ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 326, No. 2, February 15, pp. 238–242, 1996 Article No. 0071 Glucose 6-Phosphate and Mannose 6-Phosphate Are Equally and More Actively Hydrolyzed by Glucose 6- Phosphatase during Hysteretic Transition within Intact Microsomal Membrane Than after Detergent Treatment A. Ajzannay and G. Mithieux 1 Institut National de la Sante´ et de la Recherche Me´dicale, Unite´ 449, Lyon, France Received July 18, 1995, and in revised form November 6, 1995 endoplasmic reticulum. It exhibits very different ki- We have studied the rapid kinetics of glucose 6-phos- netic features according to whether it is assayed within phatase (Glc6Pase) toward glucose 6-phosphate intact microsomes or after detergent solubilization of (Glc6P) and mannose 6-phosphate (Man6P) in intact the membrane (1). In particular, it hydrolyses glucose and detergent-treated microsomes, using a radiomet- 6-phosphate (Glc6P) with a high specificity at a moder- ric assay based on the use of [U- 14 C]hexose 6-phos- ate rate when present in the intact membrane whereas phates. We show that a hysteretic transition of it hydrolyses Glc6P and other sugar 6-phosphates at Glc6Pase from a rapid hydrolytic form to a slower ki- an increased rate after detergent treatment of the netic form within the intact membrane occurs for both membrane. To interpret these features, a complex substrates with the same relaxation time. During the multicomponent protein model for the structural orga- hysteretic transition, preceding the steady-state rate nization of Glc6Pase within the membrane has been of hydrolysis, Glc6Pase is able to hydrolyze both Glc6P proposed: the so-called ‘‘substrate transport model’’ (2, and Man6P at very similar rates. Only Glc6P is signifi- 3). It predicts that the enzyme system is composed of cantly hydrolyzed at steady state. Moreover, the initial several distinct polypeptides, among them a high-rate rates of hydrolysis of both Glc6P and Man6P in intact nonspecific phosphohydrolase located inside the lumen microsomes are higher than the respective rates of hy- of microsomes, having access under these conditions to drolysis after detergent treatment of the membrane at only one of its possible substrates (Glc6P) owing to the high substrate concentrations (10 and 20 mM), while action of a rate-limiting specific transmembranous these rates are not different at lower substrate concen- Glc6P translocator. According to the model, the proper trations. These data show that the marked specificity role of the membrane in Glc6Pase features is therefore of Glc6Pase at steady state in the membrane is ac- restricted to that of a permeability barrier. Alterna- quired owing to a hysteretic transition induced by the tively, according to the ‘‘conformational’’ model, the hydrolytic phenomenon, independently of the nature presence of the surrounding membrane is crucial to of the prior phosphate donor. The role of the mem- constrain the enzyme, considered as a unique protein, brane in this phenomenon is crucial, since the transi- in a conformation characterized by a moderate rate, tion does not occur in its absence. q 1996 Academic Press, Inc. with the benefit of a high specificity toward Glc6P (4, 5). According to the latter model, the removal of sur- Glucose-6-phosphatase (Glc6Pase) 2 (EC 3.1.3.9), an rounding membrane lipids induces marked conforma- enzyme essential to the production of glucose by the tional changes of the enzyme, which hence loses its liver, is an integral membrane protein located in the high specificity toward Glc6P and gains a higher veloc- ity. Numerous arguments have been made in favor of 1 To whom correspondence should be addressed at INSERM U. the one or the other model; none has permitted a defin- 449, Faculte´ de Me´decine Alexis Carrel, rue G. Paradin. 69372 Lyon itive conclusion to be drawn (reviewed in 6). Ce´dex 08. France. Fax: (33) 78 77 87 62. Recently, an important breakthrough has been 2 Abbreviations used: Glc6Pase, glucose-6-phosphatase; Man6- achieved owing to studies reporting the rapid kinetic Pase, mannose 6-phosphatase; Glc6P, glucose 6-phosphate; Man6P, features of Glc6Pase. It was shown that the enzyme of mannose 6-phosphate; dGlc6P, 2-deoxyglucose 6-phosphate; GlcN6P, glucosamine 6-phosphate; PP i , pyrophosphate. intact microsomes does not obey a major prediction of 238 0003-9861/96 $12.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.