Peter Han

Modulation of hepatic glucose-6-phosphate dehydrogenase activity in male and female rats by estrogen

The effect of estradiol-17 beta on the activity of glucose-6-phosphate dehydrogenase was studied in both male and female rats to further characterize the sex differences in the activity of this enzyme. Four groups of intact and castrated rats were implanted subcutaneously with graded doses (2.4, 4.8 and 7.2 micrograms/day) of pelleted estradiol in a physiologically relevant experimental system. After fourteen days the rats were sacrificed and their livers were assayed for G6PD activities. The result indicated that: (i) the enzyme activity was 3-fold higher in normal adult female than in male rats, (ii) low doses of E2 (2.4, 4.8 and 7.2 micrograms/day) increased the activity of G6PD 6-fold in castrated males and over 2-fold in female castrates as well as intact rats (iii) E2 stimulation of G6PD activity appears to be more effective in castrated males than in female rats (IV) sex difference in the activity of G6PD disappeared after treatment with E2 in castrated rats. It is concluded that the activity of G6PD in rats is markedly enhanced by low doses of E2, which appears to be largely responsible for the sex differences in the activity of this enzyme in rats.

Effect of substrate and AMP on the reversal of Zn2+ inhibition of turkey liver fructose-1,6-bisphosphatase by chelators

Abstract The ability of chelators to reverse Zn 2+ inhibition of turkey liver fructose-1,6-bisphosphatase decreases greatly if substrate is first bound to the enzyme. If AMP is also present, chelators become almost completely incapable of reversing Zn 2+ inhibition when added to the enzyme after substrate. These observations indicate that the prior binding of substrate to this fructose-1,6-bisphosphatase hinders the removal of Zn 2+ from the inhibitory sites of the enzyme by chelators, especially when AMP is also present. We have also found that the initial rates of the Zn 2+ -inhibited enzyme activity show a peculiar nonlinearity and the inhibitory effects of Zn 2+ and AMP are synergistic.

Inactivation of yeast glucose-6-P dehydrogenase by aspirin

Glucose-6-P dehydrogenase is irreversibly inactivated by treatment with Na salts of aspirin. Kinetic data show that 1 molecule of aspirin reacts with each active unit when the enzyme is inactivated. The rate of inactivation is enhanced with increasing pH but is reduced in the presence of glucose-6-P or NADP+. Na salicylate fails to inactivate the enzyme.

Inhibition of liver fructose 1,6-bisphosphatase activity by Zn2+: Reversal by imidazole pyruvate

Imidazole pyruvate was found to be a very potent natural chelating agent in reversing the inhibition of liver fructose 1,6-bisphosphatase activity by Zn2+. This metabolite may play a physiological role in gluconeogenesis.

Alteration of the regulatory properties of chicken liver fructose-1,6-bisphosphatase by treatment with aspirin.

Abstract Treatment of chicken liver fructose-1,6-bisphosphatase with aspirin at pH 7.8 desensitizes the enzyme toward inhibiiton by AMP. This treatment also reduces the enzyme affinity for substrate and the enzyme sensitivity to high substrate inhibition. These altered properties remain essentially unchanged even after removal of nearly all aspirin by dialysis or ultrafiltration. They are also stable to hydroxylamine. Salicylate is ineffective in inducing these altered properties. Both the reduced affinity for substrate and the reduced sensitivity to high substrate inhibition are largely prevented by substrate, while AMP specifically prevents the desensitization to allosteric inhibition by AMP.

A simple method for removal of AMP contamination from NADP+ for the assay of muscle fructose 1,6-bisphosphatase.

Abstract Muscle d -fructose 1,6-bisphosphatase 1-phosphohydrolase (Fru-P 2 ase) (EC 3.1.3.11) is extremely sensitive to inhibition by AMP, and even a trace contamination of this inhibitor in the assay mixture may result in a significant underestimation of the enzyme activity (1,2). Currently, Fru-P 2 ase activity is most commonly assayed by measuring the rate of NADP + reduction in a coupling system (3). It was reported that commercial preparations of NADP + contained varying amounts of AMP (4). Therefore, in the process of assaying muscle Fru-P 2 ase using NADP + , extreme care must be taken to ensure that the NADP + used is free of AMP. The procedure for purification of NADP + previously described by Horecker and Kornberg (5) has been successfully employed by some researchers to remove AMP from commercial NADP + for the assay of muscle Fru-P 2 ase (2,6). In this paper we describe a much simpler method for the removal of contaminating AMP from commercial NADP + for use in the assay of this muscle enzyme.

Synergistic effect of AMP and fructose 2,6-bisphosphate on the protection of fructose 1,6-bisphosphatase against inactivation by trypsin

The rate of inactivation of chicken liver fructose, 1,6-bisphosphatase by trypsin is reduced if the digestive reaction is conducted in the presence of AMP or fructose 2,6-bisphosphate. The effects of these 2 compounds are synergistic. Although fructose 1,6-bisphosphate does not protect the enzyme against tryptic inactivation, it can enhance the effect of AMP. Selective modification of the AMP allosteric site of fructose 1,6-bisphosphatase with pyridoxal-P and NaBH4 renders the enzyme more resistant to tryptic inactivation, but the modified enzyme is no longer responsive to the protective effect of AMP.

Adenosine 5′-monophosphate-removing system in fructose-1,6-bisphosphatase assay mixture: A new approach

Abstract A simple new AMP-removing system for inclusion in the assay mixture of fructose-1,6-bisphosphatase is described. It consists of either AMP deaminase or 5′-nucleotidase, depending on the pH at which the activity of fructose-1,6-bisphosphatase is measured. This system takes advantage of the fact that AMP deaminase and 5′-nucleotidase irreversibly convert AMP to IMP and adenosine, respectively, both being inert to fructose-1,6-bisphosphatase. As compared with the widely used AMP-removing system of W. J. Black, A. Van Tol, J. Fernado, and B. L. Horecker (1972, Arch. Biochem. Biophys. 151 , 576–590), this new system is simpler and more versatile in application.

Activation of chicken liver fructose-1,6-bisphosphatase by oxidized glutathione

Treatment of chicken liver fructose‐1,6‐bisphosphatase with oxidized glutathione (GSSG) leads to an increase in activity. This activation is markedly enhanced if treatment is performed in the presence of AMP or Mn2+. The effects of AMP and Mn2+ appear to be synergistic. The maximal activation is over 13‐fold and is accompanied by the disappearance of 4 sulfhydryl groups per molecule of enzyme. Both fructose 1,6‐bisphosphate and fructose 2,6‐bisphosphate can largely prevent this activation. Activation can be reversed by dithiothreitol or cysteine. It appears that GSSG activates this enzyme by thiol/disulfide exchanges with the enzymes specific sulfhydryl groups.

Spin label studies of the essential sulfhydryl group environment in chicken liver fructose-1,6-bisphosphatase

The local environment of the essential sulfhydryl groups in chicken liver fructose‐1,6‐bisphosphatase has been investigated by ESR techniques using a series of iodoacetamide spin labels, varying in chain length between the iodoacetate and nitroxide free radical group. The ESR spectrum of spin‐labeled chicken liver fructose‐1,6‐bisphosphatase showed that the sites of labeling were highly immobilized when the enzyme was chemically modified by spin label iodoacetate, suggesting that the sulfhydryl groups of the protein are in a small, confined environment. From the change in the ESR spectra of these nitroxides as a function of chain length, we conclude that the sulfhydryl group is located in a cleft approx. 10.5 Å in depth.