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.

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 In vivo analysis reveals substrate-gating mutants of a rhomboid intramembrane protease display increased activity in living cells

2008 ◽  
Vol 389 (8) ◽  
Author(s):  
Sinisa Urban ◽  
Rosanna P. Baker
Keyword(s):  
Enzyme Activity ◽  
Active Site ◽  
Membrane Lipids ◽  
Fold Increase ◽  
Living Cells ◽  
Transmembrane Segment ◽  
Intramembrane Proteases ◽  
Gating Mechanism

Abstract Intramembrane proteases hydrolyze peptide bonds within cell membranes. Recent crystal structures revealed that rhomboid intramembrane proteases contain a hydrated active site that opens to the outside of the cell, but is protected laterally from membrane lipids by protein segments. Using Escherichia coli rhomboid (GlpG) structures as a guide, we previously took a mutational approach to identify the GlpG gating mechanism that allows substrates to enter the active site laterally from the membrane. Mutations that weaken contacts keeping the gate closed increase enzyme activity and implicate transmembrane segment 5 as the substrate gate. Since these analyses were performed in vitro with pure proteins in detergent micelles, we have now examined GlpG in its natural environment, within the membrane of live E. coli cells. In striking congruity with in vitro analysis, gate-opening mutants in transmembrane segment 5 display up to a 10-fold increase in protease activity in living cells. Conversely, mutations in other parts of the protease, including the membrane-inserted L1 loop previously thought to be the gate, decrease enzyme activity. These observations provide evidence for the existence of both closed and open forms of GlpG in cells, and show that inter-conversion between them via substrate gating is rate limiting physiologically.

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 Rapid partial purification of placental glucocerebroside β-glucosidase and its entrapment in liposomes

1977 ◽  
Vol 164 (2) ◽  
pp. 439-445 ◽  
Author(s):  
I P Braidman ◽  
G Gregoriadis
Keyword(s):  
Enzyme Activity ◽  
Organic Solvent ◽  
Affinity Chromatography ◽  
Rapid Method ◽  
Concanavalin A ◽  
Enzyme Preparation ◽  
Partial Purification ◽  
Glucosidase Activity ◽  
Beta Glucosidase

1. A glucocerebroside beta-glucosidase-rich detergent-free preparation was obtained from human placentas by a rapid method combining affinity chromatography on concanavalin A-Sepharose and organic-solvent precipitation. In a typical preparation about 11000 units of the enzyme purified 1500-fold were obtained from five placentas in 2 days. 2. The enzyme preparation also contained other hydrolases, but the extent of their purification was much smaller. 3. Studies on entrapment in liposomes showed that all glucocerebroside beta-glucosidase activity used could be incorporated in neutral egg phosphatidylcholine-cholesterol liposomes. Association with liposomes appeared to discriminate against other proteins, including some of the hydrolases, thus contributing to further purification of the enzyme. More than 95% of the liposome-associated enzyme activity was latent.

scholarly journals Dictyostelium discoideum contains three inositol monophosphatase activities with different substrate specificities and sensitivities to lithium

1996 ◽  
Vol 314 (2) ◽  
pp. 491-495 ◽  
Author(s):  
Peter VAN DIJKEN ◽  
Jan C. T. BERGSMA ◽  
Hoebert S. HIEMSTRA ◽  
Berber DE VRIES ◽  
Jeroen VAN DER KAAY ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Dictyostelium Discoideum ◽  
Mammalian Cells ◽  
Biological Effects ◽  
Fold Increase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Inositol Monophosphatase

The small ion lithium, a very effective agent in the treatment of manic depressive patients, inhibits the mammalian enzyme inositol monophosphatase, which is proposed as the biological target for the effects of lithium. In this study we investigated Dictyostelium discoideum inositol monophosphatase activity. Partial purification of the proteins in the soluble cell fraction using anion-exchange chromatography revealed the presence of at least three enzyme activities capable of degrading inositol monophosphate isomers. The first activity was similar to the monophosphatase found in mammalian cells, as it degraded Ins(4)P, Ins(1)P and to a lesser extent Ins(3)P, was dependent on MgCl2 and inhibited by LiCl in a non-competitive manner. The second enzyme activity was specific for Ins(4)P; the enzyme activity was not dependent on MgCl2 and not inhibited by LiCl. The third monophosphatase activity degraded especially Ins(3)P, but also Ins(4)P and Ins(1)P; increasing concentrations of MgCl2 inhibited this enzyme activity, whereas LiCl had no effect. In vivo, LiCl induces a reduction of inositol levels by about 20%. In [3H]inositol-labelled cells LiCl causes a 6-fold increase in the radioactivity of [3H]Ins(1)P, a doubling of [3H]Ins(4)P and a slight decrease in the radioactivity in [3H]Ins(3)P. These data indicate that the biological effects of lithium in Dictyostelium are not due to depletion of the inositol pool by inhibition of inositol monophosphatase activity.

Further Characterization of Chickpea Isoflavone 7-O-Glucoside- 6"-O-malonate: malonylesterase: Evidence for a Highly Specific, Membrane-Bound Enzyme in Roots of Cicer arietinum L.

1987 ◽  
Vol 42 (3) ◽  
pp. 251-257 ◽  
Author(s):  
Walter Hinderer ◽  
Johannes Köster ◽  
Wolfgang Barz
Keyword(s):  
Enzyme Activity ◽  
Cicer Arietinum ◽  
Biochanin A ◽  
Cicer Arietinum L ◽  
Apparent Homogeneity ◽  
Malonyl Coa ◽  
Highly Sensitive ◽  
Membrane Bound

The specific malonylesterase from chickpea (Cicer arietinum L.), hydrolyzing biochanin A 7-O- glucoside-6″-O-malonate (BGM), has been purified to apparent homogeneity and characterized recently (Hinderer et al., Arch. Biochem. Biophys. 248, 570-578 [1986]). Its substrate specificity as well as the high molecular mass of the native enzyme were further investigated. The 5-deoxy- isoflavone conjugate corresponding to BGM, the formononetin 7-O-glucoside-6,,-O-malonate (FGM), was shown to be a substrate of the malonylesterase essentially as BGM. By contrast, methyl-BGM, a diester of malonic acid was a poor substrate. The purified enzyme completely lacked thioesterase activity with malonyl-CoA as substrate. The inability of the malonylesterase to hydrolyze synthetic acetyl or propionyl esters was further demonstrated with a highly sensitive fluorometric assay using esters of 4-methylumbelliferone. The enzyme-catalyzed hydrolysis of BGM was stimulated in the presence of dissociated carboxylic acids like citrate which was most effective at 30 mM and pH 7.5-8.0. The purified malonylesterase as well as the enzyme activity in crude extracts were totally excluded in gelchromatography with Ultrogel AcA 22. The enrichment of the enzyme activity in microsomal fractions gave strong evidence that the malonylesterase is membrane-bound in vivo. Stimulation of the enzyme activity in vitro by detergents indicates the presence of lipid material in the enzyme and the activity profiles obtained after sedimentation analyses suggest that purifica­tion of a distinct membrane-protein complex had been achieved.

Half-Life of Platelet Factor 4 (PF-4) in Plasma and Platelets from Macaca Mulatta

1977 ◽  
Vol 37 (01) ◽  
pp. 073-080 ◽  
Author(s):  
Knut Gjesdal ◽  
Duncan S. Pepper
Keyword(s):  
Macaca Mulatta ◽  
Half Life ◽  
Human Platelet ◽  
Gel Filtration ◽  
Platelet Factor 4 ◽  
Active Protein ◽  
Platelet Factor ◽  
Membrane Bound

SummaryHuman platelet factor 4 (PF-4) showed a reaction of complete identity with PF-4 from Macaca mulatta when tested against rabbit anti-human-PF-4. Such immunoglobulin was used for quantitative precipitation of in vivo labelled PF-4 in monkey serum. The results suggest that the active protein had an intra-platelet half-life of about 21 hours. In vitro 125I-labelled human PF-4 was injected intravenously into two monkeys and isolated by immuno-precipita-tion from platelet-poor plasma and from platelets disrupted after gel-filtration. Plasma PF-4 was found to have a half-life of 7 to 11 hours. Some of the labelled PF-4 was associated with platelets and this fraction had a rapid initial disappearance rate and a subsequent half-life close to that of plasma PF-4. The results are compatible with the hypothesis that granular PF-4 belongs to a separate compartment, whereas membrane-bound PF-4 and plasma PF-4 may interchange.

Plasminogen Activation In Vivo upon Intravenous Infusion of DDAVP

1992 ◽  
Vol 67 (01) ◽  
pp. 111-116 ◽  
Author(s):  
Marcel Levi ◽  
Jan Paul de Boer ◽  
Dorina Roem ◽  
Jan Wouter ten Cate ◽  
C Erik Hack
Keyword(s):  
Monoclonal Antibodies ◽  
Plasminogen Activator ◽  
Plasma Levels ◽  
Fold Increase ◽  
Fibrinolytic System ◽  
Plasminogen Activation ◽  
Plasminogen Activator Activity ◽  
Insight Into

SummaryInfusion of desamino-d-arginine vasopressin (DDAVP) results in an increase in plasma plasminogen activator activity. Whether this increase results in the generation of plasmin in vivo has never been established.A novel sensitive radioimmunoassay (RIA) for the measurement of the complex between plasmin and its main inhibitor α2 antiplasmin (PAP complex) was developed using monoclonal antibodies preferentially reacting with complexed and inactivated α2-antiplasmin and monoclonal antibodies against plasmin. The assay was validated in healthy volunteers and in patients with an activated fibrinolytic system.Infusion of DDAVP in a randomized placebo controlled crossover study resulted in all volunteers in a 6.6-fold increase in PAP complex, which was maximal between 15 and 30 min after the start of the infusion. Hereafter, plasma levels of PAP complex decreased with an apparent half-life of disappearance of about 120 min. Infusion of DDAVP did not induce generation of thrombin, as measured by plasma levels of prothrombin fragment F1+2 and thrombin-antithrombin III (TAT) complex.We conclude that the increase in plasminogen activator activity upon the infusion of DDAVP results in the in vivo generation of plasmin, in the absence of coagulation activation. Studying the DDAVP induced increase in PAP complex of patients with thromboembolic disease and a defective plasminogen activator response upon DDAVP may provide more insight into the role of the fibrinolytic system in the pathogenesis of thrombosis.

Exterior and Interior Events in Early Stage of Thrombin-induced Platelet Aggregation

1977 ◽  
Vol 38 (03) ◽  
pp. 0630-0639 ◽  
Author(s):  
Shuichi Hashimoto ◽  
Sachiko Shibata ◽  
Bonro Kobayashi
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Plasma Membrane ◽  
Platelet Aggregation ◽  
Early Stage ◽  
Adenyl Cyclase ◽  
Cellular Component ◽  
Primary Event ◽  
Rabbit Platelets ◽  
Membrane Bound

SummaryTreatment of washed rabbit platelets with 1 u/ml of thrombin at 37° C resulted in a disappearance from platelets of a protein with 250,000 dalton molecular weight which was shown to be originated from plasma membrane. Parallel loss of adenyl cyclase was noted, and both reactions were complete within 30 sec. From the patterns of disc electrophoretograms, the importance of quick suppression of thrombin action in demonstrating the primary event was stressed.Thrombin induced an apparent activation of membrane bound phosphodiesterase. This reaction was also complete within 30 sec. The cellular component which contained the enzyme activity was distinct from plasma membrane. Soluble phosphodiesterase was not influenced by thrombin at all.These reactions required intact platelet cells to react with thrombin, and no reaction was detected when subcellular preparations were treated with thrombin.Possibility of collaboration of changes in externally located synthetic enzyme with those in internally located degrading enzyme in the early phase of thrombin action on platelets was suggested.

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