Hans-Ulrich Schulze

Latency studies on rat liver microsomal glucose-6-phosphatase. Correlation of membrane modification and solubilization by Triton X-114 with the enzymatic activity

Interrelationships between the catalytic properties of glucose-6-phosphatase and the membrane structure of rat liver microsomes were investigated. Membrane modification and solubilization employing the nonionic surfactant Triton X-114 were standardized and analysed by ultracentrifugation, surface tension- and turbidity measurements. The effect of Triton X-114 on the glucose-6-phosphatase activity was studied systematically and the whole magnitude of time- and temperature-dependent inactivation of this enzyme has been demonstrated. The results show that the activity measured is always a resultant of two processes, the beginning of inactivation and the release of latency. Maximal activation of about 600% (83% of apparent latency) was obtained at 0 degree C. A correlation between membrane modification and solubilization and the conditions under preincubation and test incubation reveals that studies on detergent-disrupted microsomes are performed on structures reassembled from solubilizates and this implies a modified microenvironment in the reconstitutes. Kinetic analyses suggest interrelationships between Triton X-114 and the permeability barrier of the glucose-6-phosphatase system. At 0 degree C 2-propanol and ethanol are more potent tools for membrane modification than Triton X-114 and release 88% and 86% latent activity corresponding to an activation of the glucose-6-phosphatase of about 850% and 700%, respectively. These observations suggest that detergent treatment of microsomes could not preserve the functional integrity of the glucose-6-phosphate phosphohydrolase, which is one dogma of the substrate-transport hypothesis developed by Arion and his co-workers (Arion, W.J., et al. (1975) Mol. Cell. Biochem. 6, 75-83).

Hormone-induced effects on the rat liver microsomal glucose-6-phosphatase system in vitro.

Abstract We studied the effects of various glucocorticoids, glucagon and insulin on the activity of rat liver microsomal glucose-6-phosphatase. Preincubation of microsomes with corticosterone, cortisone, cortisol and dexamethasone as well as glucagon increased the rate of glucose-6-phosphate hydrolysis by about 1.5 fold relative to the controls. The maximum activation occurred at about 10 nM steroids and 0.3 nM glucagon, respectively. On the other hand, increasing concentrations (8.3 – 50 nM) of insulin progressively inhibited glucose-6-phosphatase up to 26%; the activity of which, however, remains completely in a latent state within the microsomal membrane and can be released from it by Triton treatment. In terms of the substrate transport hypothesis, the results are interpreted as evidence that regulation of glucose-6-phosphate hydrolysis is achieved by direct interactions either of the hormones themselves or of a possible second messenger with the carrier moiety of the rat liver microsomal glucose-6-phosphatase system.

Isolation and chemical composition of the NADH: Semidehydroascorbate oxidoreductase rich membranes from rat liver

The enzymatic reduction of semidehydroascorbate to ascorbate is catalysed by NADH: semidehydroascorbate oxidoreductase (EC 1.6.5.4.) (SOR) [ 11 which have been studied both in Neurospora crassa [2] and in various subcellular particles from many mammalian organs [3] , especially in the rat liver microsome fraction [3-61. It is generally accepted that the micrdsome fraction usually isolated by differential centrifugation [7] is heterogeneous and contains plasma membranes, Golgi membranes, and endoplasmic membranes as well as low contaminations of mitochondria, lysosomes and peroxisomes. We have shown recently that the SOR does not sediment with glucose 6-phosphatase, NADH: ferricytochrome bs oxidoreductase, cytochrome bs, NADPH: ferricytochrome c oxidoreductase and cytochrome P-450 when original microsomes are fractionated by zonal centrifugation, but the SOR is bound to a membrane fraction with an especially low density [8] _ This finding is in agreement with the earlier report on our first results on the fractionation of original microsomes by differential centrifugation [8,9]. In the present paper we report the results of experiments carried out in our laboratory indicating that the SOR is localized in a hitherto not identified vesicle fraction.

Is thermostability of glucose‐6‐phosphatase indeed dependent on a stabilizing protein?

Partial purification of glucose‐6‐phosphatase from rat liver microsomes by solubilization of the membranes with the non‐ionic detergent Triton X‐144 at pH 6.5 and the removal of inactivating detergent by hydrophobic chromatography results in a thermostable enzyme protein which is not dependent on stabilizing phospholipids or proteins. The readdition of low amounts of detergent immediately causes a conversion into a thermo‐unstable phosphodydrolase protein. Thus these findings present evidence that heatinstability of partially purified glucose‐6‐phosphatase derives from traces of inactivating detergent changing the structural properties of the phosphohydrolase rather than from the absence of the postulated specific stabilizing protein.

Accessibility of glucose 6-phosphate: phosphohydrolase to antibody attack in modified microsomal vesicles.

Recent investigations supported the existence of 2 components of the endoplasmic reticulum participating in the process of glucose 6-phosphate hydrolysis: the glucose 6-phosphate-specific transporter that mediates the movement of the substrate from the cytoplasmic membrane surface into the lumen and the unspecific phosphohydrolase on the luminal side of the membrane [ 1,2]. However, this model has not been generally accepted; especially the proposed molecular arrangement of the glucose 6-phosphatase components within the membrane is contradictory [3-71. Furthermore, immunological studies [6] have suggested that the glucose 6-phosphate: phosphohydrolase is presumably not freely accessible on the luminal surface which, however, is one of the prerequisites of the substrate-transport hypothesis as described in [ 1,2]. Therefore, we have reinvestigated the transverse topology of the glucose 6-phosphatase by detailed immunological studies on detergent-modified and mechanically disrupted microsomes. These findings support our preliminary concept and demonstrate that, indeed, the glucose 6-phosphate:phosphohydrolase is not attached to the luminal membrane surface, but buried within the microsomal membrane.

Protease inhibitors but not proteases inhibit the glucose-6-phosphatase of native rat liver microsomes

Controlled proteolytic digestion by trypsin or bacterial proteases limited to the cytosolic side of the native microsomal membrane is not efficient to inhibit glucose-6-phosphate hydrolysis. Modification of the microsomes with deoxycholate prior to protease treatment is prerequisite to allow accessibility of the integral protein and inhibition of enzyme activity. Glucose-6-phosphatase of native microsomes, however, is rapidly inactivated by micromolar concentrations of TPCK as well as TLCK. In deoxycholate-modified microsomes both reagents do not affect glucose-6-phosphate hydrolysis. These results indicate that in the native, intact microsomal membrane glucose-6-phosphatase is not accessible to proteolytic attack from the cytoplasmic surface. The putative inhibitory effect of some trypsin or bacterial protease preparations on glucose-6-phosphatase of native microsomes observed most possibly is a result of contaminating agents as TPCK or TLCK.