Synaptosomal Na+,K+-ATPase as a membrane probe in studying the in vivo action of morphine

1981 ◽  
Vol 59 (8) ◽  
pp. 687-692 ◽  
Author(s):  
Ophelia Wan-Kan ◽  
E. A. Hosein
Keyword(s):  
Activation Energy ◽  
Enzyme Activity ◽  
Transition Temperature ◽  
Specific Activity ◽  
Membrane Phospholipids ◽  
Membrane Probe ◽  
Biphasic Effect ◽  
Brain Synaptosomes ◽  
Membrane Bound

The activity of membrane-bound Na+,K+-ATPase was used as a metabolic probe to study the effects of morphine in vivo in rat brain synaptosomes. Arrhenius plots were generated to study an induced perturbation within the membrane. In acute studies 0.5-h postmorphine, the drug was without effect on the basal activity of the enzyme. With dopamine-stimulated Na+,K+-ATPase morphine decreased the apparent transition temperature and specific activity of the enzyme while there was a slight stimulation in its activation energy. An increase in these parameters was observed in samples taken from animals withdrawn from the drug for 48 h. These results strongly suggest the possible involvement of the membrane phospholipids as transducer which mediates the observed biphasic effect of the drug on enzyme activity.

scholarly journals Human erythrocyte acetylcholinesterase in relation to cell age

1981 ◽  
Vol 195 (1) ◽  
pp. 221-228 ◽  
Author(s):  
D A Galbraith ◽  
D C Watts
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Kinetic Studies ◽  
Acetylcholinesterase Activity ◽  
Maximum Activity ◽  
Activity Profile ◽  
Cell Age ◽  
Cell Life ◽  
Adults And Children

Acetylcholinesterase was studied in human red cells that had been fractionated on Ficoll/Triosil density gradients into classes representing different ages in vivo. Reticulocytes have negligible acetylcholinesterase activity; this is rapidly acquired on maturation to the erythrocyte. The activity per cell reaches a maximum and then, after a constant period, declines again towards the end of cell life. The maximum activity and the rates of activity gain and loss per cell are quantitatively different in adults and children. Kinetic studies showed that Vmax. follows the same age/activity profile but Km is unaffected by cell age. The acetylcholinesterase protein content, determined by quantitative crossed immunoelectrophoresis, also shows a profile of increase and then decrease with cell age but the specific activity calculated from the protein estimate shows a reverse picture in which there is a slight decrease from young to mid-age cells followed by an increase again in older cells. These results are interpreted to indicate a complex developmental picture in which the overall cell age against enzyme activity profile is determined partly by the amount of enzyme protein present and partly from the modifying effect on the enzyme activity, of interactions with an aging cell membrane.

scholarly journals Effect of liposomal phospholipid composition on cholesterol transfer between microsomal and liposomal vesicles

1986 ◽  
Vol 238 (3) ◽  
pp. 647-652 ◽  
Author(s):  
C Bhuvaneswaran ◽  
K A Mitropoulos
Keyword(s):  
Activation Energy ◽  
Enzyme Activity ◽  
Control System ◽  
Phospholipid Composition ◽  
Acyltransferase Activity ◽  
Phosphatidylcholine Liposomes ◽  
First Order ◽  
First Order Kinetics ◽  
Phospholipid Liposomes

Preincubation of rat liver microsomal vesicles at 37 degrees C in the presence of [3H]cholesterol/phospholipid liposomes results in a net transfer of cholesterol from liposomes to microsomal vesicles. This transfer follows first-order kinetics. For similar concentrations of the donor vesicles, rates of transfer are about 6-8 times lower with cholesterol/sphingomyelin liposomes compared with cholesterol/phosphatidylcholine liposomes. Also, transfer of cholesterol from cholesterol/sphingomyelin liposomes to microsomal vesicles reveals a larger activation energy than for the process from cholesterol/phosphatidylcholine liposomes. There is a significant correlation between the amount of liposomal cholesterol transferred to microsomal vesicles during preincubation and the increase found with acyl-CoA:cholesterol acyltransferase activity in these microsomes over their corresponding controls. If, however, liposomes made solely of phospholipids are substituted for the cholesterol/phospholipid liposomes in the preincubation system containing microsomal vesicles, then the acyl-CoA:cholesterol acyltransferase activity is decreased compared with the corresponding control system. Both sphingomyelin and phosphatidylcholine liposomes are equally effective in decreasing the enzyme activity. These results offer direct kinetic evidence for the positive correlation between cholesterol and sphingomyelin found in vivo in biological membranes.

scholarly journals A major role for noncoding regulatory mutations in the evolution of enzyme activity

2019 ◽  
Vol 116 (25) ◽  
pp. 12383-12389 ◽  
Author(s):  
David W. Loehlin ◽  
Jesse R. Ames ◽  
Kathy Vaccaro ◽  
Sean B. Carroll
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Functional Divergence ◽  
Primary Source ◽  
Protein Activity ◽  
Genetic Changes ◽  
Gene Enhancer ◽  
Ethanol Toxicity ◽  
Regulatory Mutations

The quantitative evolution of protein activity is a common phenomenon, yet we know little about any general mechanistic tendencies that underlie it. For example, an increase (or decrease) in enzyme activity may evolve from changes in protein sequence that alter specific activity, or from changes in gene expression that alter the amount of protein produced. The latter in turn could arise via mutations that affect gene transcription, posttranscriptional processes, or copy number. Here, to determine the types of genetic changes underlying the quantitative evolution of protein activity, we dissected the basis of ecologically relevant differences in Alcohol dehydrogenase (Adh) enzyme activity between and within several Drosophila species. By using recombinant Adh transgenes to map the functional divergence of ADH enzyme activity in vivo, we find that amino acid substitutions explain only a minority (0 to 25%) of between- and within-species differences in enzyme activity. Instead, noncoding substitutions that occur across many parts of the gene (enhancer, promoter, and 5′ and 3′ untranslated regions) account for the majority of activity differences. Surprisingly, one substitution in a transcriptional Initiator element has occurred in parallel in two species, indicating that core promoters can be an important natural source of the tuning of gene activity. Furthermore, we show that both regulatory and coding substitutions contribute to fitness (resistance to ethanol toxicity). Although qualitative changes in protein specificity necessarily derive from coding mutations, these results suggest that regulatory mutations may be the primary source of quantitative changes in protein activity, a possibility overlooked in most analyses of protein evolution.

scholarly journals Alpha-tocopheryl succinate (α-TOS) influences cell vitality and enzyme activity in Ehrlich ascites carcinoma cells

2007 ◽  
Vol 15 (3-4) ◽  
pp. 65-68
Author(s):  
Karmen Stankov ◽  
Katica Bajin-Katic ◽  
Bojan Stanimirov ◽  
Dunja Karadzic ◽  
Zoran Kovacevic
Keyword(s):  
Enzyme Activity ◽  
Anticancer Agents ◽  
Specific Activity ◽  
Ehrlich Ascites Carcinoma ◽  
Carcinoma Cells ◽  
Malignant Cells ◽  
Ehrlich Ascites ◽  
Tocopheryl Succinate ◽  
Eac Cells

Background: One of the most important strategies in research and development of new anticancer agents is the tumor-specific induction of apoptosis. The effects of semisynthetic derivative of vitamin E, (?-TOS, D-?-tocopheryl succinate), appear to be largely restricted to malignant cells. Methods: We investigated the in vivo effects of intraperitoneally administered ?-TOS on vitality of Ehrlich ascites carcinoma cells (EAC) in mice, as well as the influence of ?-TOS on specific activity of enzymes involved in antioxidative mechanisms in EAC cells. Results: According to our results, the intraperitoneal application of ?-TOS induces the decrease of the EAC vitality, and the statistically significant alteration of the glutathione-dependent enzyme activity in EAC cells. Conclusion: We may conclude that ?-TOS is an important micronutrient, with significant impact on vitality and metabolism of malignant cells.

scholarly journals Compartmentalized system with membrane-bound glycerol kinase. Activity and product distribution versus asymmetrical substrate supply

1991 ◽  
Vol 274 (3) ◽  
pp. 819-824 ◽  
Author(s):  
A Girard ◽  
B Merchie ◽  
B Maïsterrena
Keyword(s):  
Enzyme Activity ◽  
Diffusion Layer ◽  
Cumulative Effect ◽  
Product Distribution ◽  
Substrate Supply ◽  
Enzyme Model ◽  
Membrane Bound ◽  
The Asymmetry

An artificial-membrane-bound glycerokinase chosen as a membrane-bound two-substrate-enzyme model has been used to separate two unequal compartments of a specially designed diffusion cell. An interesting feature is the asymmetry of compartments and the existence of a diffusion layer adjacent to only one face of the enzymic membrane. In such a situation the apparent enzyme activity and the product distribution in the system have been studied versus all the possibilities of combination of ATP and glycerol supply. Our approach has lead us to differentiate two different roles played by a diffusion layer adjacent to a permeable enzymic membrane. Depending on the spatial origin of the enzymic substrates (i.e. from which compartment they derive), the diffusion layer can play either the role of a passive additional resistance to that of the membrane or the role of a third compartment in which the reaction product can partially accumulate before splitting on both parts of the membrane. Our results mainly demonstrate that a membrane-bound enzyme activity and the resulting product distribution occurring in a compartmentalized system may be regulated by the cumulative effect due to the asymmetry in volumes of the compartments, the presence of a diffusion layer and the different possibilities of substrate supply. With the topography studied, which is close to that reported for many ‘in vivo’ situations, the product may be diffused lead to vectorial metabolism processes.

scholarly journals THE ENZYMATIC DEGRADATION OF HEMOGLOBIN TO BILE PIGMENTS BY MACROPHAGES

1971 ◽  
Vol 133 (6) ◽  
pp. 1264-1281 ◽  
Author(s):  
Neville R. Pimstone ◽  
Raimo Tenhunen ◽  
Paul T. Seitz ◽  
Harvey S. Marver ◽  
Rudi Schmid
Keyword(s):  
Enzyme Activity ◽  
Heme Oxygenase ◽  
Specific Activity ◽  
Indirect Evidence ◽  
Reticuloendothelial System ◽  
Bile Pigments ◽  
Absolute Requirement ◽  
Oxygenase Activity ◽  
Alternate Routes

Recent studies have identified and characterized the enzymatic mechanism by which hemoglobin-heme is converted to bilirubin. Under physiologic conditions the enzyme system, microsomal heme-oxygenase, is most active in the spleen followed by the liver and bone marrow, all of which are tissues that normally are involved in the sequestration and metabolism of red cells. Indirect evidence suggested that the reticuloendothelial system is important in this process. To test this hypothesis, conversion of heme to bilirubin was studied in macrophages obtained by chemical or immunological means from the peritoneal cavity or from the lungs of rodents. Homogenates of pure populations of these cells were devoid of heme-oxygenase activity, unless before harvesting the macrophages had been exposed to methemalbumin, microcrystalline hemin, or hemoglobin in vivo. In macrophages exposed to heme pigments, the specific activity of heme-oxygenase was far in excess of that in the spleen or liver. Enzyme activity was also present in the granulomatous tissue surrounding subcutaneous hematomas. The heme-oxygenase system in macrophages resembles that in the spleen and liver in that it is localized in the microsomal fraction, has an absolute requirement for molecular oxygen and NADPH, is inhibited by carbon monoxide, and has a similar Km. These findings indicate that cells of the reticuloendothelial system, presumably including the Kupffer cells of the liver and the macrophages of the spleen, possess the enzymatic machinery for converting hemoglobin-heme to bilirubin. The reaction is a mixed function oxidation, probably involving cytochrome P450 as the terminal oxidase. Enzyme activity in macrophages is capable of regulatory adaptation in response to substrate loads. In the standard assay system for the enzyme, disappearance of heme always was in excess of the amount of bilirubin formed, suggesting the simultaneous presence of alternate routes of heme degradation not involving bilirubin as an end product or intermediate.

scholarly journals Effects of calcium on phosphatidylserine- and saposin C-stimulated glucosylceramide β-glucosidase activity

1995 ◽  
Vol 310 (2) ◽  
pp. 571-575 ◽  
Author(s):  
E M Prence
Keyword(s):  
Enzyme Activity ◽  
Fold Increase ◽  
Sphingolipid Metabolism ◽  
Lysosomal Hydrolase ◽  
Activator Protein ◽  
Glucosidase Activity ◽  
Saposin C ◽  
Membrane Bound ◽  
Beta Glucosidase

Glucosylceramide beta-glucosidase is a membrane-bound lysosomal hydrolase that is activated by acidic lipids, the most effective of which is phosphatidylserine (PtdSer), and an activator protein, saposin C. This report documents effects of Ca2+ ions on PtdSer- and saposin C-enhanced beta-glucosidase activity. Ca2+ either increased or decreased enzyme activity, depending on (1) the concentration of phospholipid, and (2) the presence or absence of saposin C. At PtdSer concentrations between 7.6 and 76 microM, in the absence of saposin C, Ca2+ caused an increase in beta-glucosidase activity up to 3 times that measured with PtdSer alone; this was due to both an increase in Vmax, and a decrease in Km. In contrast, at PtdSer concentrations greater than 100 microM, Ca2+ inhibited beta-glucosidase activity by 50%, due to a 2-fold increase in Km. Ca2+ was inhibitory at all PtdSer concentrations tested when both PtdSer and saposin C were present in the assay. Ca2+ ions were also shown to cause changes in the aggregation states of PtdSer. These results suggest that changes in Ca2+ concentration may play a role in regulating beta-glucosidase activity in vivo, thereby modulating sphingolipid metabolism. The implications of these findings are discussed.

On the role of octopine dehydrogenase in cephalopod mantle muscle metabolism

1976 ◽  
Vol 54 (6) ◽  
pp. 871-878 ◽  
Author(s):  
Jeremy H. A. Fields ◽  
John Baldwin ◽  
Peter W. Hochachka
Keyword(s):  
Enzyme Activity ◽  
Muscle Activity ◽  
Specific Activity ◽  
Ph Dependence ◽  
Muscle Metabolism ◽  
Ph Optimum ◽  
Wet Weight ◽  
Octopine Dehydrogenase

Octopine dehydrogenases from the mantle muscle of the squid, Symplectoteuthis oualaniensis, and of the octopus, Octopus ornatus, were kinetically characterized and compared. In the squid, the specific activity of the enzyme was about 110 μmol product formed per minute per gram wet weight; in the octopus that value was over 600. Both enzymes show similar pH dependence; in the direction of octopine formation the pH optimum was about 6.5, whereas in the direction of octopine oxidation it was about 8.5. The affinities for NADH, arginine, and pyruvate were similar (Km values were about 0.04 mM, 7 mM, and 2 mM respectively). Increasing the concentration of either arginine or pyruvate increased the affinity for the cosubstrate (pyruvate or arginine), this mechanism being a means of regulating the enzyme activity in vivo. In the direction of octopine oxidation, the octopus enzyme showed a much higher affinity for octopine (Km = 0.8 mM) than did the squid enzyme (Km = 4.4 mM), suggesting that it may be better geared for reconverting octopine to arginine and pyruvate after anaerobic bursts of muscle activity.

Effect of phosphatidylinositol and phosphatidylserine on membrane-bound galactosyltransferase

1980 ◽  
Vol 58 (1) ◽  
pp. 58-66 ◽  
Author(s):  
Samuel Ratnam ◽  
Ian H. Fraser ◽  
Sailen Mookerjea
Keyword(s):  
Enzyme Activity ◽  
Phosphatidyl Serine ◽  
High Concentration ◽  
Electron Microscopic ◽  
Membrane Solubilization ◽  
Wide Range ◽  
Biphasic Effect ◽  
Solubilized Enzyme ◽  
Membrane Bound ◽  
Triton X

Membrane-bound galactosyltransferase is solubilized and activated by exogenous lysolecithin or Triton X-100. A study on the effect of different phospholipids on the lysolecithin-solubilized enzyme showed that two charged phospholipids (i.e., phosphatidylinositol and phosphatidyl-serine) inhibited the membrane-bound enzyme in the presence of a wide range of lysolecithin concentration (up to 6 μmol/mg protein). In contrast, these phospholipids produced a biphasic effect on the enzyme solubilized with Triton X-100. In lower concentration of Triton (up to 2 μmol/mg protein), the charged phospholipids somewhat reduced the enzyme activity but a reversal of this effect was observed when Triton concentration was gradually raised (from 2 to 8 μmol/mg protein). This biphasic effect of the phospholipids was also demonstrated on purified membrane-bound galactosyltransferase in presence of low and high concentration of Triton. Electron microscopic evidence suggested that an increased concentration of phosphatidylinositol prevented membrane solubilization by lysolecithin or retained the membrane vesicular organization concurrent with a restraining effect on the enzyme. The results lend support to the hypothesis that the phospholipid microenvironment of the membrane may exert a control on the membrane-bound glycosyltransferases.

scholarly journals Developmental and biochemical characteristics of the cardiac membrane-bound arginine-specific mono-ADP-ribosyltransferase

1993 ◽  
Vol 293 (3) ◽  
pp. 789-793 ◽  
Author(s):  
K K McMahon ◽  
K J Piron ◽  
V T Ha ◽  
A T Fullerton
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Developmentally Regulated ◽  
Adp Ribosylation ◽  
Cardiac Membrane ◽  
Cardiac Membranes ◽  
Membrane Bound ◽  
Species Specific ◽  
Adp Ribosyltransferase ◽  
Dose Dependent

ADP-ribosylation of protein in heart membrane preparations has been shown to be present in adult tissue but absent from early neonate tissue [Piron and McMahon (1990) Biochem. J. 270, 591-597]. To further this observation, the cardiac membrane-bound form of arginine-specific mono-ADP-ribosyltransferase (EC 2.4.2.31) has been characterized. Apparent Km values of 330 and 470 microM were found in heart membrane preparations from rat and quail respectively. The Vmax. value depended greatly on the species of animal studied, and was 1.1 and 48 nmol/min per mg in rat and quail preparations respectively. The specific activity of the enzyme was lowest in pig, intermediate in rat, dog and rabbit, and highest in mouse and quail cardiac membranes. In the rat, the ADP-ribosylation of protein and enzyme activity were very low in heart preparations from 1-15-day-old animals. Thereafter the ADP-ribosylation and enzyme activity increased gradually to adulthood. Bacillus cereus phosphatidylinositol-specific phospholipase C, known to hydrolyse glycosylphosphatidylinositol anchors of proteins, released the mono-ADP-ribosyltransferase from membrane preparations of both rat and quail in a dose-dependent, Zn(2+)-inhibited manner. Thus it appears that a membrane-bound form of arginine-specific mono-ADP-ribosyltransferase is present in heart membranes from a variety of species and is not species-specific. The activity of this ADP-ribosyltransferase appears to be developmentally regulated and to be bound to the cardiac membranes by a glycosylphosphatidylinositol anchor.

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