On the nature of the interaction between 4,4'-diisothiocyanostilbene 2,2'-disulfonic acid and microsomal glucose-6-phosphatase. Evidence for the involvement of sulfhydryl groups of the phosphohydrolase.
The effect of 4,4'-diisothiocyanostilbene 2,2'-disulfonic acid (DIDS) on microsomal glucose 6-phosphate hydrolysis has been reinvestigated and characterized in order to elucidate the topological and functional properties of the interacting sites of the glucose-6-phosphatase. The studies were performed on microsomal membranes, partially purified and reconstituted glucose-6-phosphatase preparations and show the following. (a) DIDS inhibits activity of the glucose-6-phosphatase of native microsomes as well as the partially purified glucose-6-phosphatase. (b) Inhibition is reversed when the microsomes and the partially purified phosphohydrolase, incorporated into asolectin liposomes, are modified with Triton X-114. (c) Treatment of native microsomes with DIDS and the following purification of glucose-6-phosphatase from these labeled membranes leads to an enzyme preparation which is labeled and inhibited by DIDS. (d) Preincubation of native microsomes or partially purified glucose-6-phosphatase with a 3000-fold excess of glucose 6-phosphate cannot prevent the DIDS-induced inhibition. (e) Inhibition of glucose-6-phosphatase by DIDS is completely prevented when reactive sulfhydryl groups of the phosphohydrolase are blocked by p-mecuribenzoate. (f) Reactivation of enzyme activity is obtained when DIDS-labeled microsomes are incubated with 2-mercaptoethanol or dithiothreitol. Therefore, we conclude that inhibition of microsomal glucose 6-phosphate hydrolysis by DIDS cannot result from binding of this agent to a putative glucose-6-phosphate-carrier protein. Our results rather suggest that inhibition is caused by chemical modification of sulfhydryl groups of the integral phosphohydrolase accessible to DIDS attack itself. An easy interpretation of these results can be obtained on the basis of a modified conformational model representing the glucose-6-phosphatase as an integral channel-protein located within the hydrophobic interior of the microsomal membrane [Schulze et al. (1986) J. Biol. Chem. 261, 16,571-16,578].
Speth
M
M
Biochemisches Institut der Justus-Liebig-Universität Giessen, Federal Republic of Germany.
Schulze
H U
HU
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D005952:Glucose-6-Phosphatase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008626:Mercuribenzoates / Q000494:pharmacology; D008862:Microsomes, Liver / Q000201:enzymology*; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D051381:Rats; D013050:Spectrometry, Fluorescence; D013267:Stilbenes / Q000494:pharmacology*; D013438:Sulfhydryl Compounds / Q000502:physiology*
C043407:4-mercuribenzoate; D008626:Mercuribenzoates; D013267:Stilbenes; D013438:Sulfhydryl Compounds; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D005952:Glucose-6-Phosphatase; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid