scholarly journals Histone II-A stimulates glucose-6-phosphatase and reveals mannose-6-phosphatase activities without permeabilization of liver microsomes

1995 ◽  
Vol 310 (1) ◽  
pp. 221-224 ◽  
Author(s):  
J F St-Denis ◽  
B Annabi ◽  
H Khoury ◽  
G van de Werve
Keyword(s):  
Substrate Specificity ◽  
Membrane Permeability ◽  
Phosphatase Activity ◽  
Liver Microsomes ◽  
Microsomal Membrane ◽  
Phosphatase Activities ◽  
Phosphate Hydrolysis ◽  
Mannose 6 Phosphate ◽  
Stimulation Of

The effect of histone II-A on glucose-6-phosphatase and mannose-6-phosphatase activities was investigated in relation to microsomal membrane permeability. It was found that glucose-6-phosphatase activity in histone II-A-pretreated liver microsomes was stimulated to the same extent as in detergent-permeabilized microsomes, and that the substrate specificity of the enzyme for glucose 6-phosphate was lost in histone II-A-pretreated microsomes, as [U-14C]glucose-6-phosphate hydrolysis was inhibited by mannose 6-phosphate and [U-14C]mannose 6-phosphate hydrolysis was increased. The accumulation of [U-14C]glucose from [U-14C]glucose 6-phosphate into untreated microsomes was completely abolished in detergent-treated vesicles, but was increased in histone II-A-treated microsomes, accounting for the increased glucose-6-phosphatase activity, and demonstrating that the microsomal membrane was still intact. The stimulation of glucose-6-phosphatase and mannose-6-phosphatase activities by histone II-A was found to be reversed by EGTA. It is concluded that the effects of histone II-A on glucose-6-phosphatase and mannose-6-phosphatase are not caused by the permeabilization of the microsomal membrane. The measurement of mannose-6-phosphatase latency to evaluate the intactness of the vesicles is therefore inappropriate.

scholarly journals GTP-mediated Ca2+ release in rough endoplasmic reticulum. Correlation with a GTP-sensitive increase in membrane permeability

1987 ◽  
Vol 248 (3) ◽  
pp. 741-747 ◽  
Author(s):  
C V Nicchitta ◽  
S K Joseph ◽  
J R Williamson
Keyword(s):  
Membrane Permeability ◽  
Time Course ◽  
Liver Microsomes ◽  
Subsequent Treatment ◽  
Complete Inhibition ◽  
Hill Coefficient ◽  
Release Process ◽  
Thiol Reagent ◽  
Mannose 6 Phosphate ◽  
Poly Ethylene

Guanine nucleotides have been reported to stimulate reticular Ca2+ release. By using the structure-linked latency of microsomal mannose-6-phosphate phosphatase as an index of microsomal permeability [Arion, Ballas, Lange & Wallin (1976) J. Biol. Chem. 251, 4901-4907], the effects of GTP on Ca2+ release and membrane permeability were compared in liver microsomes. In a stripped rough-microsome preparation, GTP caused a dose-dependent increase in mannose 6-phosphate permeability. Half-maximal and maximal effects were observed at 3 microM- and 10 microM-GTP respectively. The time course of the change in membrane permeability coincided with the time course of GTP-dependent Ca2+ release. This increase in microsomal permeability displayed positive to-operativity with respect to GTP (Hill coefficient = 1.8). By analogy to the GTP-dependent Ca2+ release process, guanosine 5′-[gamma-thio]triphosphate and guanosine 5′-[beta gamma-imido]-triphosphate inhibited the ability of GTP to alter microsomal permeability, but were without effect when added alone. In the presence of 50 microM-GTP, complete inhibition of the GTP-dependent increase in microsomal permeability was achieved with 10 microM-guanosine 5′-[gamma-thio]triphosphate, whereas a 25% inhibition was observed with 10 microM-guanosine 5′-[beta gamma-imido]triphosphate. In contrast with previous observations in crude microsomal preparations, GTP-dependent Ca2+ release in the stripped rough-microsome preparation did not require the addition of poly(ethylene glycol), although the latter did stimulate the rate of Ca2+ release. The ability of GTP to alter microsomal permeability was blocked by prior treatment with the thiol reagent p-hydroxymercuribenzoate; complete inhibition was observed after a 10 min exposure to 50 microM. Inhibition was reversed by subsequent treatment with dithiothreitol. The marked similarities between the two GTP-sensitive processes indicate that they may function via the same mechanism.

scholarly journals Effect of GTP on the dolichol pathway for protein glycosylation in rat liver microsomes

1993 ◽  
Vol 296 (3) ◽  
pp. 633-637 ◽  
Author(s):  
X Bossuyt ◽  
N Blanckaert
Keyword(s):  
Membrane Permeability ◽  
Rat Liver ◽  
Liver Microsomes ◽  
Protein Glycosylation ◽  
Rat Liver Microsomes ◽  
Alpha Toxin ◽  
Gtp Hydrolysis ◽  
Dolichol Pathway ◽  
Previous Proposal ◽  
Stimulation Of

Incubation of native rat liver microsomes with GTP resulted in enhanced incorporation of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc into lipid acceptors. The stimulation of GlcNAc transfer by GTP was specific for GTP; ATP exerted no effect. The GTP effect was blocked by a non-hydrolysable GTP analogue guanosine 5′-[beta gamma-imido]triphosphate, indicating that GTP hydrolysis was crucial. Though dolichyl pyrophosphate NN′-diacetylchitobiose [Dol-PP-(GlcNAc)2] was the main radiolabelled product formed upon incubation of GTP-treated microsomes with UDP-GlcNAc, GTP selectively stimulated UDP-GlcNAc:dolichyl phosphate (Dol-P) N-acetylglucosaminyl 1-phosphotransferase (N-acetylglucosaminyl 1-phosphotransferase). This conclusion was reached on the basis of experiments in which tunicamycin was used to selectively inhibit N-acetylglucosaminyl 1-phosphotransferase. The enhanced transformation of Dol-P to dolichyl pyrophosphate N-acetylglucosamine (Dol-PP-GlcNAc) by GTP ultimately led to enhanced protein glycosylation. GTP-induced stimulation of GlcNAc incorporation in lipid and protein by GTP was observed also in microsomes fully permeabilized with Staph. aureus alpha-toxin. These findings refute the previous proposal [Godelaine, Beaufay, Wibo and Ravoet (1983) J. Cell Biol. 97, 340-350] that increased membrane permeability constitutes the mechanism whereby GTP activates the reactions of the dolichol pathway.

Identification of the phosphomonoesterases that hydrolyze lysopolyphosphoinositides in rat brain and liver

1987 ◽  
Vol 65 (10) ◽  
pp. 890-898 ◽  
Author(s):  
Frederick B. St. C. Palmer
Keyword(s):  
Rat Brain ◽  
Phosphatase Activity ◽  
Divalent Cations ◽  
Liver Microsomes ◽  
Maximum Activity ◽  
Molar Ratio ◽  
Phosphatase Activities ◽  
Rat Brain And Liver ◽  
Ph Range

The phosphatase activities responsible for the sequential dephosphorylation of lysophosphatidylinositol 4,5-bisphosphate (lysoPtdIns(4,5)P2) to lysophosphatidylinositol that precedes reacylation in rat brain and liver microsomes were characterized. LysoPtdIns(4,5)P2 and the intermediate lysophosphatidylinositol 4-phosphate (lysoPtdIns4P) were hydrolyzed by two distinct phosphatase activities which were distinguishable by their substrate and cation requirements. The lysoPtdIns(4,5)P2 phosphatase activity was Mg2+ dependent and partially inhibited by Ca2+, excess Mg2+, and cationic detergent (cetyltrimethylammonium bromide). Activity was maximal at neutral (brain) or slightly alkaline (liver) pH when the Mg2+/lysoPtdIns(4,5)P2 molar ratio was 1.0 in the presence of bovine serum albumin (1 mg∙mL−1). LysoPtdIns4P phosphatase activity did not require divalent cations (not inhibited by EDTA). This activity was inhibited by Ca2+, Mg2+, and substrate concentrations above 0.2 mM. Maximum activity was observed over a broad pH range (6.0–8.5). Both activities were inhibited by lysophosphatidylinositol and lysophosphatidylcholine, but not other lysophospholipids. The lysopolyphosphoinositides are most likely hydrolyzed by the same phosphatases that act on the diacylpolyphosphoinositides, since PtdIns(4,5)P2 and PtdIns4P were also hydrolysed by Mg2+-dependent and cation-independent phosphatases, respectively. Activities with the diacylpolyphosphoinositides differed only in their requirement of detergents for maximum activity in vitro. Specific activities for the diacyl and "lyso" forms of each substrate were very similar when suitably optimized reaction mixtures were used. The subcellular distributions of the two phosphatase activities in both brain and liver were the same when acting on diacyl- or lyso-polyphosphoinositides, as was their response to inhibitors. Alkaline, acid, phosphoprotein, and inositol-1-phosphate phosphatases did not contribute substantially to the hydrolysis of either lysoPtdIns4P or lysoPtdIns(4,5)P2, since the activities were not significantly inhibited by cysteine, dithiothreitol, NaF, or LiCl. Lack of inhibition by 2,3-bisphosphoglycerate and absence of stimulation by cysteine or dithioerythritol, as well as a different subcellular distribution in liver, excluded inositol-1,4,5-trisphosphate and inositol-1,4-bisphosphate phosphatases as sources of the lysoPtdIns(4,5)P2 and lysoPtdIns4P phosphatase activities.

scholarly journals Interaction of mannose-6-phosphate with the hysteretic transition in glucose-6-phosphate hydrolysis in intact liver microsomes

FEBS Letters ◽  
1992 ◽  
Vol 302 (3) ◽  
pp. 197-200 ◽  
Author(s):  
Hubert Vidal ◽  
Alfred Berteloot ◽  
Marie-Josée Larue ◽  
Jean-François St-Denis ◽  
Gérald van de Werve
Keyword(s):  
Liver Microsomes ◽  
Phosphate Hydrolysis ◽  
Mannose 6 Phosphate

scholarly journals Histone 2A stimulates glucose-6-phosphatase activity by permeabilization of liver microsomes

2002 ◽  
Vol 367 (2) ◽  
pp. 505-510 ◽  
Author(s):  
Angelo BENEDETTI ◽  
Rosella FULCERI ◽  
Bernard B. ALLAN ◽  
Pamela HOUSTON ◽  
Andrey L. SUKHODUB ◽  
...  
Keyword(s):  
Phosphatase Activity ◽  
Liver Microsomes ◽  
Catalytic Site ◽  
Microsomal Membrane ◽  
Dose Effect ◽  
Free Access ◽  
Microsomal Enzyme ◽  
Inhibitory Effects ◽  
Effect Curve ◽  
Udp Glucuronosyltransferase

Histone 2A increases glucose-6-phosphatase activity in liver microsomes. The effect has been attributed either to the conformational change of the enzyme, or to the permeabilization of microsomal membrane that allows the free access of substrate to the intraluminal glucose-6-phosphatase catalytic site. The aim of the present study was the critical reinvestigation of the mechanism of action of histone 2A. It has been found that the dose-effect curve of histone 2A is different from that of detergents and resembles that of the pore-forming alamethicin. Inhibitory effects of EGTA on glucose-6-phosphatase activity previously reported in histone 2A-treated microsomes have been also found in alamethicin-permeabilized vesicles. The effect of EGTA cannot therefore simply be an antagonization of the effect of histone 2A. Histone 2A stimulates the activity of another latent microsomal enzyme, UDP-glucuronosyltransferase, which has an intraluminal catalytic site. Finally, histone 2A renders microsomal vesicles permeable to non-permeant compounds. Taken together, the results demonstrate that histone 2A stimulates glucose-6-phosphatase activity by permeabilizing the microsomal membrane.

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