Direct evidence for the involvement of two glucose 6-phosphate-binding sites in the glucose-6-phosphatase activity of intact liver microsomes. Characterization of T1, the microsomal glucose 6-phosphate transport protein by a direct binding assay.

S 5627 is a synthetic analogue of chlorogenic acid. S 5627 is a potent linear competitive inhibitor of glucose 6-phosphate (Glc-6-P) hydrolysis by intact microsomes (Ki = 41 nM) but is without effect on the enzyme in detergent- or NH4OH-disrupted microsomes. 3H-S 5627 was synthesized and used as a ligand in binding studies directed at characterizing T1, the Glc-6-P transporter. Binding was evaluated using Ca2+-aggregated microsomes, which can be sedimented at low g forces. Aside from a modest reduction in K values for both substrate and S 5627, Ca2+ aggregation had no effect on glucose-6-phosphatase (Glc-6-Pase). Scatchard plots of binding data are readily fit to a simple "two-site" model, with Kd = 21 nM for the high affinity site and Kd = 2 microM for the low affinity site. Binding to the high affinity site was competitively blocked by Glc-6-P (Ki = 9 microM), whereas binding was unaffected by mannose-6-phosphate, Pi, and PPi and only modestly depressed by 2-deoxy-D-glucose 6-phosphate, a poor substrate for Glc-6-Pase in intact microsomes. Thus the high affinity 3H-S 5627 binding site fits the criteria for T1. Permeabilization of the membrane with 0.3% (3-[(chloramidopropyl)-dimethylammonio]-1-propanesulfonate) activated Glc-6-Pase and broadened its substrate specificity, but it did not significantly alter the binding of 3H-S 5627 to the high affinity sites or the ability of Glc-6-P to block binding. These data demonstrate unequivocally that two independent Glc-6-P binding sites are involved in the hydrolysis of Glc-6-P by intact microsomes. The present findings are the strongest and most direct evidence to date against the notion that the substrate specificity and the intrinsic activity of Glc-6-Pase in native membranes are determined by specific conformational constraints imposed on the enzyme protein. These data constitute compelling evidence for the role of T1 in Glc-6-Pase activity.