title: Modulation of the activity of hepatic glucose-6-phosphatase by methylthioadenosine sulfoxide.
abstract: Methylthioadenosine sulfoxide (MTAS), an oxidized derivative of the cell toxic metabolite methylthioadenosine has been used in elucidating the relevance of an interrelationship between the catalytic behavior and the conformational state of hepatic glucose-6-phosphatase and in characterizing the transmembrane orientation of the integral unit in the microsomal membrane. The following results were obtained: (1) Glucose 6-phosphate hydrolysis at 37 degrees C is progressively inhibited when native microsomes are treated with MTAS at 37 degrees C. In contrast, glucose 6-phosphate hydrolysis of the same MTAS-treated microsomes assayed at 0 degrees C is not inhibited. (2) Subsequent modification of the MTAS-treated microsomes with Triton X-114 reveals that glucose-6-phosphatase assayed at 37 degrees C as well as at 0 degrees C is inhibited. (3) Although excess reagent is separated by centrifugation and the MTAS-treated microsomes diluted with buffer before being modified with Triton the temperature-dependent effect of MTAS on microsomal glucose-6-phosphatase is not reversed at all. (4) In native microsomes MTAS is shown to inhibit glucose-6-phosphatase noncompetitively. The subsequent Triton-modification of the MTAS-treated microsomes, however, generates an uncompetitive type of inhibition. (5) Preincubation of native microsomes with MTAS completely prevents the inhibitory effect of 4,4'-diisothiocyanostilbene 2,2'-disulfonate (DIDS) as well as 4,4'-diazidostilbene 2,2'-disulfonate (DASS) on glucose-6-phosphatase. (6) Low molecular weight thiols and tocopherol protect the microsomal glucose-6-phosphatase against MTAS-induced inhibition. (7) Glucose-6-phosphatase solubilized and partially purified from rat liver microsomes is also affected by MTAS in demonstrating the same temperature-dependent behavior as the enzyme of MTAS-treated and Triton-modified microsomes. From these results we conclude that MTAS modulates the enzyme catalytic properties of hepatic glucose-6-phosphatase by covalent modification of reactive groups of the integral protein accessible from the cytoplasmic surface of the microsomal membrane. The temperature-dependent kinetic behavior of MTAS-modulated glucose-6-phosphatase is interpreted by the existence of distinct catalytically active enzyme conformation forms. Detergent-induced modification of the adjacent hydrophobic microenvironment additionally generates alterations of the conformational state leading to changes of the kinetic characteristics of the integral enzyme.
authors.affiliation: Biochemisches Institut am Klinikum der Justus-Liebig-Universität Giessen, F.R.G.
authors.forename: M
authors.initials: M
authors.lastname: Speth
authors.forename: H U
authors.initials: HU
authors.lastname: Schulze
mesh_terms: D017766:Acid Anhydride Hydrolases; D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D004305:Dose-Response Relationship, Drug; D005952:Glucose-6-Phosphatase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000187:drug effects* / Q000201:enzymology; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D011487:Protein Conformation; D051381:Rats; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013696:Temperature; D013872:Thionucleosides / Q000494:pharmacology*; D014810:Vitamin E / Q000494:pharmacology
chemical_list: D013438:Sulfhydryl Compounds; D013872:Thionucleosides; D014810:Vitamin E; C071306:methylthioadenosine sulfoxide; D010744:Phosphoric Monoester Hydrolases; D005952:Glucose-6-Phosphatase; D017766:Acid Anhydride Hydrolases; C023837:nucleoside-diphosphatase; D000241:Adenosine