Doctoral Dissertations

Sung-Woo Cho

Date of Award

12-1988

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major Professor

J.G. Joshi

Abstract

This work reports studies of the effects of aluminum on glucose-6-phosphate dehydrogenase (EC 1.1.1.49) in yeast, and in human, pig, and rat brains. Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme in the pentose phosphate pathway. It catalyzes the conversion of glucose- 6-phosphate to 6-phosphoglucono-δ-lactone.

Male Sprague-Dawley rats (10-12 weeks old) were fed with 0.1 mM AICI3 in drinking water. After one year, the animals, as well as controls (those fed with tap water), were killed by decapitation and brains were dissected and homogenized in 0.1 M Tris/HCl, pH 7.4. The 800 x g supernatants were assayed for aluminum and the activities of acetylcholine esterase (AChE), hexokinase, and glucose- 6-phosphate dehydrogenase (G6PD). The concentration of aluminum in the homogenates were 40 ng and 80 ng/g wet weight, respectively. The activities of AChE were the same as in both groups but those of hexokinase and G6PD in the aluminum-fed group were about 73% and 70% of the control, respectively. Dialysis of the homogenates restored the activities of G6PD of the aluminum-fed group without changing those of the control group. However, the activities of hexokinase of both the aluminum-fed group and the control group increased by dialysis, 2.7 and 2 fold, respectively. Therefore, at this elevated level, the activities of hexokinase of the aluminum-fed group were the same as those of the control.

The concentration of aluminum during enzyme assay even for the undialyzed homogenates was too low to account for the inhibition of hexokinase. It is, therefore, suggested that dialyzable inhibitor(s) for hexokinase is normally present in rat brain and that the concentration of the inhibitor may be increased further in aluminum-fed group to inhibit the utilization of glucose.

To investigate the inhibitory effects of aluminum on G6PD, the yeast enzyme was chosen because several aspects of this enzyme was well studied. Preincubation of yeast G6PD with AICI3 produced an inactive enzyme containing 1 gram atom of aluminum per mol of enzyme subunit. None of the bound aluminum was dissociated by dialysis against 10 mM Tris/HCl, pH 7.0 with or without 0.2 mM EDTA at 4 °C for 24 h. Citrate, NADP+, EDTA, or NaF protected the enzyme against the aluminum inactivation. The aluminum-inactivated enzyme, however, was completely reactivated by citrate or NaF, and only partially by NADP"^ or malate. Modification of the histidine and lysine residues of the enzyme with diethyl pyrocarbonate and acetylsalicylic acid. respectively, inactivated the enzyme. However, the modified enzyme still bound 1 g atom of aluminum per enzyme subunit. KD value for G6PD-aluminum complex was calculated to be 4 uM using NaF. Circular dichroism studies showed that the binding of aluminum to the enzyme induced a decrease in α-helix and β-sheet and a increase in random coil. Therefore it is suggested that aluminum binding induces a conformational change unfavorable to enzyme activity.

Encouraged by these data, these studies were extended to G6PD from brain. This enzyme has been detected in the brain but it has not been purified. As a consequence, nothing is known about its molecular properties. Therefore a new procedure was developed for the purification of the brain G6PD. Human and pig brain homogenates were electrophoresed by PAGE and stained for G6PD activity. Five distinct bands were visible. Isozymes corresponding to two of those bands were purified from human and pig brain by conventional procedures. This included ammonium sulfate fractionation, hydroxylapatite chromatography, affinity chromatography with NADP-agarose and Blue-Sepharose CL-6B, and gel filtration with Sephadex S-300. The final preparations of isozymes were electrophoretically homogeneous, and had a molecular weight of 220,000 as determined by gel filtration and nondenaturing gradient polyacrylamide gel electrophoresis and a subunit molecular weight of 57,000 as determined by SDS-polyacrylamide gel electrophoresis. The isozymes were NADP+-specific. Km values for all isozymes were similar for NADP+ (6 to 8 uM) , but different for G6P (56 to 180 uM) . Interestingly, only the isozyme II in human and pig brain were active with 6-phosphogluconate as a substrate (Km = 864 and 279 uM). All isozymes were inhibited by NADPH competitively with respect to NADP+ (Ki = 15 to 30 uM) and noncompetitively with respect to G6P (Ki = 31 to 100 uM). NADH did not affect any of the isozymes. The inhibition by NADPH were abolished by GSSG and yeast glutathione reductase, but not by GSSG alone. ATP inhibited the isozymes competitively with respect to G6P (Ki = 3.5 to 4.2 mM) and noncompetitively with respect to NADP"^ (Ki = 0.9 to 1.5 mM) . Palmitoyl-CoA dissociated the active tetramers into enzymatically inactive dimeric forms as determined by nondenaturing gradient polyacrylamide gel electrophoresis. HPLC peptide maps of tryptic digest and amino acid analyses of the isozymes showed extensive homologies between the isozymes. Based upon the arginine and lysine contents of the isozymes, the expected peaks for the peptides were about 67. Under the conditions in Fig. 6, at least 62 major peaks were detected. Therefore, the native enzyme appears to be composed of four identical polypeptide chains.

Mg++ and Ca++ at 1 mM concentration activated the brain G6PD isozymes by 150 %, even though they were not necessary for the enzyme activities. The G6PD isozymes were inhibited by Zn++ and p-chloromercurybenzoic acid at 1 mM concentration, and inactivated by heat at 50 °C for 5 min. EDTA had no effect on the G6PD isozymes. Like yeast enzyme, aluminum inactivated G6PD activity of the human and pig brain isozyme I and isozyme II without affecting the 6-phosphogluconate dehydrogenase (6-PGD) activity of the G6PD isozyme II. The aluminum-inactivated enzyme contained 1 gram atom of aluminum per mol of enzyme subunit. Aluminum, bound to the G6PD isozymes, was not dissociated by dialysis against 10 mM Tris/HCl, pH 7.0, containing 0.2 mM EDTA for 16 h at 4 °C or. The dissociation constants for the enzyme-aluminum complex were calculated to be 2 to 4 uM. The aluminum inactivated enzyme was reactivated by NaF, citrate, and transferrin. These reagents also protected the G6PD isozymes against the aluminum inactivation.

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