Evoked effects of cholesterol binding on integral proteins and lipid fluidity of dog brain synaptosomal plasma membranes

1989 ◽  
Vol 67 (1) ◽  
pp. 16-24 ◽  
Author(s):  
George Deliconstantinos ◽  
Lioudmila Kopeikina ◽  
Vassiliki Villiotou
Keyword(s):  
Fluorescence Polarization ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Break Point ◽  
Hill Coefficient ◽  
Dog Brain ◽  
Integral Proteins ◽  
The Hill ◽  
Synaptosomal Plasma Membranes ◽  
Allosteric Properties

Binding of cholesterol into dog brain synaptosomal plasma membranes (SPM) within the limits of concentration used (0.5–5 μM) follows an exponential curve described by the general formula y = a∙ebx. This curve, which represents the total binding (specific and nonspecific), acquires sigmoid character in the presence of 100 μM cholesterol glucoside, with a Hill coefficient of h = 2.98 ± 0.18. The specific activity of the Na+/K+-transporting ATPase and Ca2+-transporting ATPase rose after a 2-h preincubation of SPM with cholesterol (up to 5 μM) or its glucoside (up to 50 μM) to at least 50% above their original values. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) increased with cholesterol glucoside (50 μM) incorporation. Cholesterol (5 μM) had no effect on the DPH fluorescence polarization. Arrhenius plots of Na+/K+-transporting ATPase activity exhibited a break point at 23.2 ± 1.1 °C in control SPM, which was elevated to 29.5 ± 1.4 °C in SPM treated with cholesterol glucoside (50 μM) and abolished in SPM treated with cholesterol (5 μM). The allosteric properties of SPM-bound Na+/K+-transporting ATPase inhibited by F− and Ca2+-transporting ATPase inhibited by Na+ (as reflected by changes in the Hill coefficient) were modulated by cholesterol. It could be stated that cholesterol glucoside (50 μM) produced an increased packing of the bulk lipids, while cholesterol (5 μM) increased the fluidity of the lipid microenvironment of both Na+/K+-transporting ATPase and Ca2+-transporting ATPase.Key words: cholesterol, cholesterol glucoside, (NA+/K+)ATPase, Ca2+-ATPase, membrane fluidity, fluorescence polarization.

scholarly journals Differential effect of anionic and cationic drugs on the synaptosome-associated acetylcholinesterase activity of dog brain

1985 ◽  
Vol 229 (1) ◽  
pp. 81-86 ◽  
Author(s):  
G Deliconstantinos ◽  
S Tsakiris
Keyword(s):  
Enzyme Activity ◽  
Plasma Membranes ◽  
Barbituric Acid ◽  
Acetylcholinesterase Activity ◽  
Break Point ◽  
Low Concentrations ◽  
Hill Coefficients ◽  
The Hill ◽  
Synaptosomal Plasma Membranes ◽  
Positively Charged

The evoked effects of the negatively charged drugs phenobarbital and barbituric acid, the positively charged imipramine, perphenazine and trifluoperazine, and the neutral primidone, on the synaptosome-associated acetylcholinesterase activity were studied. A marked increase in the enzyme activity was exhibited in the presence of low concentrations (up to 3 mM) of phenobarbital, barbituric acid and primidone. Higher concentrations (up to 10 mM), however, led to a progressive inhibition of the enzyme activity. However, the activity of the enzyme was not affected by imipramine, but it was decreased by perphenazine and trifluoperazine. Arrhenius plots of acetylcholinesterase activity exhibited a break point at 23.4 degrees C for the untreated (control) synaptosomes, which was shifted to around 16 degrees C in the synaptosomes treated with the charged drugs. The allosteric inhibition by F- of acetylcholinesterase was studied in control synaptosomes and in those treated with the charged drugs. Changes in the Hill coefficients in combination with changes in Arrhenius activation energy produced by the charged drugs would be expected if it is assumed that charged drugs ‘fluidize’ the synaptosomal plasma membranes.

scholarly journals Influence of phosphatidylserine on (Na+ + K+)-stimulated ATPase and acetylcholinesterase activities of dog brain synaptosomal plasma membranes

1984 ◽  
Vol 220 (1) ◽  
pp. 301-307 ◽  
Author(s):  
S Tsakiris ◽  
G Deliconstantinos
Keyword(s):  
Protein Interactions ◽  
Plasma Membranes ◽  
Physiological Processes ◽  
Maximal Stimulation ◽  
Dog Brain ◽  
The Central Nervous System ◽  
Hill Coefficients ◽  
Lipid Protein Interactions ◽  
Synaptosomal Plasma Membranes ◽  
Allosteric Properties

Phosphatidylserine (PtdSer) incubated with synaptosomal plasma membranes (SPM) of dog brain is incorporated into SPM in proportion to its concentration in the incubation medium. Low PtdSer concentrations progressively activated the SPM-associated (Na+ + K+)-stimulated ATPase and acetylcholinesterase. Increasing the PtdSer concentration above that which maximally stimulated the enzyme activities effected a progressive inhibition with respect to maximal stimulation. Arrhenius plots of (Na+ + K+ + Mg2+)-dependent ATPase and 5′-nucleotidase revealed a clear break at 23-24 degrees C for both enzymes in SPM untreated with PtdSer (controls), whereas a linear relation was obtained for SPM treated with PtdSer. Changes in the allosteric properties of (Na+ + K+)-stimulated ATPase by fluoride (F-) and/or of 5′-nucleotidase by concanavalin A (i.e. changes of Hill coefficients) indicate that PtdSer increases the membrane fluidity. These results suggest that modifications of lipid-protein interactions in SPM induced by PtdSer may have implications in the physiological processes in the central nervous system.

scholarly journals Kinetic studies on the regulation of rabbit liver pyruvate kinase

1973 ◽  
Vol 131 (2) ◽  
pp. 287-301 ◽  
Author(s):  
M. G. Irving ◽  
J. F. Williams
Keyword(s):  
Pyruvate Kinase ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Kinetic Studies ◽  
Rabbit Liver ◽  
Hill Coefficient ◽  
Saturation Curve ◽  
Low Concentrations ◽  
Ammonium Sulphate Fractionation ◽  
The Hill

Two kinetically distinct forms of pyruvate kinase (EC 2.7.1.40) were isolated from rabbit liver by using differential ammonium sulphate fractionation. The L or liver form, which is allosterically activated by fructose 1,6-diphosphate, was partially purified by DEAE-cellulose chromatography to give a maximum specific activity of 20 units/mg. The L form was allosterically activated by K+ and optimum activity was recorded with 30mm-K+, 4mm-MgADP-, with a MgADP-/ADP2- ratio of 50:1, but inhibition occurred with K+ concentrations in excess of 60mm. No inhibition occurred with either ATP or GTP when excess of Mg2+ was added to counteract chelation by these ligands. Alanine (2.5mm) caused 50% inhibition at low concentrations of phosphoenolpyruvate (0.15mm). The homotropic effector, phosphoenolpyruvate, exhibited a complex allosteric pattern (nH+2.5), and negative co-operative interactions were observed in the presence of low concentrations of this substrate. The degree of this co-operative interaction was pH-dependent, with the Hill coefficient increasing from 1.1 to 3.2 as the pH was raised from 6.5 to 8.0. Fructose 1,6-diphosphate interfered with the activation by univalent ions, markedly decreased the apparent Km for phosphoenolpyruvate from 1.2mm to 0.2mm, and transformed the phosphoenolpyruvate saturation curve into a hyperbola. Concentrations of fructose 1,6-diphosphate in excess of 0.5mm inhibited this stimulated reaction. The M or muscle-type form of the enzyme was not activated by fructose 1,6-diphosphate and gave a maximum specific activity of 0.3 unit/mg. A Michaelis–Menten response was obtained when phosphoenolpyruvate was the variable substrate (Km+0.125mm), and this form was inhibited by ATP, as well as alanine, even in the presence of excess of Mg2+.

Stimulation of Brain Synaptosome - Associated Adenylate Cyclase by Acidic Phospholipids

1984 ◽  
Vol 39 (11-12) ◽  
pp. 1196-1198 ◽  
Author(s):  
Stylianos Tsakiris
Keyword(s):  
Enzyme Activity ◽  
Adenylate Cyclase ◽  
Plasma Membranes ◽  
Protein S ◽  
Enzyme Activation ◽  
Dog Brain ◽  
The Central Nervous System ◽  
Acidic Phospholipids ◽  
Synaptosomal Plasma Membranes ◽  
Stimulation Of

Phosphatidylserine (PS), phosphatidylinositol (PIN) or phosphatidylglycerol (PGL) incubated with synaptosomal plasma membranes (SPM) of dog brain, stimulated adenylate cyclase. The enzyme activity showed a dramatic increase at around 1.6 μmol PS/mg protein, while use of higher concentrations led to inhibition of the activity with respect to the maximal percentage of stimulation. Moreover, PS stimulated the dopamine-sensitive adenylate cyclase. Solubilization of SPM by the detergent Lubrol-PX did not affect the enzyme activation induced by dopamine. The solubilization, also, showed that the enzyme activity does not change at any PS, PIN or PGL concentration used. These results indicate that acidic phospholipids do not directly act on adenylate cyclase, but indirectly, affecting the membrane fluidity probably. Such modifications of interactions through lipid-protein(s) of adenylate cyclase may have implications to physiological responses to hormones or/and neurotransm itters in the central nervous system.

Enterocyte alpha 2-adrenergic receptors: yohimbine and p-aminoclonidine binding relative to ion transport

1983 ◽  
Vol 244 (1) ◽  
pp. G76-G82 ◽  
Author(s):  
E. B. Chang ◽  
M. Field ◽  
R. J. Miller
Keyword(s):  
Plasma Membranes ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Hill Coefficient ◽  
Scatchard Analysis ◽  
Competitive Displacement ◽  
Single Receptor ◽  
Tissue Systems ◽  
The Hill ◽  
Best Fit

We previously reported that alpha 2-adrenergic agonists enhance absorption and inhibit secretion of electrolytes in small intestine. The present study was undertaken to characterize and localize the relevant receptors. Plasma membranes derived from isolated rabbit ileal epithelial cells were incubated with either [3H]yohimbine (Yo), an alpha 2-antagonist, or p-[3H]aminoclonidine (PAC), an alpha 2-agonist. Scatchard analysis of [3H]Yo binding suggests a single receptor. Competitive displacement of Yo from this receptor by other ligands had a potency order characteristic for alpha 2-receptors in other tissue systems. A Scatchard plot of [3H]PAC binding was curvilinear and best fit by assuming two independent site. Competitive displacement of [3H]PAC by PAC in the presence of 140 mM Na+ or 0.1 mM GTP increased the IC50 for PAC binding from 10 nM to 100 and 105 nM, respectively, and the Hill coefficient from 0.7 to 1.2 and 1.0, respectively. The ED50 for PAC effect on short-circuit current (200 nM) does not differ significantly from these values. We conclude that alpha 2-receptors are present on ileal enterocytes and that these receptors mediate enterocyte fluid and electrolyte transport function.

Effect of prostaglandins E2 and F2α on membrane calcium binding, Ca2+/Mg2+-ATPase activity and membrane fluidity in rat myometrial plasma membranes

1986 ◽  
Vol 110 (3) ◽  
pp. 395-404 ◽  
Author(s):  
G. Deliconstantinos ◽  
S. Fotiou
Keyword(s):  
Membrane Fluidity ◽  
Calcium Binding ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Gradient Centrifugation ◽  
Hill Coefficient ◽  
Uterine Contractions ◽  
Enzymatic Regulation ◽  
Discontinuous Sucrose Gradient ◽  
The Hill

ABSTRACT Myometrial plasma membrane (MPM) preparations from rats treated with oestradiol were obtained by discontinuous sucrose-gradient centrifugation. The preparations contained calcium-stimulated and magnesium-dependent ATPase (Ca2+/Mg2+-ATPase). A dramatic decrease in the activity of Ca2+/Mg2+-ATPase was observed when preparations were treated with 0·025–10μmol protaglandins E2 and F2α (PGE2 and PGF2α)/l. In contrast, there was a marked increase in MPM-bound 5′-nucleotidase activity at low concentrations (up to 2 μmol/l) of PGE2 and PGF2α; higher concentrations (up to 10 μmol/l), however, led to a progressive inhibition of enzyme activity. Association (specific and non-specific binding) of PGE2 and PGF2α with MPM at pH 7 was found to require Ca2+ (half-maximal concentration approximately 0·7 mmol/l). Changes in the allosteric properties of MPM-bound 5′-nucleotidase by concanavalin A (as reflected by changes in the Hill coefficient) indicated a fluidization of the membrane induced by PGE2 and PGF2α. The steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene-labelled MPM decreased in PGE2- and PGF2α-treated MPM from 1·24 ± 0·04 (s.d.) to 0·66 ± 0·01 and 0·74 ± 0·01 respectively, which is consistent with a general increase in membrane fluidity. It is suggested that PGE2 and PGF2α promote changes in the physical properties of MPM which may be relevant to the induction of uterine contractions by enzymatic regulation of intracellular calcium concentrations. J. Endocr. (1986) 110, 395–404

scholarly journals Alterations in the activities of hepatic plasma-membrane and microsomal enzymes during liver regeneration

1983 ◽  
Vol 212 (2) ◽  
pp. 445-452 ◽  
Author(s):  
G Deliconstantinos ◽  
G Ramantanis
Keyword(s):  
Plasma Membrane ◽  
Liver Regeneration ◽  
Membrane Fluidity ◽  
Plasma Membranes ◽  
Membrane Lipids ◽  
Biological Significance ◽  
Hill Coefficient ◽  
Membrane Bound ◽  
The Hill ◽  
Membrane Bound Enzymes

A marked increase in the activities of rat liver plasma-membrane (Na+ + K+)-stimulated ATPase and microsomal Ca2+-stimulated ATPase was observed 18h after partial hepatectomy. Lipid analyses for both membrane preparations reveal that in partially hepatectomized rats the cholesterol and sphingomyelin content are decreased with a subsequent decrease in the cholesterol/phospholipid molar ratio compared with those of sham-operated animals. Changes in the allosteric properties of plasma-membrane (Na+ + K+)-stimulated ATPase by F- (as reflected by changes in the Hill coefficient) indicated a fluidization of the lipid bilayer of both membrane preparations in 18 h-regenerating liver. The amphipathic dodecyl glucoside incorporated into the hepatic plasma membranes evoked a marked increase in the (Na+ + K+)-stimulated ATPase and 5′-nucleotidase activities. The lack of effect of the glucoside on the Lubrol-PX-solubilized 5′-nucleotidase indicates that changes in the activities of the membrane-bound enzymes caused by the glucoside are due to modulation of the membrane fluidity. Dodecyl glucoside appears to increase the membrane fluidity, evaluated through changes in the Hill coefficient for plasma-membrane (Na+ + K+)-stimulated ATPase. The biological significance of these data is discussed in terms of the differences and changes in the interaction of membrane-bound enzymes with membrane lipids during liver regeneration.

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