Grace Han

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.

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.

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.

ELEVATION OF INTRACELLULAR CA2+ CONCENTRATION IN RABBIT NONPIGMENTED CILIARY EPITHELIAL CELLS BY ALLICIN

A previous study has shown that allicin produces changes in aqueous humor dynamics, and this study was conducted to examine possible cellular mechanisms. In rabbit nonpigmented ciliary epithelial cells, basal levels of [Ca2+]i were determined to be 164 +/- 34 nM. Allicin, a sulfhydryl-reactive agent, induced Ca2+ transients at 0.01 mM and at 0.2 mM, the Ca2+ transient peaked at 732 +/- 35 nM. Allicin-induced Ca2+ transients were prevented by pretreatment with dithiothreitol which did not affect the basal Ca2+ levels. Allicin had only a slight, insignificant, effect on L-type Ca2+ currents, and allicin-induced Ca2+ transients were also present under extracellular Ca(2+)-free conditions. These data suggest that intracellular Ca2+ stores are the most probable source of allicins effect. Pretreatment of cells with ryanodine, an inhibitor of Ca(2+)-induced-Ca(2+)-release, inhibited allicin-induced Ca2+ transients, but the basal Ca2+ levels were unaffected by ryanodine. Thus, allicin-induced Ca2+ transients are most likely mediated through ryanodine-sensitive intracellular Ca2+ stores.

Evidence for heat-stable liver cytosol substance(s) capable of causing oxidative activation of fructose 1,6-bisphosphatase.

The endogenous fructose 1,6-bisphosphatase (FBPase) in chicken liver extract undergoes a drastic increase in activity if the pH of the extract is in the alkaline range. Greater and more consistent activation occurs when purified FBPase, placed inside dialysis sack, is incubated in liver extract. Maximal activation (over 16-fold) is accompanied by the disappearance of 4 highly reactive sulfhydryl groups (SH) per molecule of enzyme. The activating effect of the extract remains essentially unchanged after heating to 100 degrees C. Activation can be reversed by dithiothreitol. These data show the existence in liver cytosol of heat-stable substance(s) capable of activating FBPase presumably by forming disulfide bonds with the enzymes highly reactive SH groups.

Immobilization of chicken liver fructose 1,6-bisphosphatase on CNBr-activated sepharose

Chicken liver fructose 1,6-bisphosphatase is readily immobilized on CNBr-activated Sepharose. The immobilization alters some enzymatic properties. They include broader pH activity curve, loss of activation by K+ or NH 4 + , increased resistance to inactivation by trypsin, decreased sensitivity to AMP inhibition, and loss of cooperative interaction among AMP-binding sites. The immobilized enzyme retains about 38% or 19% of the specific activity of the native enzyme when the activity is measured in the absence or presence of K+, resepctively.

Use of thiopropyl sepharose for preparation of 2-nitro-5-thiobenzoic acid

Abstract A simple method is described for preparation of 2-nitro-5-thiobenzoic acid (NTB), a compound with versatile applications but is commercially unavailable. In this method, NTB is produced by the reduction of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) with thiopropyl sepharose, which is then separated from NTB by filtration. Two molecules of NTB can be generated from each molecule of DTNB when a large excess of thiopropyl sepharose is used. Thiopropyl sepharose is commercially available and can be readily regenerated for repeated use in the preparation of NTB. This method is simple and more effective than any other reported methods of NTB preparation.