A kinetic study of the effects of galactocerebroside 3-sulphate on human spleen glucocerebrosidase. Evidence for two activator-binding sites

Extraction of control human spleen glucocerebrosidase with sodium cholate and butan-l-ol reversibly inactivates the enzyme in terms of its ability to hydrolyse the water-soluble substrate 4-methylumbelliferyl beta-D-glucopyranoside (MUGlc). The acidic brain lipid galactocerebroside 3-sulphate (sulphatide) reconstitutes beta-glucosidase activity in a strongly concentration-dependent manner. In this study we show that sulphatide exhibits three critical micellar concentrations (CMCs): CMC1, 3.72 microM; CMC2, 22.6 microM; CMC3, 60.7 microM. We designate the aggregates formed at these CMCs as primary, secondary and tertiary micelles respectively. From the results of kinetic studies performed at various sulphatide concentrations (0.012-248 microM), we found that sulphatide monomers (less than 3 microM) decreased the Km (for MUGlc) of control glucocerebrosidase from 11 to 4.6 mM, and lowered the Vmax. 2-fold. However, secondary and tertiary micelles were required for expression of high control glucocerebrosidase activities. Glucocerebrosidase prepared from the spleen of a patient with non-neuronopathic type 1 Gaucher's disease exhibited a very low Km (2.8 mM) even in the absence of exogenous lipid, and sulphatide monomers had no effect on the mutant enzyme's Km or Vmax. However, secondary or tertiary micelles markedly increased the Vmax. of the type 1 glucocerebrosidase to 60% of the corresponding control enzyme value. In contrast, for the glucocerebrosidase of the neuronopathic type 2 case, although sulphatide decreased the Km from 9.2 to 1.7 mM, the Vmax. never reached more than 5% that of the control enzyme, even at high concentrations of sulphatide. In addition, we found that secondary and tertiary sulphatide micelles enhanced the rate of inactivation of all three glucocerebrosidase preparations by chymotrypsin. Collectively, these results indicate the presence of two sulphatide-binding sites on glucocerebrosidase: one that enhances substrate binding, and another that enhances catalysis.

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Bimodal Behavior of Ascorbic Acid in Musca domestica Larvae

Biointerface Research in Applied Chemistry ◽

10.33263/briac124.51995216 ◽

2021 ◽

Vol 12 (4) ◽

pp. 5199-5216

Keyword(s):

Oxidative Stress ◽

Ascorbic Acid ◽

Musca Domestica ◽

Food Supplement ◽

Water Soluble ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Dietary Antioxidant ◽

High Concentrations ◽

Normal Food

Ascorbic acid (Vitamin C) is an excellent water-soluble dietary antioxidant, well known to protect the biomolecules from oxidative stress-linked damages. It is reported to show a contrary behavior by inflicting pro-oxidant effects under varied, altered circumstances. The present work is an attempt to study the antioxidant and pro-oxidant behavior of ascorbic acid in Musca domestica larvae. Its pro-oxidant properties were tested by exposing the larvae to various concentrations of ascorbic acid. For confirming its oxidant scavenging properties, its effects on the antioxidant enzyme profiles were studied in both the normal and stress-induced M. domestica larvae. Oxidative stress was induced by adding D-Galactose (D-Gal) to the normal food supplement of the larvae. Outcomes of the study demonstrate that ascorbic acid acts as an efficient antioxidant when added in lower concentrations, but at high concentrations, it induces oxidative stress in the larvae, thus acting as a pro-oxidant. At the concentration of 10 mM, ascorbic acid significantly reduced the oxidative stress induced by D-Gal (p < 0.05) and maintained the percent pupal survival and percent eclosion. In conclusion, we suggest that ascorbic acid may function as an antioxidant and pro-oxidant in a concentration-dependent manner under normal physiological conditions.

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Neutrophil Elastase Potentiates Cathepsin G-lnduced Platelet Activation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646319 ◽

1992 ◽

Vol 68 (05) ◽

pp. 570-576 ◽

Cited By ~ 20

Author(s):

Mary A Selak

Keyword(s):

Neutrophil Elastase ◽

Cell Activation ◽

Thromboxane A2 ◽

Creatine Phosphate ◽

Dense Granule ◽

Cathepsin G ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Low Concentrations ◽

High Concentrations

SummaryWe have previously demonstrated that human neutrophil cathepsin G is a strong platelet agonist that binds to a specific receptor. This work describes the effect of neutrophil elastase on cathepsin G-induced platelet responses. While platelets were not activated by high concentrations of neutrophil elastase by itself, elastase enhanced aggregation, secretion and calcium mobilization induced by low concentrations of cathepsin G. Platelet aggregation and secretion were potentiated in a concentration-dependent manner by neutrophil elastase with maximal responses observable at 200 nM. Enhancement was observed when elastase was preincubated with platelets for time intervals of 10–60 s prior to addition of a low concentration of cathepsin G and required catalytically-active elastase since phenylmethanesulphonyl fluoride-inhibited enzyme failed to potentiate cell activation. Neutrophil elastase potentiation of platelet responses induced by low concentrations of cathepsin G was markedly inhibited by creatine phosphate/creatine phosphokinase and/or indomethacin, indicating that the synergism between elastase and cathepsin G required the participation of ADP and thromboxane A2. On the other hand, platelet responses were not attenuated by the PAF antagonist BN 52021, signifying that PAF-acether did not play a role in elastase potentiation. At higher concentrations porcine pancreatic elastase exhibits similar effects to neutrophil elastase, demonstrating that the effect of elastase was not unique to the neutrophil protease. While neutrophil elastase failed to alter the ability of cathepsin G to hydrolyze a synthetic chromogenic substrate, preincubation of platelets with elastase increased the apparent affinity of cathepsin G binding to platelets. In contrast to their effect on cathepsin G-induced platelet responses, neither neutrophil nor pancreatic elasatse potentiated aggregation or dense granule release initiated by ADP, PAF-acether, arachidonic acid or U46619, a thromboxane A2 mimetic. Moreover, unlike its effect on cathepsin G, neutrophil elastase inhibited thrombin-induced responses. The current observations demonstrate that elastase can potentiate platelet responses mediated by low concentrations of cathepsin G, suggesting that both enzymes may function synergistically to activate platelets under conditions where neutrophil degranulation occurs.

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Interactions of Dichlorodiphenyltrichloroethane (DDT) and Dichlorodiphenyldichloroethylene (DDE) With Skeletal Muscle Ryanodine Receptor Type 1

Toxicological Sciences ◽

10.1093/toxsci/kfz120 ◽

2019 ◽

Vol 170 (2) ◽

pp. 509-524

Author(s):

Kim M Truong ◽

Gennady Cherednichenko ◽

Isaac N Pessah

Keyword(s):

Skeletal Muscle ◽

Ryanodine Receptor ◽

Membrane Vesicles ◽

Receptor Type ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Voltage Gated Sodium Channels ◽

Muscle Health ◽

Living Organisms

Abstract Dichlorodiphenyltrichloroethane (DDT) and its metabolite dichlorodiphenyldichloroethylene (DDE) are ubiquitous in the environment and detected in tissues of living organisms. Although DDT owes its insecticidal activity to impeding closure of voltage-gated sodium channels, it mediates toxicity in mammals by acting as an endocrine disruptor (ED). Numerous studies demonstrate DDT/DDE to be EDs, but studies examining muscle-specific effects mediated by nonhormonal receptors in mammals are lacking. Therefore, we investigated whether o,p′-DDT, p,p′-DDT, o,p′-DDE, and p,p′-DDE (DDx, collectively) alter the function of ryanodine receptor type 1 (RyR1), a protein critical for skeletal muscle excitation-contraction coupling and muscle health. DDx (0.01–10 µM) elicited concentration-dependent increases in [3H]ryanodine ([3H]Ry) binding to RyR1 with o,p′-DDE showing highest potency and efficacy. DDx also showed sex differences in [3H]Ry-binding efficacy toward RyR1, where [3H]Ry-binding in female muscle preparations was greater than male counterparts. Measurements of Ca2+ transport across sarcoplasmic reticulum (SR) membrane vesicles further confirmed DDx can selectively engage with RyR1 to cause Ca2+ efflux from SR stores. DDx also disrupts RyR1-signaling in HEK293T cells stably expressing RyR1 (HEK-RyR1). Pretreatment with DDx (0.1–10 µM) for 100 s, 12 h, or 24 h significantly sensitized Ca2+-efflux triggered by RyR agonist caffeine in a concentration-dependent manner. o,p′-DDE (24 h; 1 µM) significantly increased Ca2+-transient amplitude from electrically stimulated mouse myotubes compared with control and displayed abnormal fatigability. In conclusion, our study demonstrates DDx can directly interact and modulate RyR1 conformation, thereby altering SR Ca2+-dynamics and sensitize RyR1-expressing cells to RyR1 activators, which may ultimately contribute to long-term impairments in muscle health.

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Binding of Dipyridamole of Human Platelets and to α-Acid Glycoprotein and its Significance for the Inhibition of Adenosine Uptake

10.1055/s-0039-1682726 ◽

1977 ◽

Author(s):

K. Subbarao ◽

B. Rucinski ◽

A. Summers ◽

S. Niewiarowski

Keyword(s):

Binding Sites ◽

Ex Vivo ◽

Human Platelets ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Acid Glycoprotein ◽

High Affinity ◽

Adenosine Uptake ◽

Adenosine Transport

The interactions of dipyridamole with α1-acid glycoprotein of plasma and with human platelets are related to inhibition of adenosine uptake by platelets. One mole of dipyridamole binds to one mole of α1-acid glycoprotein with a dissociation constant (Kd) of 1.3 μM. It was found that platelets contain both high and low affinity binding sites for the drug. The binding of dipyridamole to the high affinity sites follows a Michaelis Menten binding pattern with a Kd of 0.04 μM. Approximately 2x104 dipyridamole molecules are bound at the high affinity sites of each platelet. The lower affinity sites bind the drug with a Kd of 4 μM. In the presence of α1acid glycoprotein the binding of dipyridamole to platelets is inhibited. Correspondingly, the dipyridamole inhibition of adenosine uptake by platelets is reduced 1000-fold by α1acid glycoprotein. Binding of dipyridamole to human platelets is essential for its inhibition of adenosine uptake by platelets. Dipyridamole reduced the [14C]-ATP to [14C]-ADP ratio in the platelets. Purified α1acid glycoprotein reversed these effects of dipyridamole on adenosine metabolism of platelets in a concentration dependent manner. A correlationwas observed between the level of circulating dipyridamole in plasma and the inhibition of [14C]-adenosine uptake by platelets of PRP samples of 12 human volunteers given different amounts of dipyridamole. The in vitro and ex vivo effects of dipyridamole on the [14C]-adenosine uptake by platelets were found to be identical. Our data suggest the presence of dipyridamole binding sites in platelets that regulate adenosine transport across the cell surface.

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Activation of endothelin ETB receptors increases glomerular cGMP via an L-arginine-dependent pathway

AJP Renal Physiology ◽

10.1152/ajprenal.1992.263.6.f1020 ◽

1992 ◽

Vol 263 (6) ◽

pp. F1020-F1025 ◽

Cited By ~ 9

Author(s):

R. M. Edwards ◽

M. Pullen ◽

P. Nambi

Keyword(s):

Nitric Oxide ◽

Guanylate Cyclase ◽

Binding Sites ◽

Soluble Guanylate Cyclase ◽

Maximum Effect ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

High Affinity ◽

Dependent Pathway ◽

Stimulation Of

The effects of endothelins (ET) on guanosine 3',5'-cyclic monophosphate (cGMP) levels in intact rat glomeruli were examined. ET-3 produced a rapid approximately fivefold increase in cGMP levels with the maximum effect occurring at 1 min. The ET-3-induced increase in cGMP accumulation occurred in the absence and presence of 3-isobutyl-1-methylxanthine. ET-1, ET-2, ET-3, and the structurally related toxin, sarafotoxin S6c, all increased glomerular cGMP levels in a concentration-dependent manner and with similar potencies (EC50 approximately 15-30 nM). The L-arginine analogue, N omega-nitro-L-arginine (L-NNA), reduced basal levels of cGMP and also totally inhibited ET-induced increases in cGMP as did methylene blue, an inhibitor of soluble guanylate cyclase. The effect of L-NNA was attenuated by L-arginine but not by D-arginine. The stimulation of cGMP accumulation by ET-3 was dependent on extracellular Ca2+ and was additive to atriopeptin III but not to acetylcholine. The ETA-selective antagonist, BQ 123, had no effect on ET-3-induced formation of cGMP. Glomerular membranes displayed high-affinity (Kd = 130-150 pM) and high-density (approximately 2.0 pmol/mg) binding sites for 125I-ET-1 and 125I-ET-3. ET-1, ET-3, and sarafotoxin S6c displaced 125I-ET-1 binding to glomerular membranes with similar affinities. BQ 123 had no effect on 125I-ET-1 binding. We conclude that ET increases cGMP levels in glomeruli by stimulating the formation of a nitric oxide-like factor that activates soluble guanylate cyclase. This effect of ET appears to be mediated by activation of ETB receptors and may serve to modulate the contractile effects of ET.

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Effects of Mg2+ on Ca2+ waves and Ca2+ transients of rat ventricular myocytes

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1996.270.3.h907 ◽

1996 ◽

Vol 270 (3) ◽

pp. H907-H914 ◽

Cited By ~ 1

Author(s):

H. Terada ◽

H. Hayashi ◽

N. Noda ◽

H. Satoh ◽

H. Katoh ◽

...

Keyword(s):

Sarcoplasmic Reticulum ◽

Channel Blocker ◽

Ventricular Myocytes ◽

Inhibitory Effect ◽

Antiarrhythmic Action ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Triggered Activity ◽

Rat Ventricular Myocytes ◽

High Concentrations

It has been shown that the occurrence of the transient inward current, which is responsible for triggered activity, was often associated with propagating regions of increased intracellular Ca2+ concentration ([Ca2+]i), i.e., the “Ca2+ wave.” To investigate the mechanism of antiarrhythmic action of Mg2+, we have studied effects of high concentrations of Mg2+ on Ca2+ waves in isolated rat ventricular myocytes. [Ca2+]i was estimated using the Ca(2+)-indicating probe indo 1. Ca2+ waves in myocytes, stimulated at 0.2 Hz, were induced by perfusion of isoproterenol (10(-7) M). High Mg2+ concentration suppressed Ca2+ waves in a concentration-dependent manner (36% at 4 mM, 70% at 8 mM, and 82% at 12 mM). The Ca2+ channel blocker verapamil also suppressed Ca2+ waves in a similar way. In contrast with marked depression of Ca2+ transients by verapamil, Ca2+ transients were not affected by high Mg2+ concentration (8 mM). High Mg2+ concentration also reduced frequencies of Ca2+ waves in the absence of electrical stimulation, whereas verapamil failed to reduce frequencies of Ca2+ waves. Reduction in frequency of Ca2+ waves by high Mg2+ concentration was associated with slowing of propagation velocity of Ca2+ waves. To examine whether suppressive effects of high Mg2+ concentration on Ca2+ waves were related to an increase in intracellular Mg2+ concentration ([Mg2+]i), the effect of high-Mg2+ solution on [Mg2+]i was examined in myocytes loaded with mag-fura 2. An increase in extracellular Mg2+ concentration from 1 to 12 mM increased [Mg2+]i from 1.06 +/- 0.16 to 1.87 +/- 0.22 mM (P < 0.01) in 30 min. To examine the effect of high Mg2+ concentration on amount of releasable Ca2+ in the sarcoplasmic reticulum, the effect of high Mg2+ concentration on the Ca2+ transient induced by a rapid application of caffeine was examined. High-Mg2+ solution increased the peak of the caffeine-induced Ca2+ transient. These results suggest that the inhibitory effect of Mg2+ on Ca2+ waves was not due to inhibition of the sarcolemmal Ca2+ channel but could be due to a decreased propensity for the sarcoplasmic reticulum to divest itself of excess Ca2+.

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Intrinsic ANG II type 1 receptor stimulation contributes to recovery of postischemic mechanical function

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1998.274.5.h1524 ◽

1998 ◽

Vol 274 (5) ◽

pp. H1524-H1531 ◽

Cited By ~ 1

Author(s):

William R. Ford ◽

Alexander S. Clanachan ◽

Gary D. Lopaschuk ◽

Richard Schulz ◽

Bodh I. Jugdutt

Keyword(s):

Recovery Of Function ◽

Left Ventricular ◽

Dependent Manner ◽

Mechanical Function ◽

Ang Ii ◽

Concentration Dependent Manner ◽

Proton Production ◽

Rat Hearts ◽

Postischemic Recovery

To determine whether intrinsic angiotensin II (ANG II) type 1 receptor (AT1-R) stimulation modulates recovery of postischemic mechanical function, we studied the effects of selective AT1-R blockade with losartan on proton production from glucose metabolism and recovery of function in isolated working rat hearts perfused with Krebs-Henseleit buffer containing palmitate, glucose, and insulin. Aerobic perfusion (50 min) was followed by global, no-flow ischemia (30 min) and reperfusion (30 min) in the presence ( n = 10) or absence ( n = 14) of losartan (1 μmol/l) or the cardioprotective adenosine A1receptor agonist N 6-cyclohexyladenosine (CHA, 0.5 μmol/l, n = 11). During reperfusion in untreated hearts (controls), left ventricular (LV) minute work partially recovered to 38% of aerobic baseline, whereas proton production increased to 155%. Compared with controls, CHA improved recovery of LV work to 79% and reduced proton production to 44%. Losartan depressed recovery of LV work to 0% without altering proton production. However, exogenous ANG II (1–100 nmol/l) in combination with losartan restored recovery of LV work during reperfusion in a concentration-dependent manner, suggesting that postischemic recovery of function depends on intrinsic AT1-R stimulation.

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Increased Anti-Inflammatory Effects on LPS-Induced Microglia Cells by Spirulina maxima Extract from Ultrasonic Process

Applied Sciences ◽

10.3390/app9102144 ◽

2019 ◽

Vol 9 (10) ◽

pp. 2144 ◽

Cited By ~ 5

Author(s):

Woon Yong Choi ◽

Jae-Hun Sim ◽

Jung-Youl Lee ◽

Do Hyung Kang ◽

Hyeon Yong Lee

Keyword(s):

Mrna Expression ◽

Dependent Manner ◽

Spirulina Maxima ◽

Concentration Dependent Manner ◽

Quantitative Correlation ◽

Tnf Α ◽

Ultrasonic Process ◽

Anti Inflammatory ◽

Low Cytotoxicity ◽

High Concentrations

The Spirulina maxima exact from a non-thermal ultrasonic process (UE) contains 17.5 mg/g of total chlorophyll, compared to 6.24 mg/g of chlorophyll derived from the conventional 70% ethanol extraction at 80 °C for 12 h (EE). The UE also showed relatively low cytotoxicity against murine microglial cells (BV-2) and inhibited the production of the inflammatory mediators, NO and PGE2. The UE also effectively suppresses both mRNA expression and the production of pro-inflammatory cytokines, such as TNF-α, IL-6 and IL-1β, in a concentration-dependent manner. Notably, TNF-α gene and protein production were most strongly down-regulated, while IL-6 was the least affected by all ranges of treatment concentrations. This work first demonstrated a quantitative correlation between mRNA expression and the production of cytokines, showing that suppression of TNF-α gene expression was most significantly correlated with its secretion. These results clearly proved that the anti-inflammatory effects of Spirulina extract from a nonthermal ultrasonic process, which yielded high concentrations of intact forms of chlorophylls, were increased two-fold compared to those of conventional extracts processed at high temperature.

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Effects of amino acids and ethanolamine on choline uptake and phosphatidylcholine biosynthesis in baby hamster kidney-21 cells

Biochemistry and Cell Biology ◽

10.1139/o92-179 ◽

1992 ◽

Vol 70 (12) ◽

pp. 1319-1324 ◽

Cited By ~ 9

Author(s):

Xiliang Zha ◽

Francis T. Jay ◽

Patrick C. Choy

Keyword(s):

Amino Acids ◽

Culture Media ◽

Specific Radioactivity ◽

Kinetic Studies ◽

Choline Uptake ◽

Dependent Manner ◽

Baby Hamster Kidney ◽

Concentration Dependent Manner ◽

Cell Culture Media ◽

Phosphatidylcholine Biosynthesis

The effects of amino acids and ethanolamine on choline uptake and phosphatidylcholine biosynthesis in baby hamster kidney (BHK-21) cells were investigated. The cells were incubated with labelled choline in the presence of an amino acid or ethanolamine. The uptake of labelled choline was noncompetitively inhibited by amino acids. Glycine, L-alanine, L-serine, L-leucine, L-aspartate, and L-arginine were effective inhibitors and a maximum of 22% inhibition of choline uptake was obtained with 5 mM glycine. Analyses of the labellings in the choline-containing metabolites revealed that the conversion of choline to CDP-choline and subsequently phosphatidylcholine was not affected by the presence of amino acids. The uptake of choline was also inhibited by ethanolamine in a concentration-dependent manner. Kinetic studies on the uptake of choline indicated that the inhibition by ethanolamine was competitive in nature. Although ethanolamine is a potent inhibitor of choline kinase, analyses of the labellings in the choline-containing metabolites indicated that the conversion of choline to phosphocholine was not affected in the cells incubated with ethanolamine. Ethanolamine did not change the pool sizes of phosphocholine and CDP-choline. Based on the specific radioactivity of CDP-choline and the labelling of phosphatidylcholine, the rates of phosphatidylcholine biosynthesis were not significantly different between the control and the ethanolamine-treated cells. In view of the concentrations of amino acids (millimolar) and ethanolamine (micromolar) in most cell culture media, it appeared that only amino acids were important metabolites for the regulation of choline uptake in BHK-21 cells. We conclude that both amino acids and ethanolamine have no direct effect on the biosynthesis of phosphatidylcholine.Key words: choline uptake, phosphatidylcholine biosynthesis, amino acids, ethanolamine, BHK-21 cells.

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Organic Cosolutes as Stabilizers of Phosphoenolpyruvate Carboxylase in Storage: an Interpretation of Their Action

Functional Plant Biology ◽

10.1071/pp9870203 ◽

1987 ◽

Vol 14 (2) ◽

pp. 203 ◽

Cited By ~ 20

Author(s):

E Selinioti ◽

D Nikolopoulos ◽

Y Manetas

Keyword(s):

Phosphoenolpyruvate Carboxylase ◽

Cynodon Dactylon ◽

Dependent Manner ◽

Native Protein ◽

Complete Protection ◽

Concentration Dependent Manner ◽

Polymeric Form ◽

C4 Grass ◽

High Concentrations ◽

Protein Bovine Serum Albumin

Phosphoenolpyruvate carboxylase (EC 4.1.1.3 1) is rapidly inactivated in dilute extracts from leaves of the C4 grass Cynodon dactylon (L.) Pers. Physiological osmotica (glycerol, betaine, proline, sorbitol and sucrose) and synthetic inert polymers like polyvinylpyrrolidone and polyethylene glycol prevent inactivation in a concentration-dependent manner. In a partially purified preparation (10.5 �mol CO2 min-1 mg-1), lability to inactivation and the minimum levels of cosolvents needed for complete protection become higher as the protein is diluted. Exogenous protein (bovine serum albumin) was much less effective than native protein in stabilising activity. The results could be interpreted on the basis of the exclusion volume theory and the assumption that the catalytic activity is mainly due to a polymeric form which is maintained only at high concentrations of the enzymic protein.

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