Intramembraneous localization of rat liver microsomal hexose-6-phosphate dehydrogenase and membrane permeability to its substrates.

A method for purifying hexose-6-phosphate dehydrogenase (beta-D-glucose: NAD(P) -oxidoreductase, EC 1.1.1.47) from rat liver microsomes is described. The purified enzyme was shown to be homogeneous by sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis. It is shown that the enzyme is bound to the inner surface of microsomal membranes, and that glucose 6-phosphate, but not NADP, penetrates almost freely into the membranes at 37 degrees C.

Metabolism of calf trabecular (reticular) meshwork.

It has been found that calf eyes are an excellent source of trabecular meshwork tissue for biochemical studies. Homogenates of pooled meshwork were centrifuged at 27K X g and 1.5K X g. The high-speed supernatants produced lactate at 0.35 mumole/min/gm tissue in the presence of hexokinase-saturating concentrations of glucose (10 mM) at pH 7. The optimum pH was 7.6. In the absence of ammonia, lactate could be produced from fructose 1,6-diphosphate but not from glucose or glucose 6-phosphate. The optimum ammonia concentration was 1 to 2 mM. Lactate was produced at an even greater rate from fructose, but only poorly from sorbitol or galactose (all at 10 mM). The activity of hexokinase, glucose 6-phosphate dehydrogenase and UDPG dehydrogenase was measured. Fructokinase could not be detected. The low-speed supernatant readily oxidized succinate, malate, and glutamate at about 0.012 muAtO/min/gm tissue. The oxidative rate in vivo is estimated to be about one third of this. These results demonstrate that knowledge of the normal metabolism of calf trabecular meshwork may be obtained with relative ease, with possible important implications for understanding the disease of glaucoma.

Fractionation and biochemical properties of the mother cell and forespore functions from sporulating cells of Bacillus subtilis.

A lysozyme-detergent method was developed for the fractionation of sporulating cells of B. subtilis 168 wild type into mother cell and forespore fractions. The method is very mild and is reproducible with optimum concentrations of Brij-58, deoxycholic acid and sucrose. The results were confirmed by application of the method to temperature sensitive mutants. Ts-1 and Ts-3. The amounts of proteins, and the activities of protease, alkaline phosphatase and glucose dehydrogenase were about 55, 56, 91, and 40%, respectively, in the mother cell fraction, and about 45, 44, 9, and 60%, respectively, in the forespore fraction, taking the totals for the combined fractions as 100%. Slab gel electrophoretic patterns indicated that many species of proteins with different molecular weights were present in the two fractions. Pulse-labeling with [3H]UTP was carried out in vivo at stage III, and 35.2 and 64.8% of the [3H]UMP incorporated into RNAs were distributed in the mother cell and forespore fractions, respectively. The results indicate that more RNA synthesis occurs in the forespores than in the mother cells of sporulating cells.

Glucose dehydrogenase of bovine heart: activity, purification and properties of the enzyme.

The activity of glucose dehydrogenase (EC 1.1.1.47; GDH; glucose NAD(P) oxidoreductase) was demonstrated in the supernatant obtained after centrifugation (1500 g) of bovine heart homogenate. More than 50% of GDH activity was found in the microsomal fraction. The optimum pH for the microsomal enzyme was 8.9. About 200-fold purification of GDH was achieved by successive application of ultracentrifugation in 0.25 M mannitol, treatment with solid ammonium sulphate, and CM-32 cellulose chromatography. The purified preparation oxidized not only glucose but also D-glucosamine, N-acetylglucosamine and xylose in 87, 63 and 23%, respectively. Purified GDH was inhibited by p-chloromercuribenzoate and glucose 6-phosphate.

Construction and characterization of heterodimeric soluble quinoprotein glucose dehydrogenase.

In order to study in greater detail the subunit interaction of the homodimeric soluble quinoprotein glucose dehydrogenase (PQQGDH-B), we developed an effective method of creating heterodimeric PQQGDH-B. Two different homodimers are combined, one of which has a polyarginine tail (Arg-tail), and subjected to a protein dissociation/redimerization procedure. Separation of the mixture by cation exchange chromatography results in three peaks showing GDH activity, eluting at 133, 231 and 273 mM NaCl concentration. These peaks were determined to correspond to the Arg-tailless homodimer, heterodimer, and Arg-tailed homodimer, respectively. To test this approach, we constructed and characterized heterodimeric PQQGDH-B composed of native (wild-type) and inactive mutant (His168Gln) subunits. The heterodimeric wild-type-His168Gln showed slightly decreased GDH activity and almost identical substrate specificity profile to the wild-type enzyme. Moreover, the Hill coefficient of the heterodimer was calculated as 1.13, indicating positive cooperativity.

Alcohol dehydrogenases, aldehyde dehydrogenases, and related enzymes.

Several new structures have recently been determined for dehydrogenases which are involved in alcohol metabolism. These structures give new insight into catalytic properties, structure-function relationships, and evolutionary connections. They also explain the structural basis for known metabolic deviations. Among alcohol dehydrogenases, the first primary structures for human enzyme forms have been reported (the beta 1, gamma 1, and "atypical" beta-chains). These structures explained functional properties and enzymatic differences, showed separate and parallel events of isozyme divergence, and suggested differential gene activations. Similarly, for aldehyde dehydrogenases, the first cytoplasmic isozyme structures have been reported in man and horse. The data showed positions of functionally important residues, and established clear differences between the mitochondrial and cytoplasmic isozymes of aldehyde dehydrogenase. For sorbitol dehydrogenase, the first complete structure which establishes a relationship with other "long" alcohol dehydrogenases in a scheme exhibiting both structural divergence and functional convergence has also been reported. Finally, glucose dehydrogenase has been structurally linked with sorbitol dehydrogenase, extending the scheme and adding further enzymes to the group of "short" alcohol dehydrogenases.

An automatic dehydrogenase-based flow-injection system: application for the continuous determination of glucose and lactate in mammalian cell-cultures.

A concept for the development of an automatic flow-injection analyzer with integrated dehydrogenase columns and its application in the control of industrial processes is presented. The system is based upon a kernel consisting of a nested-loop injection unit, pumps for the filling of the injection loops and the transport of buffer and values for switching on the one hand between sample and standard solutions and on the other hand between different enzyme columns. A Microsoft Windows 3.x application 'WIN-FIA' controls interactively the whole system and can be easily adapted to a specific solution of an analytical problem. As an example, the flow-injection system was used for the continuous determination of glucose and lactate, using glucose dehydrogenase (GDH) and lactate dehydrogenase (LDH) as indicator enzymes, in a mammalian cell-culture fermentation process. The resulting concentration values are in good agreement with those obtained by discontinuously taken standard spectrophotometric enzyme assays.

Glucose metabolism in batch and continuous cultures of Gluconacetobacter diazotrophicus PAL 3.

Periplasmic glucose oxidation (by way of a pyrrolo-quinoline-quinone [PQQ]-linked glucose dehydrogenase [GDH]) was observed in continuous cultures of Gluconacetobacter diazotrophicus regardless of the carbon source (glucose or gluconate) and the nitrogen source (N(2) or NH(3)). Its synthesis was stimulated by conditions of high energetic demand (i.e., N(2)-fixation) and/or C-limitation. Under C-excess conditions, PQQ-GDH synthesis increased with the glucose concentration in the culture medium. In batch cultures, PQQ-GDH was actively expressed in very early stages with higher activities under conditions of N(2)-fixation. Hexokinase activity was almost absent under any culture condition. Cytoplasmic nicotinamide adenine dinucleotide (NAD)-linked glucose dehydrogenase (GDH) was expressed in continuous cultures under all tested conditions, and its synthesis increased with the glucose concentration. In contrast, low activities of this enzyme were detected in batch cultures. Periplasmic oxidation, by way of PQQ-GDH, seems to be the principal pathway for metabolism of glucose in G. Diazotrophicus, and NAD-GDH is an alternative route under certain environmental conditions.

[Urine glucose testing in children with diabetes mellitus--obsolete as a daily control measure?]. / Uringlucosediagnostik bei Kindern mit Diabetes mellitus--als tägliche Kontrollmassnahme veraltet?

Sixty-four children with type 1 diabetes (33 girls and 31 boys with a mean age of 11 1/4 years) were studied in order to ascertain whether urine sugar measurements performed by the patients or their parents can provide adequate monitoring of treatment. The results of two home-testing methods for urine glucose were compared with subsequent glucose dehydrogenase assays in the laboratory. Over the range from 0 to 5 g/dl the results of home testing disagreed with the laboratory checks by an average of one concentration grade, and displayed wide scatter. Only in the concentration range of 0.5 g/dl were there differences between the nonspecific reduction test (Clinitest) and the specific enzymatic assay. Glucose concentrations in urine specimens passed at 7 am, 12 noon and 6 pm (home testing) were compared with blood glucose concentrations checked at the same times. Correlation was not very close (correlation coefficients between 0.3 and 0.64). Correlation between pooled urine collections and 24 hour blood sugar profiles was equally poor (0.5-0.6). The author concludes that urine sugar testing is unsatisfactory for home monitoring of children with diabetes and the results are inadequate for diagnostic or therapeutic purposes.

Sporulation of bacterial species in the presence of metabisulphite.

The effect of metabisulphite on the sporulating ability of Bacillus subtilis E52 and B. cereus W18 was studied. Whereas metabisulphite concentrations of 500 and 600 micrograms ml-1 prevented sporulation of B. subtilis and B. cereus, respectively, lower concentrations caused reductions in their percentage sporulations. Glucose dehydrogenase (GDH) and alkaline phosphatase (ALP) activities of both sporulating bacteria were not detected at the stated metabisulphite concentrations. A relationship between the percentage sporulation of the bacteria and the activity of the enzymes in the presence of metabisulphite was exhibited. ALP activity may serve as an index of the effectiveness of the antisporulating activity of metabisulphite. Both enzymes are likely to be among the few targets for the antisporulating activity of metabisulphite.

Cloning, nucleotide sequences, and enzymatic properties of glucose dehydrogenase isozymes from Bacillus megaterium IAM1030.

Bacillus megaterium is known to have several genes that code for isozymes of glucose dehydrogenase. Two of them, gdhI and gdhII, were cloned from B. megaterium IAM1030 in our previous work (T. Mitamura, R. V. Evora, T. Nakai, Y. Makino, S. Negoro, I. Urabe, and H. Okada, J. Ferment. Bioeng. 70:363-369, 1990). In the present study, two new genes, gdhIII and gdhIV, were isolated from the same strain and their nucleotide sequences were identified. Each gene has an open reading frame of 783 bp available to encode a peptide of 261 amino acids. Thus, a total of four glucose dehydrogenase genes have been cloned from B. megaterium IAM1030. In addition, this strain does not seem to have other glucose dehydrogenase genes that can be distinguished from the four cloned genes so far examined by Southern hybridization analysis. The two newly cloned genes were expressed in Escherichia coli cells, and the products, GlcDH-III and GlcDH-IV, were purified and characterized and compared with the other isozymes, GlcDH-I and GlcDH-II, encoded by gdhI and gdhII, respectively. These isozymes showed different mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (GlcDH-I greater than GlcDH-III = GlcDH-IV greater than GlcDH-II), although they have the same number of amino acid residues. Double-immunodiffusion tests showed that GlcDH-I is immunologically different from the other isozymes and that GlcDH-III and GlcDH-IV are identical to one another but a little different from GlcDH-II. These glucose dehydrogenases were stabilized in the presence of 2 M NaCl. The effect of NaCl was especially large for GlcDH-III, which is most unstable enzyme. Kinetic studies showed that these isozymes are divided into two groups with respect to coenzyme specificity, although they can utilize both NAD and NADP: GlcDH-III and GlcDH-IV prefer NAD, and GlcDH-I and GlcDH-II prefer NADP. The phylogenic relationship of these glucose dehydrogenase genes is also discussed.

An improved assay for hexokinase activity in human tissue homogenates.

An improved method has been developed for the assay of hexokinase (EC 2.7.1.1) levels in human tissue homogenates. The enzyme is quantitated by the spectrophotometric measurement, at 340 nm, of NADPH formed according to the reaction scheme: [formula: see text] In tissue homogenates a number of enzymes are present which can interfere with the assay by reacting with substrates or products of the assay reactions. In the described procedure hexokinase is assayed directly in homogenates under conditions in which the effect of possible contaminating enzymes (glucose dehydrogenase, EC 1.1.1.47; glucose 6-phosphatase, EC 3.1.3.9; glucose phosphate isomerase, EC 5.3.1.9; 6-phosphogluconate dehydrogenase EC 1.1.1.44; and NADP-reducing enzymes) are eliminated. Precision studies on the assay gave within-day reproducibility of 4.3% (CV) on a tissue having a mean activity of 1.68 U/g of tissue, and day-to-day variability of 15% (CV) for a tissue averaging 1.83 U/g of tissue.

Dehydrogenase patterns in the taxonomy of Bacteroides.

The malate dehydrogenase (MDH) electrophoretic mobilities of 128 strains of bacteroides belonging to 17 species, including three subspecies of Bacteroides melaninogenicus and two subspecies of Bacteroides ruminicola, were examined. Amongst the pigmented bacteroides, the migration of this enzyme correlated well with recognized taxa, and only one strain, VPI 9085 was clearly different. Other species such as B. oralis, B. buccalis, B. denticola, B. pentosaceus, B. bivius, B. disiens and B. ruminicola were delineated by the combined use of MDH and glutamate dehydrogenase. Forty-three strains belonging to the 'B. fragilis group' differed from the above species in possessing glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, and reference strains as well as fresh isolates were assigned to the correct species by the mobility pattern of these two enzymes. Other properties of MDH such as the pH optima for the oxidation of malate or the reduction of oxaloacetate were of limited taxonomic value. However, the alkaline stability of this enzyme at pH 9, 10 and 11 clearly differentiates the saccharolytic from the non-saccharolytic species of pigmented bacteroides with the latter showing highly stable enzymes with a half life greater than 50 min.

[Variations of placental enzymes in hypoxia (author's transl)]. / Veränderungen plazentarer Enzyme unter Sauerstoffmangel.

Tissue of normal-term placentas, after normal pregnancy and spontaneous delivery, was incubated under normoxic and hypoxic conditions. Placental tissue samples were taken under sterile conditions, immediately after delivery, and incubated six hours in a medium to which six per cent or 26 per cent oxygen were supplied. After incubation, the tissue was homogenised, and the following enzymes were determined in the supernatant: aldolase, lactate-dehydrogenase, alkaline phosphatase, acid phosphatase, and glucose-6-dehydrogenase. - The activities of aldolase, glucose-6-dehydrogenase, and acid phosphatase increased and those of lactate-dehydrogenase and acid phosphatase decreased under conditions of oxygen deficit. Such changes in enzyme activity seem to suggest that in hypoxia anaerobic glycolysis is likely to increase, while maternal-foetal exchange drops, all accompanied by beginning compensatory proliferation of the trophoblast.

Characterization of the developmentally regulated Bacillus subtilis glucose dehydrogenase gene.

The DNA sequence of the structural gene for glucose dehydrogenase (EC 1.1.1.47) of Bacillus subtilis was determined and comprises 780 base pairs. The subunit molecular weight of glucose dehydrogenase as deduced from the nucleotide sequence is 28,196, which agrees well with the subunit molecular weight of 31,500 as determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the 49 amino acids at the NH2 terminus of glucose dehydrogenase purified from sporulating B. subtilis cells matched the amino acid sequence derived from the DNA sequence. Glucose dehydrogenase was purified from an Escherichia coli strain harboring pEF1, a plasmid that contains the B. subtilis gene encoding glucose dehydrogenase. This enzyme has the identical amino acid sequence at the NH2 terminus as the B. subtilis enzyme. A putative ribosome-binding site, 5'-AGGAGG-3', which is complementary to the 3' end of the 16S rRNA of B. subtilis, was found 6 base pairs preceding the translational start codon of the structural gene of glucose dehydrogenase. No known promoterlike DNA sequences that are recognized by B. subtilis RNA polymerases were present immediately preceding the translational start site of the glucose dehydrogenase structural gene. The glucose dehydrogenase gene was found to be under sporulation control at the trancriptional level. A transcript of 1.6 kilobases hybridized to a DNA fragment within the structural gene of glucose dehydrogenase. This transcript was synthesized 3 h after the cessation of vegetative growth concomitant to the appearance of glucose dehydrogenase.

Enzymatic properties of isozymes and variants of glucose dehydrogenase from Bacillus megaterium.

Three glucose dehydrogenases (GlcDH) from Bacillus megaterium, GlcDH-I, GlcDH-II and GlcDH-IWG3, were purified from Escherichia coli cells harboring one of the hybrid plasmids, pGDK1, pGDK2 and pGDA3, respectively, pGDK1 and pGDK2 contain two isozyme genes, gdhI and gdhII, respectively, from B. megaterium IAM 1030 and pGDA3 contains an isozyme gene from B. megaterium IWG3; GlcDH-IWG3 is a variant of GlcDH-I. GlcDH-I and GlcDH-II have similar pH/activity profiles and the profile for GlcDH-IWG3 is identical to that of GlcDH-I. The pH/stability profiles of these enzymes show that GlcDH-IWG3 is the most stable enzyme in the acidic region, while GlcDH-II is the most stable in the alkaline region, and GlcDH-I is the most unstable throughout the entire pH range examined. As for thermostability, GlcDH-II is the most resistant against heat inactivation at pH 6.5. The values of the first-order rate constant for heat inactivation at 50 degrees C are 0.27 min-1, 0.05 min-1 and 0.11 min-1 for GlcDH-I, GlcDH-II and GlcDH-IWG3, respectively. Kinetic studies show that these enzymes have similar kinetic constant values except that there are some differences in Kia for NAD(P) and Ka (the limiting Michaelis constant) for NAD; the values of the ratio of Kia for NAD and NADP are 11,340 and 8.7 for GlcDH-I, GlcDH-II and GlcDH-IWG3, respectively. GlcDH-I and GlcDH-IWG3 have very similar substrate specificities and GlcDH-II has a slightly higher specificity for D-glucose and 2-deoxy-D-glucose than the others. The results are discussed on the basis of the amino acid substitutions between the enzymes.

Selection of glucose-assimilating variants of Acinetobacter calcoaceticus LMD 79.41 in chemostat culture.

Glucose metabolism has been studied in two strains of Acinetobacter calcoaceticus. Strain LMD 82.3, was able to grow on glucose and possessed glucose dehydrogenase (EC 1.1.99.17). Glucose oxidation by whole cells was stimulated by PQQ, the prosthetic group of glucose dehydrogenase. PQQ not only increased the rate of glucose oxidation and gluconic acid production but also shortened the lag phase for growth on glucose. Strain LMD 79.41 also possessed glucose dehydrogenase but was unable to grow on glucose. Batch cultures and carbon-limited chemostat cultures growing on acetate in the presence of glucose oxidized the sugar to gluconic acid, which was not further metabolized. However, after prolonged cultivation on mixtures of acetate and glucose, carbon-limited chemostat cultures suddenly acquired the capacity to utilize gluconate. This phenomenon was accompanied by the appearance of gluconate kinase and a repression of isocitrate lyase synthesis. In contrast to the starter culture, cells from chemostats which had been fully adapted to gluconate utilization, were able to utilize glucose as a sole carbon and energy source in liquid and solid media.

Glucose metabolism in Xanthomonas campestris and influence of methionine on the carbon flow.

The glucose flow in Xanthomonas campestris was investigated with radio-labelled glucose and by enzymological studies. Only 7% of the radioactivity was incorporated into the cell material, but 41% was oxidized to carbon dioxide and 28% transformed to xanthan. Up to 16% of cell dry weight consisted of the polysaccharide glycogen. In the presence of 2.7 mM methionine, which is an inhibitor of xanthan formation, increased carbon dioxide formation (51%) occurred. This increase was in accordance with a twofold increase in the NAD-dependent isocitrate dehydrogenase activity. The other carbon dioxide liberating enzyme, 6-P-gluconate dehydrogenase, was not influenced by methionine, but its occurrence indicates the presence of an active pentose phosphate pathway in X. campestris. Among the other enzymes detected in X. campestris was glucose dehydrogenase. The presence of this enzyme together with hexokinase indicates the operation of two different glucose metabolizing steps: one oxidative, the other phosphorylative. Only the latter directly provides phosphorylated glucose as a precursor for the activated sugars required for xanthan synthesis.

An NADH-linked luciferase assay for glycogen: the preparation of glycogen-free, viable human eccrine sweat glands.

A glycogen assay based on bacterial NADH luciferase is described. It is free of tissue interference. The detection limit is 0.12 nmol glycogen, and the coefficient of variation is 5.5%. A method of depleting human eccrine sweat glands while retaining their viability is described. This depends on their incubation in 10(-5) M acetylcholine and 1 mM pyruvate. This method may be applicable to other tissues. The evidence for the viability of glycogen-depleted human eccrine sweat glands is reported and includes tissue contents of ATP and the rates of oxidation of glucose, pyruvate, beta-hydroxybutyrate, and palmitate.

Characterization studies of glucose dehydrogenase.

Porcine liver beta-D-glucose dehydrogenase has been isolated using Triton X-114 to release it from the endoplasmic reticulum. The purified enzyme contains a limited amount (1.7%) of lipid material, including cholesterol, fatty acids, mono and diglycerides, phosphatidylcholine, phosphatidylethanolamine, and cholesterol esters. This enzyme is a tetrameric protein containing an extensive number of hydrophobic residues. This form of glucose dehydrogenase is capable of turning over both beta-D-glucose and alpha-D-glucose-6-phosphate in vivo as indicated from a steady state kinetic analysis at 37 degrees C.