Darold Holten

Relationships among the multiple molecular forms of rat liver glucose 6-phosphate dehydrogenase

Abstract 1. 1.|An improved procedure is described for the purification of rat liver glucose 6-phosphate dehydrogenase( d -glucose 6-phosphate:NADP + oxidoreductase, EC 1.1.1.49). 2. 2.|The active enzyme is formed from inactive subunits of mol. wt. 64 000. 3. 3.|The purified enzyme exists in at least three different dimeric forms of mol. wt. 130 000 which account for 90% of the total activity. These dimers can then aggregate to form tetrameric and hexameric forms which account for almost all of the remaining activity. 4. 4.|Each of the dimeric forms of the enzyme shows immunological identity with the other dimeric forms and with the higher molecular weight aggregates of these dimers when using double diffusion plates and an antiserum against the purified rat liver enzyme.

Regulation of pentose phosphate pathway dehydrogenases by NADP+/NADPH ratios.

Abstract Equilibrium dialysis indicates that rat liver glucose-6-P dehydrogenase binds two molecules of NADP + per subunit with a dissociation constant of 0.6 × 10 −6 M. The NADP + free enzyme will not bind glucose-6-P indicating a compulsory order of substrate binding. Development of an isotopic assay allowed a direct measurement of the effect of physiological alterations in the NADP + /NADPH ratio on the activity of glucose-6-P and 6-phosphogluconate dehydrogenases. A combination of enzyme induction and altered NADP + /NADPH ratios could produce 30–50 fold changes in the capacity of these enzymes to produce NADPH during alterations in the nutritional state of the animal.

The effect of unsaturated fatty acids on the rate of synthesis of rat liver glucose-6-phosphate dehydrogenase

Abstract 1. 1. Rat liver glucose-6-phosphate dehydrogenase has been used to test the hypotheses that unsaturated fatty acids inhibit this enzyme in vivo or act as co-pressors regulating the synthesis of lipogenic enzymes. When rats are switched from a non-fat diet to one containing 15% fatty acid there is an 8-fold decrease in the level of glucose-6-phosphate dehydrogenase. Quantitative precipitation of the enzyme with an antiserum against glucose-6-phosphate dehydrogenase established that this 8-fold decrease was due to a decrease in the amount of enzyme protein rather than an inhibition of preexisting enzyme. 2. 2. A kinetic method was used to determine the rate of glucose-6-phosphate dehydrogenase synthesis and degradation in the presence of dietary fatty acids. It is concluded that dietary fatty acids decrease the rate of synthesis of glucose-6-phosphate dehydrogenase. Alterations in the levels of glucose and fatty acid in the diet lead to the conclusion that fatty acids act indirectly by decreasing the appetite. These results do not support the hypothesis that polyunsaturated fatty acids act as co-repressors to regulate the synthesis of lipogenic enzymes.

The effect of dietary carbohydrate and fat on the synthesis of rat liver 6-phosphogluconate dehydrogenase.

Abstract 1. 1. A kinetic method was used to estimate the rates of 6-phosphogluconate dehydrogenase synthesis and degradation in vivo in rats fed diets containing various levels of carbohydrate and fat. 2. 2. Switching rats from a commercial pellet diet to a synthetic diet containing 60% carbohydrate resulted in a io -fold increase in the rate of synthesis and a 6-fold increase in the rate of degradation of this enzyme. 3. 3. The extent of this induction and the increase in the rate enzyme synthesis is correlated with the caloric consumption of carbohydrate. 4. 4. Insulin and dietary fat increase and decrease, respectively, the rate of enzyme synthesis without effecting the rate of enzyme degradation and both appear to act indirectly by affecting the appetite.

Effect of dietary cholesterol on apolipoprotein E synthesis in the rabbit

Female New Zealand White rabbits were fed either rabbit chow or rabbit chow plus 1% (w/w) cholesterol for 14 days. The chow-fed rabbits had normal plasma lipoprotein profiles on agarose gel electrophoresis, 59 +/- 5 mg of cholesterol and 5.5 +/- 0.4 mg of apolipoprotein E (apoE) per dl of serum. The cholesterol-fed rabbits had significant amounts of beta-VLDL in their serum, 1870 +/- 140 mg of cholesterol and 96 +/- 12 mg of apoE per dl of serum. Relative rates of apoE synthesis were determined by incubating hepatocytes in culture medium containing [3H]leucine for 15 min at 37 degrees C and expressing the radioactivity incorporated into immunoprecipitable apoE as a percentage of the radioactivity incorporated into total protein. Hepatocytes from cholesterol-fed rabbits had twice the relative rate of apoE synthesis (1.05 +/- 0.18%) of hepatocytes from chow-fed rabbits (0.55 +/- 0.07%). This increase in synthesis could be a major contributor to the 17-fold increase in serum apoE levels in the cholesterol-fed rabbit.

Regulation of glucose-6-P dehydrogenase activity in primary rat hepatocyte cultures

Abstract The glucose-6-P dehydrogenase specific activity in rat hepatocytes increases approximately 10-fold when the cells are placed into culture for three days. The induction requires insulin with maximum enzyme levels occurring at 10 −7 M. Pulse-labeling experiments revealed a 10-fold increase in the enzymes relative rate of synthesis after only 8 hours in culture.

Quantitation of apolipoprotein E in rabbit sera with a competitive enzyme-linked immunosorbent assay.

A competitive enzyme-linked immunosorbent assay was developed to quantitate apoE levels in normal and cholesterolemic rabbit serum. The assay can detect 1 ng of apoE and has an interassay coefficient of variation of 5.1%. The assays antigen specificity was established by the generation of a competitive displacement curve with rabbit apoE, but not with rabbit albumin nor with rabbit apoC. Nonimmune serum was not able to produce a detectable response in the assay. Lipid-protein interactions did not interfere with the assay and color development was linear throughout the incubation time. Rabbits fed a normal diet had 5.3 +/- 0.4 mg of apoE/dl serum. Rabbits fed a diet containing 1% (w/w) cholesterol for 14 days had 86 +/- 11 mg of apoE/dl serum.

Kinetics for changes in enzyme synthesis and mRNA content and hormones required for induction of 6-phosphogluconate dehydrogenase in hepatocytes

Rats fasted for 2 days were refed a 60% glucose diet for varying periods of time in order to follow the kinetics for changes in 6-phosphogluconate dehydrogenase synthesis and mRNA content. Hepatocytes isolated from control or induced rats were incubated with actinomycin D and the rate of decline in 6-phosphogluconate dehydrogenase mRNA was determined by translating RNA in a nuclease-treated reticulocyte lysate. The half-life for 6-phosphogluconate dehydrogenase mRNA under both of these conditions was about 2 h. Thus, increases in transcription or the processing of nuclear RNA may increase 6-phosphogluconate dehydrogenase mRNA during the dietary induction of this enzyme. Hepatocytes prepared from fasted rats were cultured with 5% serum and various hormones and energy sources. If hepatocytes were isolated from thyroidectomized rats and cultured in serum from a thyroidectomized calf, the 4-fold induction of 6-phosphogluconate dehydrogenase was primarily dependent upon added insulin. In the presence of optimal insulin concentrations (10(-7) M) triiodothyronine slightly stimulated 6-phosphogluconate dehydrogenase induction. The gut hormones somatostatin and secretin had no effect on 6-phosphogluconate dehydrogenase induction in cultured hepatocytes. Hepatocytes cultured in carbohydrate-free medium and 5% serum required added insulin for maximal induction. 8-Br-cGMP did not significantly affect 6-phosphogluconate dehydrogenase induction in hepatocytes either in the presence or absence of added insulin. Dibutyryl cAMP did not alter the time course or extent of 6-phosphogluconate dehydrogenase induction in cultured hepatocytes. We have concluded that under these conditions insulin is a potent signal regulating the levels of 6-phosphogluconate dehydrogenase mRNA and that this induction is not mediated by cyclic nucleotides.

Regulation of glucose-6-P dehydrogenase synthesis in rat epididymal fat pads☆

Abstract The relative rate of synthesis of glucose-6-P dehydrogenase increases up to 8-fold when fasted rats are fed a 60% carbohydrate, fat-free diet for 3 days but the specific activity of the enzyme only increases 2 to 3 fold. This suggests that the high carbohydrate diet also causes a 2 to 3 fold increase in the rate of glucose-6-P dehydrogenase degradation. The nutritional induction of this enzyme in adipose tissue is primarily due to a large increase in the rate of its synthesis.

Solution hybridization quantitation of G6PD mRNA in rat epididymal fat pads

A solution hybridization assay is systematically characterized and used to quantitate glucose-6-phosphate dehydrogenase (G6PD) mRNA from epididymal fat pads in fasted and glucose-induced rats. G6PD mRNA and specific activity increase 9-fold and 2-fold, respectively. The 9-fold increase in G6PD synthesis reported previously (Wolfe et al. (1979) Biochem. Biophys. Res. Commun. 89, 108-115) can, therefore, be accounted for by the increase in G6PD mRNA. This solution hybridization assay is sensitive enough to quantitative levels of G6PD mRNA in total liver RNA from a fasted rat, one of the least abundant sources of this mRNA. It can, therefore, be used to answer several questions about the regulation of G6PD synthesis in rat tissues. Preliminary results suggest that the dietary regulation of G6PD mRNA in rat liver is much larger than previously reported.