Dehydrogenase Patterns in the Study of Bacteroidaceae

Enzyme patterns were obtained by starch-gel electrophoresis of cell-free extracts from organisms in the family Bacteroidaceae. Esterases, phosphatases, and lactic and succinic dehydrogenases were detected but offered no possibility for classification purposes. Alpha-glycerophosphate dehydrogenase and 6-phosphogluconate dehydrogenase were not detected. Zymograms of malic, glutamic, and glucose-6-phosphate dehydrogenases separated Bacteroides species from Sphaerophorus and Fusobacterium species. Malic dehydrogenase and glucose-6-phosphate dehydrogenase were found in Bacteroides but not Sphaerophorus and Fusobacterium. These dehydrogenase zymograms placed three gram-negative, nonsporeforming anaerobic rod isolates with the Bacteroides species. A close correlation was found between the classification of Bacteroidaceae by zymogram analysis and a numerical taxonomy scheme previously published from this laboratory.     

Genetic Mapping of Loci for Glucose-6-Phosphate Dehydrogenase, Gluconate-6-Phosphate Dehydrogenase, and Gluconate-6-Phosphate Dehydrase in Escherichia coli

The loci on the Escherichia coli genome of mutations affecting the constitutive enzymes glucose-6-phosphate dehydrogenase (zwf) and gluconate-6-phosphate dehydrogenase (gnd), and the inducible enzyme gluconate-6-phosphate dehydrase (edd), were determined by conjugation and transduction experiments, chiefly by three-factor crosses. They are in the same region of the chromosome, and their order is gnd-his-(edd, zwf)-aroD; gnd and his are cotransduceable, as are zwf and edd. The position of gnd in Salmonella typhimurium was shown to be similar to that in E. coli.     

Selection of Escherichia coli mutants lacking glucose-6-phosphate dehydrogenase or gluconate-6-phosphate dehydrogenase

Glucose is metabolized in Escherichia coli chiefly via the phosphoglucose isomerase reaction; mutants lacking that enzyme grow slowly on glucose by using the hexose monophosphate shunt. When such a strain is further mutated so as to yield strains unable to grow at all on glucose or on glucose-6-phosphate, the secondary strains are found to lack also activity of glucose-6-phosphate dehydrogenase. The double mutants can be transduced back to glucose positivity; one class of transductants has normal phosphoglucose isomerase activity but no glucose-6-phosphate dehydrogenase. An analogous scheme has been used to select mutants lacking gluconate-6-phosphate dehydrogenase. Here the primary mutant lacks gluconate-6-phosphate dehydrase (an enzyme of the Enter-Doudoroff pathway) and grows slowly on gluconate; gluconate-negative mutants are selected from it. These mutants, lacking the nicotinamide dinucleotide phosphate-linked glucose-6-phosphate dehydrogenase or gluconate-6-phosphate dehydrogenase, grow on glucose at rates similar to the wild type. Thus, these enzymes are not essential for glucose metabolism in E. coli.     

An Electrophoretic Analysis of Variation in the Glucose-6-Phosphate Dehydrogenase and Malate Dehydrogenase of Vibrio cholerae, Vibrio parahaemolyticus and Vibrio fluvialis

A total of 59 isolates of Vibrio cholerae, V. parahaemolyticus and V. fluvialis were studied using the techniques of enzyme electrophoresis. The enzymes used were malate dehydrogenase (E.C.I. 1.1.37) and glucose-6-phospate dehydrogenase (E.C.I. 1.1.49). The results in general confirmed classical methods of vibrio taxonomy. The three species could be separated from each other and identified by their enzyme variant types.
The term zymovar, a group of strains having similar enzyme variants, is introduced. In the V. cholerae isolates six zymovars were found. All V. cholerae serovar 01 strains were classified in the same zymovar except for some strains of environmental origin, which occurred in another zymovar. In V. fluvialis two zymovars were detected corresponding with biovars 1 and 2 of this organism. All isolates of V. parahaemolyticus gave the same enzyme type.
The technique of enzyme electrophoresis appears to be a useful tool in vibrio taxonomy and used in conjunction with other methods may aid in the elucidation of the systematics of the genus.     

Selective neutrality of glucose-6-phosphate dehydrogenase allozymes in Escherichia coli

Six naturally occurring alleles representing four electromorphs of the enzyme glucose-6-phosphate dehydrogenase were transferred by P1- mediated transduction from natural isolates of Escherichia coli into the genetic background of E. coli K12 and were studied in pairwise competition in chemostats limited for glucose in order to estimate differences in growth rate associated with the alleles. Although the level of resolution of such experiments is a growth rate differential of approximately 0.002 h-1, no significant differences among the strains were found. Studies of apparent Km and Vmax in crude enzyme extracts of the strains also failed to reveal any significant differences among the electromorphs. These results support the view that the alleles are selectively neutral or nearly neutral under these conditions.      

Genetic relationships among the oral streptococci.

Genetic relationships and species limits among the oral streptococci were determined by an analysis of electrophoretically demonstrable variation in 16 metabolic enzymes. Fifty isolates represented 40 electrophoretic types, among which the mean genetic diversity per locus was 0.857. Mannitol-1-phosphate dehydrogenase was not detected in isolates of the sanguis species complex, and glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were absent in species of the mutans complex. Clustering from a matrix of Gower's coefficient of genetic similarity placed the 40 electrophoretic types in 10 well-defined groups corresponding to the Streptococcus species S. mutans, S. sobrinus, S. cricetus, S. rattus, S. ferus, S. oralis (mitior), two distinct assemblages of S. sanguis strains, and two subdivisions of "S. milleri." The assignments of isolates to these groups were the same as those indicated by DNA hybridization experiments, and the coefficient of correlation between genetic distance estimated by multilocus enzyme electrophoresis and genetic similarity indexed by DNA hybridization was -0.897 (P less than 0.001) for 50 pairwise combinations of isolates. S. ferus, which is widely believed to be a member of the mutans complex, was shown to be phylogenetically closer to species of the sanguis complex.     

Purification and Characterization of the Pseudomonas multivorans Glucose-6-Phosphate Dehydrogenase Active with Nicotinamide Adenine Dinucleotide

The Pseudomonas multivorans glucose-6-phosphate dehydrogenase (EC 1.1.1.49) active with nicotinamide adenine dinucleotide, which is inhibitable by adenosine-5'-triphosphate, was purified approximately 1,000-fold from extracts of glucose-grown bacteria, and characterized with respect to subunit composition, response to different inhibitory ligands, and certain other properties. The enzyme was found to be an oligomer composed of four subunits of about 60,000 molecular weight. Reduced nicotinamide adenine dinucleotide phosphate, but not reduced nicotinamide adenine dinucleotide, was found to be a potent inhibitor of its activity. The range of concentrations of reduced nicotinamide adenine dinucleotide phosphate over which inhibition occurred was about 100-fold lower than that for adenosine-5'-triphosphate. The data suggest that reduced nicotinamide adenine dinucleotide phosphate may play an important role in regulation of hexose phosphate metabolism in P. multivorans. Antisera prepared against the purified enzyme strongly inhibited its activity, but failed to inhibit the activity of the nicotinamide adenine dinucleotide phosphate-specific glucose-6-phosphate dehydrogenase which is also present in extracts of this bacterium. Immunodiffusion experiments confirmed the results of the enzyme inhibition studies, and failed to support the idea that the two glucose-6-phosphate dehydrogenase species from P. multivorans represent different oligomeric forms of the same protein.     

Screening and Mutagenesis of Aspergillus niger for the Improvement of Glucose-6-Phosphate Dehydrogenase Production

The Aspergillus niger strain ZBY-7 was selected as the original strain of glucose-6-phosphate dehydrogenase production. After mutagenesis of the strain by means of UV irradiation and nitrosoguanidine, mutants of Aspergillus niger resistant to a certain metabolic inhibitor were obtained. Five of the mutants showed increased glucose-6-phosphate dehydrogenase production. The mutant resistant to antimycin A (Aspergillus niger AM-23) produced the highest level of glucose-6-phosphate dehydrogenase (695.9% of that produced by the original strain).     

Molecular cloning of DNA sequences complementary to rat liver glucose-6-phosphate dehydrogenase mRNA. Nutritional regulation of mRNA levels

The nutritional regulation of rat liver glucose-6-phosphate dehydrogenase was studied using a cloned DNA complementary to glucose-6-phosphate dehydrogenase mRNA. The recombinant cDNA clones were isolated from a double-stranded cDNA library constructed from poly(A+) RNA immunoenriched for glucose-6-phosphate dehydrogenase mRNA. Immunoenrichment was accomplished by adsorption of polysomes with antibodies directed against glucose-6-phosphate dehydrogenase in conjunction with protein A-Sepharose and oligo(dT)-cellulose chromatography. Poly(A+) RNA encoding glucose-6-phosphate dehydrogenase was enriched approximately 20,000-fold using these procedures. Double-stranded cDNA was synthesized from the immunoenriched poly(A+) RNA and inserted into pBR322 using poly(dC)-poly(dG) tailing. Escherichia coli MC1061 was transformed, and colonies were screened for glucose-6-phosphate dehydrogenase cDNA sequences by differential colony hybridization. Plasmid DNA was purified from clones which gave positive signals, and the identity of the glucose-6-phosphate dehydrogenase clones was verified by hybrid-selected translation. A collection of glucose-6-phosphate dehydrogenase cDNA plasmids with overlapping restriction maps was obtained. Northern blot analysis of rat liver poly(A+) RNA using nick-translated, 32P-labeled cDNA inserts revealed that the glucose-6-phosphate dehydrogenase mRNA is 2.3 kilobases in length. RNA blot analysis showed that refeeding fasted rats a high carbohydrate diet results in a 13-fold increase in the amount of hybridizable hepatic glucose-6-phosphate dehydrogenase mRNA which parallels the increase in enzyme activity. These results suggest that the nutritional regulation of hepatic glucose-6-phosphate dehydrogenase occurs at a pretranslational level.     

Linkage of two enzyme loci in the fish genus Poecilia (Teleostei:Poeciliidae).

The linkage of loci coding for glucose-6-phosphate dehydrogenase (G6PD) and phosphogluconate dehydrogenase (PGD) is described in fish of the genus Poecilia (Teleostei:Poeciliidae) and designated Poecilia linkage group I. These two loci were shown to assort independently from six other informative markers (peptidase S, malate dehydrogenase 2 [soluble], mannose phosphate isomerase, parvalbumin 2, phosphoglucomutase, and glyceraldehyde-3-phosphate dehydrogenase 2) within the limits of the data obtained. Data for the linkage analyses were generated by scoring starch-gel electrophoretic phenotypes of the eight loci in reciprocal backcross hybrids obtained from matings between Poecilia perugiae and P. vittata. The linkage chi 2 for G6PD-PGD locus pairs was significant (P less than 0.001) in all reciprocal backcross hybrid broods (22.7% recombinants in the combined data), indicating linkage in both parental species. The linkage of G6PD and PGD in gene maps of the poeciliid genera Xiphophorus and Poeciliopsis documents homology of this linkage within the family. Linkages in salmonid and centrarchid fishes suggest conservation of this linkage group in most or all teleosts. The six additional indpendently assorting loci have been assigned to independent linkage groups in Xiphophorus; thus, no example of poeciliid linkage group divergence has yet been identified.     

基于己糖激酶与葡萄糖-6-磷酸脱氢酶共表达的辅酶NADPH高效再生

【目的】构建己糖激酶与葡萄糖-6-磷酸脱氢酶的大肠杆菌共表达体系,以葡萄糖为底物实现辅酶NADPH的高效再生。【方法】通过分子生物学方法,克隆己糖激酶HKgs、HKpp基因,并于Escherichia coli BL21(DE3)中表达,再将己糖激酶HKgs、HKpp分别与葡萄糖-6-磷酸脱氢酶Gpd PP共表达,实现NADPH的原位再生。比较两个共表达工程菌的辅酶再生效果,并针对催化活力较高的工程菌BL21(HKgs+Gpd PP)进行表达条件优化。【结果】NADPH再生活力达到856 U/L。该辅酶再生体系与醇脱氢酶Adh R联合催化,使不对称还原4-氯乙酰乙酸乙酯的催化活力提高至原始值的2.5倍。【结论】通过己糖激酶与葡萄糖-6-磷酸脱氢酶在大肠杆菌中的共表达,构建了一个新的NADPH高效再生体系,并用于醇脱氢酶催化的不对称还原反应。     

Enzyme variation in Eimeria species of the chicken.

A method for the biochemical identification of protozoa belonging to the genus Eimeria is described for the first time. Starch gel electrophoresis of the enzymes lactate dehydrogenase, glucose phosphate isomerase, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase from parasite extracts revealed both intra- and inter-species differences when 11 strains representative of 6 species of Eimeria were examined. Oocysts were the most accessible parasite stage for investigation but sporozoites and merozoites of an embryo-adapted strain of E. tenella were also examined for enzyme activity.     

Identification of oral Neisseria species of animals

Ninety-seven strains of presumptive Neisseria spp. obtained from the dental plaque of a wide variety of animals and 21 strains from culture collections were compared by physiological tests, enzyme electrophoresis and isoelectric focusing of proteins. Three physiological groups based on the fermentation of maltose and production of extracellular polysaccharide were established. The ability of strains within these groups to reduce nitrate and nitrite differed. The electrophoretic mobility of dehydrogenases and isoelectric focusing patterns of proteins were not, however, characteristic of species or physiological groups. It is difficult, therefore, to identify new isolates of Neisseria because the criteria for describing species overlap. A rapid spot test was devised to distinguish Branhamella catarrhalis from other Neisseria by the absence of glucose-6-phosphate dehydrogenase.     

L-Sorbose metabolism in Klebsiella pneumoniae and Sor+ derivatives of Escherichia coli K-12 and chemotaxis toward sorbose.

L-Sorbose degradation in Klebsiella pneumoniae was shown to follow the pathway L-sorbose leads to L-sorbose-1-phosphate leads to D-glucitol-6-phosphate leads to D-fructose-6-phosphate. Transport and phosphorylation of L-sorbose was catalyzed by membrane-bound enzyme IIsor of the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system, specific for and regulated by this ketose and different from all other enzymes II described thus far. Two soluble enzymes, an L-sorbose-1-phosphate reductase and a D-glucitol-6-phosphate dehydrogenase, were involved in the conversion of L-sorbose-1-phosphate to D-fructose-6-phosphate. This dehydrogenase was temperature sensitive, preventing growth of wild-type strains of K. pneumoniae at temperatures above 35 degrees C in the presence of L-sorbose. The enzyme was distinct from a second D-glucitol-6-phosphate dehydrogenase involved in the metabolism of D-glucitol. The sor genes were transferred from the chromosome of nonmotile strains of K. pneumoniae by means of a new R'sor+ plasmid to motile strains of Escherichia coli K-12. Such derivatives not only showed the temperature-sensitive Sor+ phenotype characteristic for K. pneumoniae or Sor+ wild-type strains of E. coli, but also reacted positively to sorbose in chemotaxis tests.     

Cytoplasmic NADP-linked dehydrogenase activities in avian tissues

Cytoplasmic activities of NADP-linked malic enzyme (E.C. 1.1.1.40), glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) and NADP-linked isocitrate dehydrogenase (E.C. 1.1.1.42) were determined in tissues of selected avian species, and compared with those in mammals. Malic enzyme was generally more active in avian liver and kidney than in the corresponding mammalian tissues. Hepatic activities as high as 200 units/g wet wt and 100 units/g wet wt were recorded in the Nectariniidae and the Ploceidae respectively. Glucose-6-phosphate dehydrogenase was generally less active in avian tissues than malic enzyme. In passerine birds activities were very low indeed, and in most cases spectrophotometrically undetectable. Malic enzyme and glucose-6-phosphate dehydrogenase were highly active in the adipose tissue of mammals but were inactive in the adipose tissue of birds. Marked increases in hepatic malic enzyme and glucose-6-phosphate dehydrogenase activities were associated in birds with premigratory fattening. Activities of isocitrate dehydrogenase were comparable in the corresponding avian and mammalian tissues, including adipose tissue.     

A Quantitative Cytochemical Study of Glutamate and Glucose-6-Phosphate Dehydrogenase Activities during Chilling Injury in Tubers of Dioscorea rotundata Poir.

Onyia, G. O. C. and Gahan, P. B. 1985. A quantitative cytochemicalstudy of glutamate and glucose-6-phosphate dehydrogenase activitiesduring chilling injury in tubers of Dioscorea rotundala Poir.—J.exp. Bot. 36: 1249–1256. The response of glucose-6-phosphate dehydrogenase and glutamatedehydrogenase activities in healthy Jamaican Dioscorea rotundalatubers and those chilled at 3 ?C for 1,2,3,4, and 7 d at 70%r.h. were assessed by quantitative cytochemical assays. Bothenzymes in chill-damaged tuber tissues showed a substantiallyhigher activity than did those of the healthy tubers. An early,sharp increase in the response of the NADP-tetrazolium reductasesystem of damaged tuber tissue was significantly higher (P =0.001) than that of healthy tubers or those chilled but ableto recover. This response may be used as an early marker ofchilling injury in the yam tuber. Key words: Dioscorea rotundata Poir, quantitative cytochemistry, yam tuber, glucose-6-phosphate, dehydrogenase, glutamate dehydrogenase, NADPitetrazolium reductase     

Glucose-6-phosphate dehydrogenase isozymes in fish--a comparative study

The electrophoretic distribution and substrate specificities of isozymes of glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) were studied in seven species of teleost fish. The fish examined included two species of bonefish, Albula neoguinaica and A. glossodonta (Albulidae, Anquilliformes) (Shaklee and Tamaru, '81), and five representatives of the order Perciformes: two species of butterflyfish, Chaetodon miliaris and C. aurega (Chaetodontidae); a goatfish, Upeneus arge (Mullidae); a goby, Bathygobius fuscus (Gobiidae); and a snapper, Pristipomoides filamentosus (Lutjanidae). After horizontal starch gel electrophoresis, gel slices were stained using a variety of substrates and cofactors. In all species except the goby, two groups of isozymes were distinguished, corresponding to the mammalian G6PD (specific for glucose-6-phosphate (G6P) and NADP+) and H6PD (capable of utilizing galactose-6-phosphate and in certain cases other monosaccharide phosphates in addition to G6P). None of the five visible isozymes in the goby was specific for G6P. In each of the other species a single G6P- and NADP+-specific isozyme was noted, having the most rapid mobility toward the anode. In addition, it was found that all of the isozymes in all of the fish examined could catalyze the oxidation of fructose-6-phosphate at a rate comparable to that for G6P, suggesting that glucose-6-phosphate dehydrogenase can obviate the role of glucosephosphate isomerase in monosaccharide metabolism.     

Purification and Kinetic Characterization of Glucose-6-phosphate Dehydrogenase from Dictyostelium discoideum

Reimers, J. M., Huang, Q., Albe, K. R., and Wright, B. E. 1993. Purification and kinetic characterization of glucose-6-phosphate dehydrogenase from Dictyostelium discoideum. Experimental Mycology 17, 1-6. Glucose-6-phosphate dehydrogenase from Dictyostelium discoideum was purified 650-fold and kinetically characterized. The enzyme catalyzed the conversion of G6P + NADP to 6PG + NADPH stoichiometrically and irreversibly in vitro . The purified enzyme is specific for NADP. Michaelis constants for G6P and NADP were 0.040 and 0.011 mM, respectively. NADPH was found to be a competitive inhibitor with respect to NADP with a K_i of 0.006 mM and a noncompetitive inhibitor with respect to G6P. The data from initial velocity and product inhibition studies were consistent with a sequential mechanism.