Description
A metabolic route which converts glucose to glycolate and pyruvate, without the intermediacy of phosphorylated compounds, may occur in animal tissues. Two of the component enzymes of the proposed pathway, glucose dehydrogenase and D-arabinose dehydrogenase, have been partially purified and characterized. We present evidence for the existence of D-arabinose isomerase and a separate glucono-δ-lactonase in mammalian tissue. These enzymes, together with a previously identified gluconate dehydrogenase, provide a metabolic route culminating in the formation of D-arabonate. The latter is apparently converted into glycolate and pyruvate in the presence of NAD⁺ and the liver soluble fraction. Glucose dehydrogenase is an enzyme which is associated with the microsomal fraction of the cell. It has been purified up to 1500 fold from the livers of 16 vertebrate species and about 66 fold from sheep kidney. Each of the hepatic enzymes has a molecular weight of about 240,000, operates most efficiently in the pH range 8-9, and displays a relatively high Kₘ value for glucose. Nevertheless, both hepatic and renal glucose dehydrogenase catalyzes glucose oxidation with either NAD⁺ or NADP⁺ at a rate which may be physiologically significant. Substrate for the enzymes, in addition to glucose, are 6-deoxyglucose, 6-fluoroglucose, and D-xylose. The animal cells containing glucose dehydrogenase activity, in order of decreasing level, are: liver > kidney > ovaries >> placenta, lung, Erlich ascites cells > testes >> brain, spleen, and rat intestinal mucosa. D-arabinose dehydrogenase is a NADP⁺ specific enzyme found for the most part in the soluble fraction of cell homogenates. The enzyme has about the same distribution in mammalian organs as glucose dehydrogenase. D-arabinose dehydrogenase has been purified 60 fold from rat liver. 2-deoxyribose and D-lyxose are the only other substrates found, yielding about 20% of the activity of D-arabinose. The enzymatic activity is reversibly lost upon dilution; the rate of activity loss is slowed by the presence of NAD⁺ or NADP⁺. This, and the partial separation of the two isoenzymes, suggests that D-arabinose dehydrogenase is composed of subunits. D-arabinose dehydrogenase concentrations are lowered in the livers of fasted or alloxan-diabetic rats. Neither of these conditions affects the glucose dehydrogenase level. Evidence suggesting that the proposed non-phosphorylated glucose oxidative pathway is operative in vivo is provided by experiments using rat liver or kidney homogenates and [¹⁴C]glucose. The amount of ¹⁴CO₂ expired from these systems was studied in the presence of unlabeled glucose-6-P, 6-phosphogluconate, 3-phosphoglycerate, glucosamine-6-P, and 2-deoxyglucose-6-P. Each of these compounds, especially the latter two, stimulated glucose oxidation, particularly in the presence of exogenous NADP⁺. The equivalent non-phosphorylated compounds elicited no effect on ¹⁴CO₂ production, while the addition of ATP to the homogenate system in some cases caused dilution of the CO₂ label. [¹⁴C]fructose and [¹⁴C]glucose-6-P behaved as expected (dilution of the ¹⁴CO₂ label) under the above conditions. Mitochondria could be removed from the homogenates without abolishing the stimulation of glucose oxidation by the above sugar phosphates. However, the presence of the microsomal fraction was essential to observation of the increased ¹⁴CO₂ production with increasing phosphate ester concentration. This is the expected result if the glucose dehydrogenase-initiated pathway plays a role in cellular glucose metabolism.
