酸脱氢酶（IDH）及戊糖途径（PPP）的磷酸葡萄糖脱氢酶（G6PDH）、磷酸葡萄糖酸脱氢酶（6PGDH）和5一磷酸核糖异构酶（RSPI）的活性。结果表明：可育花药的5种酶活性皆高于同期不育花药;而幼穗中,TCA途径中的MDH和IDH在不育系与保持系之间无差异,PPP途径的G6PDH和6PGDH及R5PI则保持系高于不育系。这说明不育系中PPP发生的变化早于TCA途径,PPP途径的改变可能与小孢子败育有着更为直接的关系。     

Pentose phosphate metabolism during dormancy breakage in Corylus avellana L.

Commercially obtained fruits of Corylus avellana exhibit the characteristic loss of dormancy of this seed following chilling under moist conditions. The activities of cytosolic and organellar enzymes of pentose phosphate pathway in cotyledonary tissue were assayed throughout stratification and over a similar period in damp vermiculite at 20° C. Glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconic acid dehydrogenase (6PGDH) were both found in cytosolic extracts in all treatments; only 6PGDH was present in the organellar fraction.The enzyme activities monitored in seeds at 20° C remained relatively constant over the course of the investigation except in the case of cytosolic 6PGDH where it is suggested an inhibitor of the enzyme accumulated. This inhibitor was removed by the partial purification procedure. Increases in the activities of the enzymes occurred during stratification, the major increase coinciding exactly with dormancy breakage but prior to the initiation of germination. The marked increase in G6PDH and 6PGDH concurrent with the change in germination potential of the chilled seed may have considerable biochemical significance in breaking down the dormant state.Abbreviations G6P glucose-6-phosphate - G6PDH glucose-6 phosphate dehydrogenase - NADP nicotinamide adenine dinucleotide phosphate - 6 PGDH 6-phosphogluconic acid dehydrogenase - PPP pentose phosphate pathway     

ACTIVATION OF HEXOSE MONOPHOSPHATE PATHWAY IN BRAIN BY ELECTRICAL STIMULATION IN VITRO

Abstract— The incubation of cerebral cortical slices for 15 min in Krebs-Ringer-tris (pH 7.6) solution at 37°C with [1-¹⁴C]glucose or [6-¹⁴C]glucose as substrates yielded a C-1:C-6 ¹⁴CO₂ ratio of 1.21, whereas this ratio increased to 3.01 after the application of electrical stimulation (ES). Tissue levels of 6-phosphoglu-conate (6PG) and glucose 6-phosphate (G6P), intermediary metabolites of hexose monophosphate (HMP) pathway, were 7 and 180 nmol/g tissue following 15 min incubation, and increased by 33 and 45 per cent respectively following the application of ES. Activities of 6-phosphogluconate dehydrogenase (6PGDH, 6-phospho- d -gluconate: NADP⁺ 2-oxidoreductase, EC 1.1.1.44) and glucose-6-phosphate dehydrogenase (G6PDH, d -glucose-6-phosphate: NADP⁺ 1-oxidoreductase, EC 1.1.1.49), important enzymes in regulating the activity of HMP pathway, in cerebral cortical slices were 689 and 907 pmol/mg protein/min and were increased by 66 and 25 per cent respectively by the application of ES. Synaptosomal G6PDH and 6PGDH activities were maximally activated by the addition of 40 m m -Na⁺ to the reaction mixture, whereas no activation by Na⁺ was observed in microsomal G6PDH and 6PGDH. Amobarbital inhibited more strongly the Embden–Meyerhof (EM) pathway than the HMP pathway, while imipramine had a stronger inhibitory effect on HMP pathway than on EM pathway in the electrically stimulated cerebral tissues.
The present results indicate that the HMP shunt pathway in the cerebral cortex is activated by the application of ES in vitro , possibly at synaptic regions and may play an important metabolic and functional role in the brain.     

Changes in activity of glucose-6-phosphate and 6-phosphogluconate dehydrogenase isozymes upon potato virus Y infection in tobacco leaf tissues and protoplasts

The changes in the activity of glucose-6-phosphate dehydrogenase (G6PDH) (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH) (EC 1.1.1.44) in leaf tissues and the subcellular localisation of their isozymes in protoplasts derived from healthy and potato virus Y (PVY) infected plants of Nicotiana tabacum L. cv. Samsun were determined. The activities of G6PDH and 6PGDH were markedly increased in virus-infected leaves during the acute phase of infection both in crude homogenate and partial purificate (when compared with the values found in healthy control plants) and correlated with the multiplication curve of PVY. Intact chloroplasts and soluble cytosolic proteins were obtained from whole plants upon the culmination of the multiplication curve of PVY and upon the enhancement of the activity of both dehydrogenases by means of differential centrifugation of broken protoplasts. The chloroplastic fraction from infected protoplasts (based on chlorophyll content or NADP⁺-triosephosphate dehydrogenase activity) showed an enhanced activity of G6PDH (1.81 times that of healthy protoplasts), and 6PGDH (1.77 times). Cytosol from infected protoplasts (based on phosphoenolpyruvate carboxylase activity) contained only slightly enhanced activities of G6PDH and 6PGDH (only 1.26 and 1.16 times, respectively).     

高等植物葡萄糖-6- 磷酸脱氢酶与6- 磷酸葡萄糖酸脱氢酶基因的不同进化起源

葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶是植物戊糖磷酸途径中的两个关键酶。在克隆了水稻质体葡萄糖-6-磷酸脱氢酶基因OsG6PDH2和质体6-磷酸葡萄糖脱氢酶基因Os6PGDH2基础上,分析比较了水稻胞质和质体葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因的基因结构、表达特性和进化地位。结合双子叶模式植物拟南芥两种酶基因的分析结果,认为高等植物葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因在进化方式上截然不同,葡萄糖-6-磷酸脱氢酶的胞质基因与动物和真菌等真核生物具有共同的祖先;6-磷酸葡萄糖酸脱氢酶的胞质酶和质体酶基因都起源于原核生物的内共生。讨论了植物葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶基因可能的进化模式,为高等植物及质体的进化起源提供了新的资料。     

In situ kinetic parameters of glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase in different areas of the rat liver acinus

The reaction velocity of glucose-6-phosphate dehydrogenase (G6PDH) and phosphogluconate dehydrogenase (PGDH) was quantified with a cytophotometer by continuous monitoring of the reaction product as it was formed in liver cryostat sections from normal, young mature female rats at 37 degrees C. Control incubations were performed in media lacking both substrate and coenzyme for G6PDH activity and lacking substrate for PGDH activity. All reaction rates were non-linear but test minus control reactions showed linearity with incubation time up to 5 min using Nitro BT as final electron acceptor. End point measurements after incubation for 5 min at 37 degrees C revealed that the highest specific activity of G6PDH was present in the intermediate area (Vmax = 7.79 +/- 1.76 mumol H2 cm-3 min-1) and of PGDH in the pericentral and intermediate areas (Vmax = 17.19 +/- 1.73 mumol H2 cm-3 min-1). In periportal and pericentral areas, Vmax values for G6PDH activity were 4.48 +/- 1.03 mumol H2 cm-3 min-1) and 3.47 +/- 0.78 mumol H2 cm-3 min-1), respectively. PGDH activity in periportal areas showed a Vmax of 10.84 +/- 0.33 mumol H2 cm3 min-1. Variation of the substrate concentration for G6PDH activity yielded similar KM values of 0.17 +/- 0.07 mM, 0.15 +/- 0.13 mM and 0.22 +/- 0.11 mM in periportal, pericentral and intermediate areas, respectively. KM values of 0.87 +/- 0.12 mM in periportal and of 1.36 +/- 0.10 mM in pericentral and intermediate areas were found for PGDH activity. The significant difference between KM values for PGDH in areas within the acinus support the hypothesis that PGDH is present in the cytoplasmic matrix and in the microsomes. A discrepancy existed between KM and Vmax values determined in cytochemical assays using cryostat sections and values calculated from biochemical assays using diluted homogenates. In cytochemical assays, the natural microenvironment for enzymes is kept for the demonstration of their activity and thus may give more accurate information on enzyme reactions as they take place in vivo.     

高等植物葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶基因的不同进化起源

葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶是植物戊糖磷酸途径中的两个酶.在克隆了水稻质体葡萄糖-6-磷酸脱氢酶基因OsG6PDH2和质体6-磷酸葡萄糖脱氢酶基因Os6PGDH2基础上,分析比较了水稻胞质和质体葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因的基因结构、表达特性和进化地位.结合双子叶模式植物拟南芥两种酶基因的分析结果,认为高等植物葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因在进化方式上截然不同,葡萄糖-6-磷酸脱氢酶的胞质基因与动物和真菌等真核生物具有共同的祖先;6-磷酸葡萄糖酸脱氢酶的胞质酶和质体酶基因都起源于原核生物的内共生.讨论了植物葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶基因可能的进化模式,为高等植物及质体的进化起源提供了新的资料.     

Coenzyme specificity of enzymes in the oxidative pentose phosphate pathway of Gluconobacter oxydans

The coenzyme specificity of enzymes in the oxidative pentose phosphate pathway of Gluconobacter oxydans was investigated. By investigation of the activities of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) in the soluble fraction of G. oxydans, and cloning and expression of genes in Escherichia coli, it was found that both G6PDH and 6PGDH have NAD/NADP dual coenzyme specificities. It was suggested that the pentose phosphate pathway is responsible for NADH regeneration in G. oxydans.     

Aluminum resistance in wheat (Triticum aestivum) is associated with rapid, Al-induced changes in activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in root apices

We have investigated the effect of aluminum (Al) on the activity of glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) isolated from 5-mm root apices of 4-day-old wheat ( Triticum aestivum ) cultivars differing in resistance to Al. Rapid increases in G6PDH and 6PGDH activities were observed in Al-resistant cultivars (PT741 and Atlas 66) during the first 10 h of treatment with 100 μ M Al, while no change in the activity of either enzyme was observed in Al-sensitive cultivars (Katepwa and Neepawa) during a 24-h exposure to Al. The Al-induced increases in enzyme activities observed in the Al-resistant PT741 appear to reflect an induction of protein synthesis since the increases were completely abolished by 1 m M cycloheximide. No differences in G6PDH and 6PGDH activities were observed between the Al-sensitive and the Al-resistant genotypes when Al was supplied in vitro. Under these conditions, an increase in Al concentration from 0 to 1.4 m M caused a gradual decrease in activity of both enzymes, irrespective of the Al-resistance of whole seedlings. Aluminum-sensitive and aluminum-resistant cultivars also differed in the rate and extent of accumulation of slowly-exchanging Al in 5-mm root apices. During the first 6 h of Al treatment, Al accumulation was only 10% more rapid in Katepwa than in PT741. After 24-h exposure, accumulation in the Al-sensitive Katepwa, was two-fold higher. A decline in Al accumulation in a slowly-exchanging compartment as well as a decrease in activities of G6PDH and 6PGDH were found in the Al-resistant PT741, when seedlings were transferred to Al-free treatment solutions after 16-h exposure to 100 μ M Al. These results suggest that rapid induction of G6PDH and 6PGDH in the Al-resistant line PT741 by Al may play a role in the mechanism of Al resistance, possibly by regulation of the pentose phosphate pathway.     

Aluminum resistance in wheat (Triticum aestivum) is associated with rapid, Al-induced changes in activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in root apices

We have investigated the effect of aluminum (Al) on the activity of glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) isolated from 5-mm root apices of 4-day-old wheat ( Triticum aestivum ) cultivars differing in resistance to Al. Rapid increases in G6PDH and 6PGDH activities were observed in Al-resistant cultivars (PT741 and Atlas 66) during the first 10 h of treatment with 100 μ M Al, while no change in the activity of either enzyme was observed in Al-sensitive cultivars (Katepwa and Neepawa) during a 24-h exposure to Al. The Al-induced increases in enzyme activities observed in the Al-resistant PT741 appear to reflect an induction of protein synthesis since the increases were completely abolished by 1 m M cycloheximide. No differences in G6PDH and 6PGDH activities were observed between the Al-sensitive and the Al-resistant genotypes when Al was supplied in vitro. Under these conditions, an increase in Al concentration from 0 to 1.4 m M caused a gradual decrease in activity of both enzymes, irrespective of the Al-resistance of whole seedlings. Aluminum-sensitive and aluminum-resistant cultivars also differed in the rate and extent of accumulation of slowly-exchanging Al in 5-mm root apices. During the first 6 h of Al treatment, Al accumulation was only 10% more rapid in Katepwa than in PT741. After 24-h exposure, accumulation in the Al-sensitive Katepwa, was two-fold higher. A decline in Al accumulation in a slowly-exchanging compartment as well as a decrease in activities of G6PDH and 6PGDH were found in the Al-resistant PT741, when seedlings were transferred to Al-free treatment solutions after 16-h exposure to 100 μ M Al. These results suggest that rapid induction of G6PDH and 6PGDH in the Al-resistant line PT741 by Al may play a role in the mechanism of Al resistance, possibly by regulation of the pentose phosphate pathway.     

Heterotrophy and nitrate metabolism in a cyanobacteriumPhormidium uncinatum

Nitrate-supported heterotrophic growth ofPhormidium uncinatum was achieved after repeated exposure to glucose in the presence of a photosystem (PS) II inhibitor. Nitrate and glucose utilization as well as activities of their metabolizing enzymes were measured comparatively in photoautotrophic and heterotrophic cells. Nitrate and glucose were taken up together at the ratio of 1:8 (molar basis) and glucose catabolism via glucose-6-phosphate dehydrogenase (G6PDH), and 6-phosphogluconate dehydrogenase (6PGDH) activities transferred desired electrons for nitrate reduction to ammonia through coupled ferredoxin-NADP⁺ reductase (FNR) activity. Ammonia thus generated was assimilated mainly by NADPH-glutamate dehydrogenase (GDH) activity. These data demonstrate an operation of nitrate assimilation in this cyanobacterium under heterotrophic conditions.     

The correlation between glycogen–glycolysis metabolite pyruvate and vancomycin antibiotic productions of Amycolatopsis orientalis grown in glucose medium

The effect of glucose concentration in the growth medium on the relationship between glycolysis, glycogen accumulation and vancomycin production of Amycolatopsis orientalis was investigated depending on the incubation time. After a lag phase, bacterial growth of A. orientalis began and biomass concentration increased continuously up to 36th or 48th hours while glucose concentration in the culture medium was consumed rapidly in the same time of incubation. In addition, increase in glucose concentrations of the growth medium lead to increase intracellular glucose as well as glycerol levels. Intracellular pyruvate levels increased significantly up to 15 g/L while extracellular pyruvate levels with respect to increases in glucose concentration. A positive correlation between glucose kinase activities and glucose concentration was determined during the incubation period. Pyruvate kinase activity increased up to 15 g/L glucose and 48th hour of incubation. As a glycopeptide antibiotic, vancomycin production increased with the increases in glucose concentrations up to 15 g/L. These results indicated that glycogen accumulation with respect to glucose concentration of the growth medium was concomitant with the sporulation of A. orientalis. When the initial glucose concentration exceeded 15 g/L, pyruvate excretions as well as intracellular glycogen and glycerol productions were supported in spite of repression in vancomycin production of A. orientalis.     

Uridine and cytidine nucleotide synthesis in renal hypertrophy: Biochemical differences in response to the growth stimulus of diabetes and unilateral nephrectomy

The effects of unilateral nephrectomy (UN) and streptozotocin (STZ) diabetes on the activities of enzymes involved in uridine and cytidine synthesis in early renal growth (3–14 days after stimulus to growth) have been compared. Measurements were also made of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) and of glucose 6-phosphate (G6P), UDP-glucose, and glycogen, in relation to phosphoribosyl pyrophosphate, ribonucleotide, and complex carbohydrate formation. There were striking differences in the activities of CTP synthetase, G6PDH, and 6PGDH in the two conditions, with a three-fold increase in all three enzymes at 3 and 5 days and a two-fold increase above basal values at 14 days of STZ diabetes. The UN group showed no significant change in CTP synthetase at any stage and the activity of G6PDH and 6PGDH only kept pace with renal growth. Changes in routes of uridine synthesis were less marked, with a more rapid rise in carbamoyl-phosphate synthetase (glutamine) and a lesser response of dihydroorotate dehydrogenase in the UN relative to the STZ-diabetic groups. The enzymes of complex II and of uracil phosphoribosyltransferase showed essentially similar patterns during renal hypertrophy in UN and STZ diabetes. The parallel increase in CTP synthetase, G6PDH, and 6PGDH in the kidney in diabetes, also known to increase in growth situations in hepatomas and in renal tumors, is discussed in relation to hormone signals involved in renal growth. The importance of the concentration of CTP, and thus of CTP synthetase, in the CTP-cytidyltransferase reaction, an enzyme with a high K_m for CTP, makes the present observation of the striking increase in CTP synthetase in STZ diabetes of particular interest in relation to phosphatidylcholine formation and hormone signal transduction.     

Variations in the kinetic behaviour of the NADPH-production systems in different tissues of the trout when fed on an amino-acid-based diet at different frequencies

We have studied the effects of feeding an amino-acid-based diet (ABD) at different frequencies upon growth and several NADPH-production systems in the rainbow trout (Oncorhynchus mykiss). The kinetic behavior of glucose 6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), malic enzyme (ME) and NADP-linked isocitrate dehydrogenase (NADP-IDH) was followed in the liver, kidney and adipose tissue.The kinetic parameters of NADP-IDH alone remained unaltered by either ABD or changes in feeding frequency. Maximum-velocity and catalytic-efficiency values of hepatic G6PDH and ME increased significantly when fed four times a day compared to twice a day with both the control diet and ABD, although these parameters for ME were significantly lower with ABD than with the control diet at both frequencies. In the kidney the activity and catalytic efficiency of G6PDH and 6PGDH increased significantly with high-frequency feeding on ABD. The activities of these enzymes in adipose tissue were much lower than in hepatic tissue. In the liver, maximum velocity and the catalytic efficiency of G6PDH, 6PGDH and ME increased significantly with the control diet at high-frequency feeding whereas they decreased significantly with ABD, especially with high-frequency feeding. Neither the Michaelis constant nor the activity ratios varied.Both feeding frequency and free amino acid altered the activity of the most important cytosolic NADPH-production systems. The varying response to nutritional stimuli of NADP-linked enzymes in fish tissues shows that they have independent physiological and metabolic roles and that their regulatory mechanisms respond to changes in nutritional and metabolic factors.     

Coenzyme Specificity of Enzymes in the Oxidative Pentose Phosphate Pathway of Gluconobacter oxydans

The coenzyme specificity of enzymes in the oxidative pentose phosphate pathway of Gluconobacter oxydans was investigated. By investigation of the activities of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) in the soluble fraction of G. oxydans, and cloning and expression of genes in Escherichia coli, it was found that both G6PDH and 6PGDH have NAD/NADP dual coenzyme specificities. It was suggested that the pentose phosphate pathway is responsible for NADH regeneration in G. oxydans.     

Carbohydrate deprivation reduces NADPH-production in fish liver but not in adipose tissue

Little is known about the way in which carnivorous fish such as salmonids mobilise and metabolise dietary carbohydrates, which are essential to lipid metabolism. Thus we have studied changes caused by the absence of dietary carbohydrates to the kinetics and molecular behaviour of the four cellular NADPH-production systems [glucose 6-phosphate dehydrogenase (G6PDH); 6-phosphogluconate dehydrogenase (6PGDH); malic enzyme (ME); and isocitrate dehydrogenase NADP-dependent (NADP-IDH)] in the liver and adipose tissue of rainbow trout (Oncorhynchus mykiss). We used spectrophotometry to study enzyme kinetics and nucleic acid concentrations, and immunoblot analysis to determine specific protein concentrations. The absence of carbohydrate reduced specific enzyme activity, maximum rate and catalytic efficiency by almost 65% in G6PDH and 6PGDH, by more than 50% in ME, and by almost 25% in NADP-IDH but caused no significant changes in the K(m) values or activity ratios in any of these hepatic enzymes. Molecular analysis clearly showed that this kinetic behaviour reflected concomitant changes in intracellular enzyme concentrations, produced by protein-induction/repression processes rather than changes in the activity of pre-existing enzymes. We conclude that the absence of carbohydrates significantly reduces intracellular concentrations of G6PDH, ME and NADP-IDH in trout liver in percentages similar to those recorded for enzyme activity. We found no such variations in the concentrations of any of these enzymes in adipose tissue and no change in the levels of their activity, suggesting that the liver and adipose tissues are subject to different regulation systems with regard to carbohydrates and play distinct roles in lipid metabolism.     

Dietary induction of hepatic glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and malic enzyme in lean and obese female Zucker rats

Responses of the hepatic lipogenic enzymes, glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), and malic enzyme (ME) to starvation refeeding and diet shifting were determined in lean and obese female Zucker rats. Rats were either fed nonpurified diet, starved 48 hr, and then refed nonpurified diet or one of the refined carbohydrate diets containing either glucose, fructose, cornstarch, or sucrose for 72 hr, or shifted from nonpurified diet directly to one of the refined carbohydrate diets for 72 hr. Initial activities were greater in obese than lean rats for all three enzymes studied. Similar to other strains of female rats, lean Zucker rats failed to demonstrate a starve-refeed response when refed nonpurified diet. Obese female littermates showed a statistically significant increase in enzymes when refed a nonpurified diet. Both lean and obese female Zucker rats demonstrated increases in enzyme activities above controls when starved and refed any of the refined carbohydrate diets. The greatest responses were observed when female rats were starved and refed sucrose; activities increased 2.6- to 3.5-fold in lean and 3.0- to 4.3-fold in obese Zuckers. In lean females 50-70% of the starve-refeed response observed with G6PDH and ME can be accounted for by simply shifting from a nonpurified diet to the respective refined carbohydrate diet, whereas in obese females only 33-55% of the increase could be attributed to diet shifting. Plasma testosterone/estrogen ratios were consistently 1.5 times higher in obese than in lean female rats. This phenotypic difference may potentiate the heightened starve-refeed overshoot response observed in obese rats.