Immunological titration of rat liver glucose-6-phosphate dehydrogenase from animals fed high and low carbohydrate diets.

Recent work (Hizi and Yagil [1974] Eur. J. Biochem. $\text{45}$: 211–221, and Kelly $\text{et. al.}$ [1975] Fed. Proc. $\text{34}$: 881) suggests that the marked increase in rat liver glucose-6-phosphate dehydrogenase activity which is observed upon feeding an animal a high carbohydrate diet does not involve an increase in the total amount of enzyme present. In contrast, the data presented herein involving immunological titrations of rat liver glucose-6-phosphate dehydrogenase indicates that the increase in enzyme activity resulting from feeding a high carbohydrate diet does involve an increase in the total amount of enzyme present.     

The role of cyclic AMP in neoplastic cell growth and regression. II. Growth arrest and glucose-6-phosphate dehydrogenase isozyme shift by dibutyryl cyclic AMP

N⁶,O²′-Dibutyryl cyclic adenosine 3′,5′-monophosphate (DBcAMP) injected into rats bearing MTW9 mammary carcinoma resulted in an early disappearance of tumor microsomal glucose-6-phosphate dehydrogenase activity while mitochondrial and supernatant isozyme activities were not affected. Prolonged DBcAMP treatment of rats bearing 5123 hepatoma significantly decreased all glucose-6-phosphate dehydrogenase isozyme activities but did not alter host liver isozyme activities or liver regeneration. Since DBcAMP treatment arrested growth of these tumors, the loss of microsomal glucose-6-phosphate dehydrogenase may be an early event in the inhibition of tumor growth $\text{in vivo}$.     

An electron cytochemical study of 6-phosphogluconate dehydrogenase activity in infected erythrocytes during malaria

The distribution of 6-phosphogluconate dehydrogenase (6-PGD) was examined by an electron microscopic technique in erythrocytes of rodents infected with $\text{Plasmodium berghei}$, chickens with $\text{P. gallinaceum}$ and rhesus monkeys with $\text{P. knowlesi}$. Unlike glucose-6-phosphate dehydrogenase, which is restricted to the host erythrocyte, 6-PGD was found to be present in the parasite as well as the host erythrocyte in all infections studied. The implications of these findings are discussed in relation to the metabolism of malaria parasites.     

Thalidomide metabolite inhibits tumor cell attachment to concanavalin A coated surfaces

The inhibitory effect of drug treatment on tumor cell attachment to plastic surfaces coated with concanavalin A correlates well with the $\text{in}\text{vivo}$ teratogenicity of the drug. Using attachment as an assay, the effects of thalidomide and some of its metabolites have been examined for inhibitory activity. While thalidomide and its hydrolysis products did not alter attachment, metabolites of thalidomide produced by incubation of the drug with murine liver microsomes were inhibitory. Generation of inhibitory products required the presence of glucose-6-phosphate, NADP, glucose-6-phosphate dehydrogenase and magnesium chloride. The degree of inhibition was dependent on the duration of incubation at 37°C. These results suggest a model for the teratogenic action of thalidomide in which metabolites of the drug alter cell surface function leading to interference with normal morphogenic cell to cell interactions.     

Purification and molecular weight determination of glucose-6-phosphate dehydrogenase and malic enzyme from mouse and Drosophila.

A facile two-step procedure was employed for simultaneous purification of glucose-6-phosphate dehydrogenase and malic enzyme from mouse (strain $\text{DBA}\text{2J}$) and Drosophila melanogaster. This involved the use of an 8-(6-aminohexyl)-amino-2′,5′-ADP-Sepharsoe affinity column chromatography followed by DEAE-Sephadex chromatography. The native and subunit molecular weights of these two homogeneous enzymes were determined by gel-filtration chromatography and SDS-polyacrylamide gel electrophoresis. From this study, it was concluded that the two enzymes are tetrameric and have native molecular weights between 200,000 and 280,000 in both species.     

Quantitative requirements for NADPH in the support of aromatization by human placental microsomes

Suitable incubation conditions were developed for reduced pyridine nucleotide protection and regeneration to permit quantitative assessment of the NADPH requirement for steroid aromatization by human placental microsomes. 10 mM dithiothreitol was found to protect NADP(H) from microsomal nucleotide pyrophosphatase and 2 mM nicotinamide mononucleotide was utilized to control nucleotide glycohydrolase activity. Under these assay conditions, the initial rates of aromatization obtained with restricted NADPH levels were critically dependent upon both the amount and the source of exogenous NADPH-regenerating dehydrogenase system. With excess $\text{Leuconostoc mesenteroides}$ glucose-6-phosphate dehydrogenase, an apparent Km for NADPH of 0.20 μM was observed for aromatization which is significently below all previous estimates of the NADPH requirement and which is at greatest only one-tenth the Km value for NADPH utilization by NADPH-cytochrome $\text{c}$ reductase. These findings suggest a potential regulatory role for both NADPH-generating and NADPH-accepting enzymes in the support of estrogen biosynthesis.     

Stereoselective preparation of deuterated reduced nicotinamide adenine nucleotides and substrates by enzymatic synthesis.

A-Side (4-R)-(4-²H)-reduced nicotinamide adenine dinucleotide (NADD) was prepared by a stepwise oxidation of ethanol-d₆ to acetate in the presence of NAD, alcohol dehydrogenase, and aldehyde dehydrogenase. The B-side (4-S) isomer of NADD was prepared using the glucose dehydrogenase activity of glucose-6-phosphate dehydrogenase to oxidize to oxidize glucose-1-d in 40% dimethyl aulfoxide. Subsequent purifieation of the reduced nucleotides was achieved using a column of strongly basic polystyrene macroporous resin (AG MP-1) eluted with 0.2 m LiCl, pH 10, and applying the pooled NADD peak to a polyacrylamide gel (Bio-Gel P-2) column. The final $\text{A}{}_{260}\text{A}{}_{340}$ ratio obtained for these preparations was below 2.3. Preparation of the deuterated reduced nucleotides in this manner allows production of specifieally deuterated substrates by coupled enzymatic synthesis. L-Malate-2-d was prepared by coupled synthesis of A-side NADD to the reduction of oxaloacetate by the A-side enzyme malate dehydrogenase.     

Initial characterization of sucrose-6-phosphate hydrolase from Streptococcus mutans and its apparent identity with intracellular invertase.

An intracellular enzyme catalyzing the hydrolysis of sucrose-6-phosphate to glucose-6-phosphate and fructose has been identified in extracts of $\text{Streptococcus}\text{mutans}$ 6715-10. The preparation was purified chromatographically and found to have an apparent molecular weight of 42,000. The enzyme has as a K_m for sucrose-6-phosphate of 0.21 mM, a pH optimum of 7.1, is quite stable and requires no added cofactors or metal ions. Sucrose is a competitive inhibitor of sucrose-6-phosphate hydrolysis (K_i = 8. 12 mM). A previously described intracellular invertase copurifies with the enzyme and could not be separated from it by disc gel electrophoresis. It is concluded that intracellular invertase is a sucrose-6-phosphate hydrolase with a low catalytic activity for hydrolysis of sucrose.     

Covalent binding of proteins and glucose-6-phosphate dehydrogenase to cellulosic carriers activated with s-triazine trichloride

A method was developed for covalently binding proteins and enzymes to cellulosic carriers such that the enzymes retained high specific activity. Optimal conditions for activating the carriers with s-triazine trichloride were found to be: (a) pretreatment of cellulose with 3 m NaOH; and (b) reaction with 5% ($\text{w}\text{w}$) s-triazine trichloride in dioxane-xylene (1:1 $\text{w}\text{w}$) for 30 min at room temperature. All proteins tested bound most readily at pH values below pH 7. Extensive investigation of immobilized glucose-6-phosphate dehydrogenase showed that: (a) over 80% of the specific activity of the enzyme was retained; and (b) the pH optimum and K_m values were not altered significantly from that of the free enzyme. The binding method has been applied successfully to hexokinase, phosphorylase and pronase.     

Effects of chronic ethanol ingestion on glucose homeostasis in males and females

Liver glycogen levels were decreased about 61% in both males and females after chronic ethanol ingestion. In contrast, blood glucose concentration was decreased only in males. In an attempt to explain these data, six enzymes involved with the metabolism of glucose and glycogen were measured. Sex differences were found in the effects of ethanol on the activities of glucose-6-phosphate dehydrogenase, UDPG- pyrophosphorylase and phosphoglucomutase. Glucose-6-phosphate dehydrogenase and UDPG-pyrophosphorylase were decreased by ethanol in males 42 and 62%, respectively. Females were not changed. Phosphoglucomutase was decreased 29% in females, and males were not changed. Glucose-6-phosphatase was increased and hexokinase and phosphorylase $\text{a}$ were decreased about the same extent in both sexes by ethanol. The data suggest that both sexes have been altered in such a manner as to keep liver glucose in the unphosphorylated form and thus readily available to other tissues; however, the mechanism appears to be different between the sexes. The decrease in liver glycogen may be explained by an increased utilization of glucose by peripherial tissues; however, the lack of change in blood glucose in females argues against this.     

The transition from embryonic to adult isozyme expression in reaggregating cell cultures of mouse brain

The transition from embryonic to adult l-glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) was examined in reaggregating cell cultures of mouse brain. Culture conditions were selected that enabled viable aggregates to be produced from neural cells of mice that ranged in age from the 12th day of gestation to 6 days of postnatal age. In all cultures, an increase in the specific and total activity of l-glycerol 3-phosphate dehydrogenase was observed and this was due to an increase in the adult isozyme. The cultures from the older cells, i.e., cerebellar aggregates from mice 6 days of age, expressed approximately a 50-fold increase in activity, whereas cerebellar aggregates from newborn mice exhibited about a three-fold increase in activity. In both cases, the time course for the developmental increase followed the normal temporal sequence. The amount of l-glycerol 3-phosphate dehydrogenase activity in a cerebellar aggregate from a 6-day-old mouse was greater than 1% of the soluble cellular protein, which is about seven times higher than the specific activity determined in the cerebellums of adult mice. Alleles at the structural locus for l-glycerol 3-phosphate dehydrogenase, the Gdc-1 locus, and those alleles that control the activity levels in the cerebellums of mice were expressed in aggregates from $\text{BALB}\text{cBy}$ and $\text{C57BL}\text{6J}$ neonates. However, although the expected structural allele was expressed in all cultures, the expression of activity differences in cerebellar aggregates depended on the age of the mouse from which the cerebellar cells were isolated. Acetylcholinesterase (EC 3.1.1.7) was also measured in these aggregates; the specific activities were highest in aggregates from mice in the 16th day of gestation and least in the cerebellar aggregates of neonates, a trend that was opposite to that of l-glycerol 3-phosphate dehydrogenase.     

Establishment and characterization of a cell line from embryos of the Indianmeal moth,Plodia interpunctella

A cell line, UMN-PIE-1181, initiated in November, 1981, from embryos of a malathion-resistant strain of Indianmeal moth, Plodia interpunctella, was in the 83rd passage on January 28, 1985. The line consists of single, small, fibroblastlike cells that are polyploid with chromosome numbers ranging from 56 to 180. Growth rate is dependent on seeding density, there being no growth at or below seeding densities of $\text{2 ×}\text{10}{}^5\text{5}\text{,}\text{ml}$; optimum growth requires a fetal bovine serum concentration of at least 5%. Twenty-nine isozymes were examined. Five enzymes from the cell lines resolved well and subsequently were compared to enzymes extracted from 4-day-old embryos and other life stages of the insects. Phosphomannose isomerase, malic enzyme, malate dehydrogenase, phosphoglucose isomerase, and glucose-6-phosphate dehydrogenase in extracts from the cultured cells and from the insects had identical patterns. Two bands for glutamate-oxalacetate transaminase, present in the cell line, were not observed in the tissue extracts. Furthermore, lactate dehydrogenase from the cultured cells appeared as four bands but was not detectable in any of the samples run from the various life stages of the insects.     

Bakers' yeast uridine nucleosidase is a regulatory copper containing protein.

The enzymatic properties of homogeneous bakers' yeast uridine nucleosidase, prepared as previously described (G. Magni et al., J. Biol. Chem. 1975 $\text{250}$, 9–13) have been further investigated, and in addition to glucose-6-phosphate and ribose the enzyme activity was inhibited by ribose-5-phosphate and ribulose-5-phosphate. The curves describing this inhibitions were sigmoidal and when the data were plotted according to Hill, n′ values different from 1 were observed suggesting the existence of interactions among the inhibitory molecules binding sites. Furthermore the percentage of inhibition exerted by glucose-6-phosphate, ribose and ribose-5-phosphate on the enzyme activity varied at different pH values. The addition of various chelating agents to the activity assay mixture caused a strong inhibition of the enzyme activity and metal analysis by atomic absorption spectrophotometry, colorimetric methods and electronic paramagnetic resonance, indicated the presence of 1 copper atom per enzyme molecule. Finally the inhibition exerted by metal ions on the enzyme activity was described.     

Evidence that ATP exerts control of the rate of glucose utilization in the intact Escherichia coli cell by altering the cellular level of glucose-6-P, an intermediate known to inhibit glucose transport in vitro

The effects of treating nitrogen-starved cultures of $\text{Escherichia coli}$ W4597 (K) with various doses of 2,4-dinitrophenol include increases in the rates of glucose utilization, decreases in ATP and glucose-6-P and maintenance of the level of fructose-1, 6-P₂. A quantitative correlation was observed between the increases in the rates of glucose utilization and decreases in glucose-6-P in agreement with the observation made $\text{in vitro}$ that glucose-6-P inhibits glucose transport in $\text{E. coli}$. A quantitative correlation was also observed between glucose-6-P and ATP indicating that the fall in glucose-6-P is effected by the fall in ATP which indirectly signals increased glucose utilization and increased ATP production.     

Synthesis and antiestrogenic activity of 2,4-bis(bromomethyl)-estrone methyl ether1: A bifunctional alkylating steroid

The synthesis of 2,4-bis(bromomethyl)estrone methyl ether was accomplished by reacting estrone methyl ether with formaldehyde and hydrogen bromide in s-tetrachloroethane. The compound readily reacts with cysteine, tryptophan and histidine in 0.05 $\text{M}$ phosphate buffer pH 7.0. It also reacts with compounds containing sulfhydryl groups, such as 2-mercaptoethanol and the reduced form of 5,5'dithiobis(2-nitrobenzoic acid). Structural analysis and stoichiometry of the reaction of the bromomethylsteroid with cysteine indicates that the title compound contains two reactive bromomethyl groups. The biological activity of the bromomethylsteroid was evaluated by analysis of its effect on uterine glucose-6-phosphate dehydrogenase activity after administration into the uterine lumen of ovariectomized rats. While it lacked estrogenic activity, the compound did display an antiestrogenic effect that was both greater and more persistent than that of an equimolar quantity of estrone methyl ether.     

Increased synthesis of L-glycerol 3-phosphate dehydrogenase during in vitro differentiation of reaggregating cerebellar cells

Reaggregating cell cultures of neonatal mouse cerebellar cells express many of the differentiated properties of normal developing cerebellum, including the transition for the embryonic and adult isozymes of l-glycerol 3-phosphate dehydrogenase (EC 1.1.1.8). In order to determine the mechanism leading to increased levels of adult isozyme, aggregates in culture from 2 to 17 days were labeled with radioactive leucine and the relative rate of enzyme synthesis was measured after purification of the enzyme by affinity chromatography on Blue Sepharose 6B. During the course of in vitro differentiation, the relative rate of synthesis increased 100-fold, such that it represented 0.5% of the total protein synthesized in the cytoplasmic fraction of the cell. In vivo, $\text{BALB}\text{cBy}$ mice have twice the level of enzyme activity in the cerebellum as do $\text{C57BL}\text{6J}$ mice. Reaggregating cell cultures of cerebellar cells from these strains of mice also express a difference in the activity level, but only when the cerebellar cells are taken from mice 4 days of age or less. When the relative rates of synthesis of l-glycerol 3-phosphate dehydrogenase were measured in cultures expressing the strain-dependent difference in activity, these rates were found to be approximately twofold greater in cultures of $\text{BALB}\text{cBy}$ cells. In contrast, estimates of the relative rate of enzyme degradation by the double-isotope labeling technique indicate that neither specific enzyme degradation nor degradation of total protein is different in aggregates from the two strains of mice. The results suggest that the genetic mechanisms controlling the levels of l-glycerol 3-phosphate dehydrogenase in the cerebellum during development are intrinsic to the cells and, with the exception of serum factors, are independent of systemic influences.     

Evidence for an allosteric control of the pentose phosphate pathway in Candida utilis

Glucose 6-phosphate dehydrogenase from $\text{Candida utilis}$ exists in two interconvertible species having molecular weights of 110,000 and 220,000, respectively. By use of sodium dodecyl-sulfate the enzyme can be dissociated into 2 electrophoretically separable subunits. The native enzyme was shown to be strongly inhibited by low concentrations of erythrose 4-phosphate and glyceraldehyde 3-phosphate, respectively. Since both these sugar phosphates are metabolites in the later course of the oxidative pathway this regulation may be considered as a negative feedback control of the pentose phosphate cycle.     

The stereospecificity of D-glucose-6-phosphate: 1L-myo-inositol-1-phosphate cycloaldolase on the hydrogen atoms at C-6

D-Glucose-6-phosphate: 1L-$\text{myo}$-Inositol-1-phosphate cycloaldolase from rat testis or mammary gland removed stereospecifically the pro-S hydrogen atom at C-6 from D-glucose-6-phosphate. The pro-R hydrogen at C-6 remained in the product, 1L-$\text{myo}$-Inositol-1-phosphate and evidence is given that it is the hydrogen at C-1 of 1L-$\text{myo}$-Inositol-1-phosphate. The possible mechanism of cyclization is discussed.     

Cyclic AMP inhibition of reactions of glyceraldehyde 3-phosphate dehydrogenase

Adenosine 3′,5′-monophosphate (cyclic AMP) is an inhibitor of the reaction of d-glyceraldehyde 3-phosphate dehydrogenase with glyceraldehyde 3-phosphate and benzaldehyde. Inhibition appears to be competitive toward glyceraldehyde 3-phosphate and of a mixed type toward NAD⁺. In the absence of arsenate a plot of $\text{1}\text{V}$ vs (I) is sigmoidal at constant concentrations of glyceraldehyde 3-phosphate and NAD⁺ and linear at constant concentrations of benzaldehyde and NAD⁺. Thus, sigmoidal inhibition plots are dependent on the nature of the aldehyde substrate as was found previously to be the case with inhibition of these reactions by highly branched acyl phosphates. In the presence of 0.013 m arsenate the plots of $\text{1}\text{V}$ vs [I] are linear.     

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).