Identification and purification of a liver microsomal glucose 6-phosphatase.

1. Hepatic glucose 6-phosphatase activity was purified 65-fold in good yield over that in cholate-solubilized microsomal fractions. 2. This preparation still contained five major polypeptides and numerous minor contaminants. 3. The smallest of the five major polypeptides (Mr approx. 18 500) could be purified from heat-treated microsomal fractions. 4. Antisera raised against the heat-stable protein doublet was used to immunoprecipitate specifically glucose 6-phosphatase activity from cholate-solubilized microsomal fractions. 5. This work indicates that hepatic microsomal glucose 6-phosphatase appears to be one or both of the low-molecular-weight heat-stable polypeptides.     

Tautomeric energetics of xanthine oxidase substrates: xanthine, 2-oxo-6-methylpurine, and lumazine

The stability of the tautomers of each of the three important substrates of xanthine oxidase, xanthine, 2-oxo-6-methylpurine, and lumazine, was examined by quantum mechanical calculations. The geometries of these tautomers were optimized at the AM1, Hartree-Fock (HF/6-31G), and hybrid Hartree-Fock/density functional theory (B3LYP/6-31G(d)) levels of theory. The single point energies of some of the more stable tautomers for each of the substrates were calculated at the B3LYP/6-311 +G(2d,p) level of theory. The Conductor Polarized Continuum Model (CPCM) was used to evaluate the solvent effects on the relative stabilities of these tautomers. The calculations clearly identify the lowest energy tautomeric form for xanthine and lumazine. On the other hand, there appear to be three tautomers for 2-oxo-6-methylpurine, with only minor energetic differences in vacuo. In water, however, only one of them predominates. The lowest energy tautomers presumably represent the predominant tautomeric forms at the molybdenum center of xanthine oxidase during catalysis. Implications of these computational results are discussed in the context of enzyme catalysis.     

Reversibility of lysosomal and glucose 6-phosphatase changes produced in the rat liver by dimethylnitrosamine

The present investigation was undertaken to discover whether repeated doses of dimethylnitrosamine (DMNA) could produce a cumulative toxic effect on the rat liver. For this purpose doses were selected at a level just too low to produce cytopathological changes, as indicated by depression of glucose-6-phosphatase and induction of autophagic vacuoles (AV) in hepatocytes, when given once only. Single subcutaneous injections of 10 or 3 mg/kg induced these cytopathological changes in the centrilobular (CLB) hepatic cells but when the dose was reduced to l mg/kg no such changes were seen.

After daily administration of l mg/kg for 4 or 8 weeks we observed both glucose-6-phosphatase depression and autophagy, and in addition there was marked hypertrophy of the rough endoplasmic reticulum, nucleolar microsegregation and the appearance of distorted, often ring-shaped mitochondria with shortened cristae. Kupffer cells exhibited a marked increase in lysosomal activity. With the exception of mitochondrial changes and Kupffer cell activity this same picture was observed, although in milder form, when the dose administered was 0.3 or 0,1 mg/kg daily for the same period. When treatment was continued for 12 weeks, however, the only differences from control rats were the presence of hypertrophied rough endoplasmic reticulum (RER) at all three dose levels, nucleolar microsegregation at the upper two dose levels, and pronounced Kupffer cell activity at the top dose. These findings indicate that cumulative cytopathologic effects occur only up to 8 weeks at the dose levels studied but hypertrophy of RER and increased Kupffer cell activity persist up to 12 weeks.     

Heterogeneous distribution of glucose 6-phosphatase in rat liver microsomal fractions as shown by adaptation of a cytochemical technique

1. A novel technique for the subfractionation of rat liver smooth and rough microsomal fractions according to their content of glucose 6-phosphatase is described. This technique, based on the Gomori lead histochemical procedure, involves incubation of smooth and rough microsomal fractions with low concentrations of Pb(NO(3))(2) and glucose 6-phosphate. Control experiments, in which enzyme was assayed in the presence of various amounts of Pb(NO(3))(2) or in which microsomal fractions were reisolated after incubation with low concentrations of Pb(NO(3))(2) and glucose 6-phosphate, showed that lead does not interfere with glucose 6-phosphatase activity. 2. Discontinuous sucrose-density-gradient centrifugation of microsomal fractions which had previously been incubated with various amounts of Pb(NO(3))(2) and glucose 6-phosphate showed that it is possible to subfractionate both smooth- and rough-microsomal fractions into several bands, owing to a differential modification of the density of the microsomal vesicles by the trapping of lead phosphate within them. 3. When the material in the bands obtained by density-gradient centrifugation of incubated microsomal fractions was assayed for glucose 6-phosphatase activity, it was found that the modification of the density of the microsomal fractions was directly related to their relative enrichment in glucose 6-phosphatase activity. Control experiments, in which microsomal fractions were incubated with Pb(NO(3))(2) and glucose 6-phosphate and then treated with EDTA, showed that the subfractionation was not due to aggregation of microsomal vesicles, lead and glucose 6-phosphate. Thus the resolution of microsomal preparations into subfractions with different glucose 6-phosphatase activities is interpreted as indicating heterogeneity of glucose 6-phosphatase distribution in the microsomal vesicles. 4. Electron micrographs of both smooth- and rough-microsomal subfractions show deposits of lead phosphate within the microsomal vesicles. The frequency and extent of these deposits correlate with the different amounts of glucose 6-phosphatase activity measured biochemically. 5. The nature of the heterogeneous distribution of glucose 6-phosphatase is discussed and the more general applicability of the technique for studying membrane fractions containing a heterogeneous distribution of phosphatases is indicated.     

Inhibition of microsomal glucose 6-phosphatase by unsaturated aliphatic aldehydes and ketones.

Aldehydes and ketones with one double bond conjugated to the carbonyl group inhibited the enzyme glucose 6-phosphatase, which is embedded in the microsomal membrane. The Michaelis constant, Km and the maximal rate of reaction, V, were affected in a way dependent on the inhibitor's chain-length: trans-2-pentenal and 1-penten-3-one increased Km linearly with concentration and had almost no effect on V, whereas trans-2-nonenal caused a large increase in V but only a small and non-linear change in Km. The effect of the short-chain aldehydes on the kinetic parameters increased with chain-length, but pentenone increased Km more than did trans-2-heptenal and conjugated dienals did not act as inhibitors. Therefore, sterical effects apparently are of importance. Washing the microsomes after incubation with hexenal or heptenal did not substantially decrease the inhibition, but with nonenal the inhibition was reduced by washing. Inhibition by the SH-group blocking reagent p-hydroxymercuribenzoate was competitive to inhibition by the alkenals. It is concluded that the alpha-beta unsaturated oxo-compounds inhibit glucose 6-phosphatase by binding covalently to an important mercapto group and that perturbation of the enzyme's membrane environment also plays a part in the inhibition.     

Histo-chemical mapping of phosphomonoesterases in the gonads of Notopterus notopterus and Colisa fasciatus during different seasons. Part II. Adenosine triphosphatase and glucose-6-phosphatase

Histochemical techniques described by McManus (1960) have been applied in the fishes, Notopterus notopterus and Colisa fasciatus, for the study of Glucose-60phosphatase and adenosine triphosphatase in the four stages of gonads in different seasons. It has been observed that the activity of adenosine triphosphatase is more intense in comparison to the activity of Glucose-6-phosphatase in all the stages i.e. I (immature), II (maturing), III (mature) and IV (spent) of the gonads in both the fishes. The general tendency of the adenosine triphosphatase and Glucose-6-phosphatase distribution in the gonads are much more remarkable in stage II in comparison to stage I, III and IV. The stage I seems to be the stage of synthesis of these enzymes. In stage III and IV, these enzymes show the tendency of declination with the time period. The possible role of these enzymes seems to be the transport of glucose across the cell membrane involving phosphorylation and dephosphorylation which depend on the different stages of gonad maturation.     

In vitro effects of corticosteroids, DDT, and 4, 9-dichlorodibenzodioxin on rat liver xanthine oxidase activity. Interactions between xanthine oxidase and cytochrome P450 in rat liver in vivo

It was established that deoxycorticosterone, cortisol, dexamethasone, DDT, and 4,9-dichlorodibenzodioxin inhibit in vitro binding of xanthine to highly purified rat liver xanthine oxidase. They are suggested to be allosteric inhibitors. The corresponding inhibition and binding constants were estimated. Also established was that cortisol and DDT, in dose 20 mg per kg of weight, inhibit xanthine oxidase and increase microsomal cytochrome P450 level in rat liver. There is an inverse relationship between xanthine oxidase activity and cytochrome P450 level in rat liver.