Abstract
Rat liver glucose-6-phosphate dehydrogenase (G6PD) can be induced by entirely different stimuli, i. e. refeeding high carbohydrate diets and liver injuries caused by CCl4 and other hepatotoxic agents. This led us to examine a possible involvement of different molecular species of the enzyme in the induction under these experimental conditions.
G6PD in the soluble fraction of rat liver was resolved into three major components, I, II and III, in the order of decreasing mobility on polyacrylamide gel disc electrophoresis. Fresh liver supernatants from normal rats had only II and III while those from CCl4-injured and glucose-casein-refed rats revealed all of the three components. Artifactual resolution could be excluded for the present experiment with G6PD by demonstrating an identical electrophoretic pattern of these forms on both Cellogel and pre-washed or riboflavin-polymerized polyacrylamide gel. Molecular weights of these forms were found similar, and the resolution on disc electrophoresis appeared to be mainly due to charge differences. Each of three forms was identically precipitated in the Ouchterlony test by rabbit antiserum prepared against purified G6PD, which was composed of I exclusively.
Conversion of slower migrating forms into faster ones, finally to sole Component I, without any significant change in activity was demonstrated by treatment with p-chloromercuribenzoate(pCMB) or HgCl2. Upon treatment with high concentrations of these sulfhydryl reagents or N-ethylmaleimide (NEM), however, decrease in intensity of Component I from refed rat livers was accompanied by loss of the enzyme activity. Therefore, at least two readily reactive but catalytically not essential sulfhydryl groups are present per a dimer (mol. wt.=110,000) and are involved in sequential interconversion of I, II and III forms of G6PD. A similar sequential transformation of slower moving components into faster ones occurred upon depletion of GSH in liver supernatants by treatment with diamide, a specific GSH oxidant, at 0° and its reversal was demonstrated by regenerati on of GSH following incubation of the diamide-treated supernatants at 37°. Liver supernatants prepared from CCl4-treated rats showed decrease in liver GSH concentration with concomitant increase of the faster moving G6PD bands. Addition of GSH to the supernatants restored the normal disc electrophoretic pattern having the components II and III. The increased intensity of the faster moving components observed in liver and other tissues under varying in vivo conditions was found to be closely associated with the increased ratio of G6PD activity to GSH level.
Among three interconvertible forms of X-linked human G6PD (type B+),B+-I, B+-II and B+-III in the order of decreasing mobility on disc electrophoresis, only two components,B+-II and B+-III, were demonstrated in fresh supernatants of intact livers. Faster moving components of G6PD increased in intensity in livers from patients with chronic hepatitis, liver cirrhosis and primary hepatoma, resulting in appearance of component B+-I in some of these cases. The altered G6PD pattern was closely associated with concomitant increase in G6PD activity and decrease in GSH level of liver, hence with the increased ratio of G6PD/GSH. The most marked changes inthese parameters were found in a case of primary hepatoma with elevated concentration of serum α-fetoprotein. Hepatitis and cirrhosis patients with increased faster migrating components of G6PD were characterized by extensive necrosis and degeneration of parenchymal liver cells and by relatively high levels of serum α-fetoprotein. Concentration of GSH, activity of G6PD and its electrophoretic pattern in erythrocytes were not significantly altered inthese subjects with liver diseases.
