Glucose-6-phosphatase: is activity regulated by phosphorylation–dephosphorylation?

1983 ◽  
Vol 61 (10) ◽  
pp. 1085-1089 ◽  
Author(s):  
Jasbir Singh ◽  
Richard E. Martin ◽  
Robert C. Nordlie
Keyword(s):  
Protein Kinase ◽  
Protein Phosphorylation ◽  
Alternative Explanation ◽  
Potent Inhibitor ◽  
Liver Microsomes ◽  
Time Dependent ◽  
Rat Liver Microsomes ◽  
Phosphoprotein Phosphatase ◽  
Dependent Inactivation ◽  
Competitive Inhibitors

Incubation of rat liver microsomes with ATP and Mg2+ in the absence or presence of an exogenous protein kinase showed no changes in the activity of glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9). These observations confirm the recent findings of the Burchells and colleagues and refute on methodological grounds the earlier conclusions of Begley and Craft implicating regulation of this enzyme by protein phosphorylation–dephosphorylation. In other studies, the time-dependent inactivation of microsomal glucose-6-phosphatase by incubation with deoxycholate was used to obtain the inactive enzyme which in the presence of a protein kinase, ATP, and Mg2+ could not be restored to its original level. A number of substrates and competitive inhibitors of glucose-6-phosphatase, most notably vanadate which is the most potent inhibitor of the enzyme identified, stabilized this enzyme against its time-dependent inactivation in the presence of detergent as effectively as did fluoride and molybdate which are also effective competitive inhibitors of glucose-6-phosphatase. An alternative explanation to the involvement of a phosphoprotein phosphatase, as discussed by the Burchells, in the time-dependent inactivation of glucose-6-phosphatase is thus suggested.

Isolation and properties of a protein kinase from rat liver microsomes

1969 ◽  
Vol 178 (1) ◽  
pp. 199-201 ◽  
Author(s):  
L.A. Pinna ◽  
B. Baggio ◽  
V. Moret ◽  
N. Siliprandi
Keyword(s):  
Protein Kinase ◽  
Rat Liver ◽  
Liver Microsomes ◽  
Rat Liver Microsomes

Non-Stick Sugars: Synthesis of Difluorosugar Fluorides as Potential Glycosidase Inactivators

2009 ◽  
Vol 62 (6) ◽  
pp. 590 ◽  
Author(s):  
Brian P. Rempel ◽  
Stephen G. Withers
Keyword(s):  
Active Site ◽  
Time Dependent ◽  
Enzyme Active Site ◽  
Radical Bromination ◽  
Dependent Inactivation ◽  
Competitive Inhibitors

Four new difluorosugar fluorides, 2-deoxy-2,5-difluoro-α-l-idopyranosyl fluoride, 1,5-difluoro-d-glucopyranosyl fluoride, 1,5-difluoro-l-idopyranosyl fluoride, and 2-deoxy-1,2-difluoro-d-glucopyranosyl fluoride, were synthesized from known precursors by a radical bromination/fluoride displacement sequence, followed by deprotection. The compounds were tested as time-dependent inactivators of the β-glucosidase from Agrobacterium sp. (Abg, EC 3.2.1.21) and, while they were shown to bind to the enzyme active site as reversible competitive inhibitors, the only time-dependent inactivation observed was traced to the presence of an extremely small amount (<0.1%) of a highly reactive contaminating impurity.

scholarly journals Time-dependent inactivation of chick-embryo prolyl 4-hydroxylase by coumalic acid. Evidence for a syncatalytic mechanism

1987 ◽  
Vol 242 (1) ◽  
pp. 163-169 ◽  
Author(s):  
V Günzler ◽  
H M Hanauske-Abel ◽  
R Myllylä ◽  
J Mohr ◽  
K I Kivirikko
Keyword(s):  
Chick Embryo ◽  
Competitive Inhibition ◽  
Enzymic Activity ◽  
Time Dependent ◽  
Alpha Subunit ◽  
Prolonged Incubation ◽  
Dependent Inactivation ◽  
Competitive Inhibitors ◽  
Inactivation Constant ◽  
High Concentrations

From the structure-activity relationships of known competitive inhibitors, coumalic acid (2-oxo-1,2H-pyran-5-carboxylic acid) was deduced to be a potential syncatalytic inhibitor for chick-embryo prolyl 4-hydroxylase. The compound caused time-dependent inactivation, the reaction rate being first-order. The inactivation constant was 0.094 min-1, the Ki 17 mM and the bimolecular rate constant 0.09 M-1 X S-1. Human prolyl 4-hydroxylase and chick embryo lysyl hydroxylase were also inactivated, though to a lesser extent. Inactivation could be prevented by adding high concentrations of 2-oxoglutarate or its competitive analogues to the reaction mixture. In Lineweaver-Burk kinetics, coumalic acid displayed S-parabolic competitive inhibition with respect to 2-oxoglutarate. The inactivation reaction had cofactor requirements similar to those for the decarboxylation of 2-oxoglutarate. Enzymic activity was partially preserved in the absence of iron, but the rescue was incomplete, owing to decreased stability of the enzyme under this condition. Coumalic acid also decreased the electrophoretic mobility of the alpha-subunit, but the beta-subunit was not affected. Prolonged incubation of coumalic acid above pH 6.8 led to loss of its inactivating potency, owing to hydrolysis. It is concluded that the inactivation of prolyl 4-hydroxylase by coumalic acid is due to a syncatalytic mechanism. The data also suggest that the 2-oxoglutarate-binding site of the enzyme is located within the alpha-subunit.

Regulation of rat liver phosphatidylethanolamine N-methyltransferase by cytosolic factors. Examination of a role for reversible protein phosphorylation

1986 ◽  
Vol 64 (6) ◽  
pp. 565-574 ◽  
Author(s):  
Steven L. Pelech ◽  
Nilay Ozen ◽  
François Audubert ◽  
Dennis E. Vance
Keyword(s):  
Protein Kinase ◽  
Rat Liver ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Methyltransferase Activity ◽  
Dependent Protein Kinase ◽  
Catalytic Subunits ◽  
Phosphatase Inhibitors ◽  
Dependent Protein ◽  
Cytosolic Factors

The nature of cytosolic factors which modulate the activity of rat liver phosphatidylethanolamine (PE) methyltransferase was investigated. The combined additions of cytosol, Mg∙ATP, and NaF to incubations with rat liver microsomes produced a 1.6-fold activation of the methyltransferase at pH 9.2 and a 1.3-fold stimulation at pH 7.0. Nonhydrolyzable 5′ -adenylylimidodiphosphate could not substitute for ATP, although GTP could. The activation was time dependent, stable to reisolation of the microsomes by ultracentrifugation, and partially preventable by other cytosolic components. Despite these indications that PE methyltransferase might be a substrate for cytosolic protein kinases, cAMP and Ca2+–calmodulin exerted little influence on the activation reaction. Furthermore, microsomal PE methyltransferase activity was unaffected by purified preparations of cAMP-dependent protein kinase, calmodulin–dependent protein kinase, and casein kinase II, nor was methyltransferase activity influenced by the purified catalytic subunits of protein phosphatases 1 and 2A. Cytosol also contained inhibitors of PE methyltransferase which could overcome the Mg∙ATP∙NaF-mediated activation of the enzyme, but were not affected by the thermostable phosphatase inhibitors 1 and 2. Part of this inhibitory activity (apparent molecular mass of 15 × 103 daltons) was insensitive to trypsin and chymotrypsin, stimulated by Mn2+, and partly inhibited by NaF. Therefore, regulation of methyltransferase by reversible phosphorylation, while still a tenable hypothesis, is apparently more complex than previously proposed.

scholarly journals Inhibition of cholesterol biosynthesis by Δ22-unsaturated phytosterols via competitive inhibition of sterol Δ24-reductase in mammalian cells

2002 ◽  
Vol 366 (1) ◽  
pp. 109-119 ◽  
Author(s):  
Carlos FERNÁNDEZ ◽  
Yajaira SUÁREZ ◽  
Antonio J. FERRUELO ◽  
Diego GÓMEZ-CORONADO ◽  
Miguel A. LASUNCIÓN
Keyword(s):  
Mammalian Cells ◽  
Competitive Inhibition ◽  
Cholesterol Biosynthesis ◽  
Liver Microsomes ◽  
Side Chain ◽  
Rat Liver Microsomes ◽  
Cholesterol Lowering ◽  
Alkyl Groups ◽  
Competitive Inhibitors ◽  
Physiological Substrates

Dietary phytosterols are cholesterol-lowering agents that interfere with the intestinal absorption of cholesterol. In the present study, we have studied their effects on cholesterol biosynthesis in human cells, particularly in the sterol-conversion pathway. For this, both Caco-2 (intestinal mucosa) and HL-60 (promyelocytic) human cell lines were incubated with [14C]acetate, and the incorporation of radioactivity into sterols was determined using HPLC and radioactivity detection online. Sterols containing a double bond at C-22 in the side chain (stigmasterol, brassicasterol and ergosterol) dramatically inhibited the activity of sterol Δ24-reductase, as indicated by the decrease in radioactivity incorporation into cholesterol and the accumulation of its precursors (mainly desmosterol). Phytosterols with the saturated side chain (β-sitosterol and campesterol) were inactive in this regard. The inhibition of sterol 24-reductase was confirmed in rat liver microsomes by using 14C-labelled desmosterol as the substrate. The 22-unsaturated phytosterols acted as competitive inhibitors of sterol 24-reductase, with Ki values (41.1, 42.7 and 36.8μM for stigmasterol, brassicasterol and ergosterol respectively) similar to the estimated Km for desmosterol (26.3μM). The sterol 5,22-cholestedien-3β-ol, an unusual desmosterol isomer that lacks the alkyl groups characteristic of phytosterols, acted as a much stronger inhibitor of 24-reductase (Ki = 3.34μM). The usually low intracellular concentrations of the physiological substrates of 24-reductase explains the strong inhibition of cholesterol biosynthesis that these compounds exert in cells. Given that inhibition of sterol 24-reductase was achieved at physiologically relevant concentrations, it may represent an additional mechanism for the cholesterol-lowering action of phytosterols, and opens up the possibility of using certain 22-unsaturated sterols as effective hypocholesterolaemic agents.

Time dependent changes occurring in rat liver microsomes upon lipid peroxidation

Lipids ◽  
1989 ◽  
Vol 24 (10) ◽  
pp. 905-908 ◽  
Author(s):  
Fumiaki Ifoh ◽  
Yoshiyuki Minamide ◽  
Toshiharu Horie ◽  
Shoji Awazu
Keyword(s):  
Lipid Peroxidation ◽  
Rat Liver ◽  
Liver Microsomes ◽  
Time Dependent ◽  
Rat Liver Microsomes
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