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 Medawar’s PostEra: Galectins Emerged as Key Players During Fetal-Maternal Glycoimmune Adaptation

2021 ◽  
Vol 12 ◽  
Author(s):  
Ellen Menkhorst ◽  
Nandor Gabor Than ◽  
Udo Jeschke ◽  
Gabriela Barrientos ◽  
Laszlo Szereday ◽  
...  
Keyword(s):  
Immune Cell ◽  
Successful Pregnancy ◽  
Fetal Health ◽  
The Past ◽  
Mammalian Embryogenesis ◽  
Carbohydrate Ligands ◽  
Membrane Bound

Lectin-glycan interactions, in particular those mediated by the galectin family, regulate many processes required for a successful pregnancy. Over the past decades, increasing evidence gathered from in vitro and in vivo experiments indicate that members of the galectin family specifically bind to both intracellular and membrane bound carbohydrate ligands regulating angiogenesis, immune-cell adaptations required to tolerate the fetal semi-allograft and mammalian embryogenesis. Therefore, galectins play important roles in fetal development and placentation contributing to maternal and fetal health. This review discusses the expression and role of galectins during the course of pregnancy, with an emphasis on maternal immune adaptions and galectin-glycan interactions uncovered in the recent years. In addition, we summarize the galectin fingerprints associated with pathological gestation with particular focus on preeclampsia.

scholarly journals Role of glycosylation in transport and enzymic activity of neutral endopeptidase-24.11

1994 ◽  
Vol 302 (2) ◽  
pp. 451-454 ◽  
Author(s):  
M H Lafrance ◽  
C Vézina ◽  
Q Wang ◽  
G Boileau ◽  
P Crine ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Cell Surface ◽  
Intracellular Transport ◽  
Neutral Endopeptidase ◽  
Soluble Form ◽  
Cell Extracts ◽  
Glycosylation Sites ◽  
Sugar Addition ◽  
Membrane Bound

Neutral endopeptidase (NEP, EC 3.4.24.11) is a major ectoenzyme of the brush-border membrane. The ectodomain of NEP contains five putative N-glycosylation sites. In order to determine the role of the addition of sugar moieties on the activity and intracellular transport of NEP, we have used site-directed mutagenesis to remove all or some of the five potential sites of sugar addition in membrane-bound and secreted forms of the enzyme. Expression of NEP glycosylation mutants in COS-1 cells showed that all five sites are used for sugar addition. Immunoblotting of NEP in COS-1 cell extracts or culture media indicated that total expression of normal membrane-bound NEP was not affected by mutations at glycosylation sites, whereas this expression level appeared to be strictly dependent on the number of glycosylation sites retained on the soluble form. The transport to the cell surface was also reduced by decreased glycosylation, but again the phenomenon appeared more drastic in the case of the soluble form than for the membrane-bound enzyme. Enzyme activity was decreased by deglycosylation. However, the presence of either of two crucial sites (sites 1 and 5; numbered from the N-terminus of the protein) was sufficient to recover close-to-normal enzymic activities. Transport to the cell surface and enzyme activity of NEP are thus both dependent on sugar residues, probably through different conformational constraints. These constraints seem to be local for enzyme activity but more global for transport to the cell surface.

scholarly journals Differential CYP 2D6 Metabolism Alters Primaquine Pharmacokinetics

2015 ◽  
Vol 59 (4) ◽  
pp. 2380-2387 ◽  
Author(s):  
Brittney M. J. Potter ◽  
Lisa H. Xie ◽  
Chau Vuong ◽  
Jing Zhang ◽  
Ping Zhang ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Antimalarial Activity ◽  
Wild Type ◽  
Phenolic Metabolites ◽  
Species Specific ◽  
Human And Mouse ◽  
Different Levels ◽  
Cyp 2D6

ABSTRACTPrimaquine (PQ) metabolism by the cytochrome P450 (CYP) 2D family of enzymes is required for antimalarial activity in both humans (2D6) and mice (2D). Human CYP 2D6 is highly polymorphic, and decreased CYP 2D6 enzyme activity has been linked to decreased PQ antimalarial activity. Despite the importance of CYP 2D metabolism in PQ efficacy, the exact role that these enzymes play in PQ metabolism and pharmacokinetics has not been extensively studiedin vivo. In this study, a series of PQ pharmacokinetic experiments were conducted in mice with differential CYP 2D metabolism characteristics, including wild-type (WT), CYP 2D knockout (KO), and humanized CYP 2D6 (KO/knock-in [KO/KI]) mice. Plasma and liver pharmacokinetic profiles from a single PQ dose (20 mg/kg of body weight) differed significantly among the strains for PQ and carboxy-PQ. Additionally, due to the suspected role of phenolic metabolites in PQ efficacy, these were probed using reference standards. Levels of phenolic metabolites were highest in mice capable of metabolizing CYP 2D6 substrates (WT and KO/KI 2D6 mice). PQ phenolic metabolites were present in different quantities in the two strains, illustrating species-specific differences in PQ metabolism between the human and mouse enzymes. Taking the data together, this report furthers understanding of PQ pharmacokinetics in the context of differential CYP 2D metabolism and has important implications for PQ administration in humans with different levels of CYP 2D6 enzyme activity.

scholarly journals Morphogenic Effects of Ezrin Require a Phosphorylation-Induced Transition from Oligomers to Monomers at the Plasma Membrane

2000 ◽  
Vol 150 (1) ◽  
pp. 193-204 ◽  
Author(s):  
Alexis Gautreau ◽  
Daniel Louvard ◽  
Monique Arpin
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Wild Type ◽  
Erm Proteins ◽  
Threonine Residue ◽  
Phosphorylation Event ◽  
Membrane Bound

ERM (ezrin, radixin, moesin) proteins act as linkers between the plasma membrane and the actin cytoskeleton. An interaction between their NH2- and COOH-terminal domains occurs intramolecularly in closed monomers and intermolecularly in head-to-tail oligomers. In vitro, phosphorylation of a conserved threonine residue (T567 in ezrin) in the COOH-terminal domain of ERM proteins disrupts this interaction. Here, we have analyzed the role of this phosphorylation event in vivo, by deriving stable clones producing wild-type, T567A, and T567D ezrin from LLC-PK1 epithelial cells. We found that T567A ezrin was poorly associated with the cytoskeleton, but was able to form oligomers. In contrast, T567D ezrin was associated with the cytoskeleton, but its distribution was shifted from oligomers to monomers at the membrane. Moreover, production of T567D ezrin induced the formation of lamellipodia, membrane ruffles, and tufts of microvilli. Both T567A and T567D ezrin affected the development of multicellular epithelial structures. Collectively, these results suggest that phosphorylation of ERM proteins on this conserved threonine regulates the transition from membrane-bound oligomers to active monomers, which induce and are part of actin-rich membrane projections.

Plastoquinone-9 biosynthesis in cyanobacteria differs from that in plants and involves a novel 4-hydroxybenzoate solanesyltransferase

2012 ◽  
Vol 442 (3) ◽  
pp. 621-629 ◽  
Author(s):  
Radin Sadre ◽  
Christian Pfaff ◽  
Stephan Buchkremer
Keyword(s):  
Biosynthetic Pathway ◽  
In Vitro Assays ◽  
Transport Chain ◽  
In Vivo Labelling ◽  
Membrane Bound ◽  
Transformation Processes ◽  
Synechocystis Sp

PQ-9 (plastoquinone-9) has a central role in energy transformation processes in cyanobacteria by mediating electron transfer in both the photosynthetic as well as the respiratory electron transport chain. The present study provides evidence that the PQ-9 biosynthetic pathway in cyanobacteria differs substantially from that in plants. We identified 4-hydroxybenzoate as being the aromatic precursor for PQ-9 in Synechocystis sp. PCC6803, and in the present paper we report on the role of the membrane-bound 4-hydroxybenzoate solanesyltransferase, Slr0926, in PQ-9 biosynthesis and on the properties of the enzyme. The catalytic activity of Slr0926 was demonstrated by in vivo labelling experiments in Synechocystis sp., complementation studies in an Escherichia coli mutant with a defect in ubiquinone biosynthesis, and in vitro assays using the recombinant as well as the native enzyme. Although Slr0926 was highly specific for the prenyl acceptor substrate 4-hydroxybenzoate, it displayed a broad specificity with regard to the prenyl donor substrate and used not only solanesyl diphosphate, but also a number of shorter-chain prenyl diphosphates. In combination with in silico data, our results indicate that Slr0926 evolved from bacterial 4-hydroxybenzoate prenyltransferases catalysing prenylation in the course of ubiquinone biosynthesis.

scholarly journals Desensitization of Capsaicin-Sensitive Afferents Accelerates Early Tumor Growth via Increased Vascular Leakage in a Murine Model of Triple Negative Breast Cancer

2021 ◽  
Vol 11 ◽  
Author(s):  
Noémi Bencze ◽  
Csaba Schvarcz ◽  
Gábor Kriszta ◽  
Lea Danics ◽  
Éva Szőke ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Enzyme Activity ◽  
Tumor Growth ◽  
Triple Negative ◽  
Vascular Leakage ◽  
Size Difference ◽  
Control Group ◽  
Protease Enzyme

There is growing interest in the role of nerve-driven mechanisms in tumorigenesis and tumor growth. Capsaicin-sensitive afferents have been previously shown to possess antitumoral and immune-regulatory properties, the mechanism of which is currently poorly understood. In this study, we have assessed the role of these terminals in the triple negative 4T1 orthotopic mouse model of breast cancer. The ultrapotent capsaicin-analogue resiniferatoxin (RTX) was used for the selective, systemic desensitization of capsaicin-sensitive afferents. Growth and viability of orthotopically implanted 4T1 tumors were measured by caliper, in vivo MRI, and bioluminescence imaging, while tumor vascularity and protease enzyme activity were assessed using fluorescent in vivo imaging. The levels of the neuropeptides Calcitonin Gene-Related Peptide (CGRP), Substance P (SP), and somatostatin were measured from tumor tissue homogenates using radioimmunoassay, while tumor structure and peritumoral inflammation were evaluated by conventional use of CD31, CD45 and CD3 immunohistology. RTX-pretreated mice demonstrated facilitated tumor growth in the early phase measured using a caliper, which was coupled with increased tumor vascular leakage demonstrated using fluorescent vascular imaging. The tumor size difference dissipated by day seven. The MRI tumor volume was similar, while the intratumoral protease enzyme activity measured by fluorescence imaging was also comparable in RTX-pretreated and non-pretreated animals. Tumor viability or immunohistopathological profile was measured using CD3, CD31, and CD45 stains and did not differ significantly from the non-pretreated control group. Intratumoral somatostatin, CGRP, and SP levels were similar in both groups. Our results underscore the beneficial, antitumoral properties of capsaicin sensitive nerve terminals in this aggressive model of breast cancer, which is presumed to be due to the inhibition of tumor vascular bed disruption. The absence of any difference in intratumoral neuropeptide levels indicates non-neural sources playing a substantial part in their expression.

scholarly journals Functional Characterization of Membrane Sialyltransferase Activity of Human Platelets

1977 ◽  
Author(s):  
K. K. Wu ◽  
C. Ku ◽  
C. Smith
Keyword(s):  
Enzyme Activity ◽  
Functional Characterization ◽  
Adenosine Diphosphate ◽  
Normal Subjects ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Transferase Activity ◽  
Platelet Surface

To evaluate the role of membrane sialyltransferase in the initiation of platelet aggregation, we studied the stimulatory effect of epinephrine and adenosine diphosphate and the inhibitory effect of aspirin on the platelet surface sialyl transferase activity. The enzyme activity was assayed under optimal conditions as determined previously. The assay mixture consisted of intact washed human platelets, CMP-14C-sialic acid, desialated fetuin, Mn2+ and buffer to a final volume of 1 ml. The enzyme activity was enhanced to 172% of control by 1μH, 152% by 5μM and 146% by 10μM epinephrine. Adenosine diphosphate enhanced the enzyme activity to a lesser extent:103% at 1μM and 113% at 5μM. In contrast, aspirin inhibited the enzyme activity to 46% of control when 10μg/ml of aspirin was used. Higher concentrations of aspirin failed to cause further inhibition. In the in-vivo experiment, 600 mg aspirin was given to normal subjects and the surface enzyme activity was determined 12 hours later. The enzyme activity reduced to 43% following aspirin administration. Furthermore, we studied the enzyme activity in a patient with “aspirin-like” release disorder. While the mean surface enzyme activity of 10 normal subjects was 1.56 + 0.21 (S. D.) pmole-hr-1 per 108 platelets, the enzyme activity of the patient was only 0.91 pmole.hr-l. The results strongly suggest that the membrane sialyltransferase plays an important part in the initiation of platelet release reaction.

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 ACETYLCHOLINESTERASE, AS A POTENTIAL BIOMARKER OF NAPHTHALENE TOXICITY IN DIFFERENT TISSUES OF FRESHWATER TELEOST, ANABAS TESTUDINEUS

2021 ◽  
Vol 29 (4) ◽  
pp. 403-409
Author(s):  
Susri Nayak ◽  
Lipika Patnaik
Keyword(s):  
Enzyme Activity ◽  
Control Group ◽  
Anabas Testudineus ◽  
Potential Biomarker ◽  
Maximum Inhibition ◽  
Polycyclic Aromatic ◽  
Acetylcholinesterase Enzyme ◽  
Different Tissues

Naphthalene, a Polycyclic Aromatic Hydrocarbon is widely used as a fumigant and disinfectant despite its toxic effect and is ranked as the ninth most threatening compound. The present study was carried out to determine the in vivo effect of naphthalene at different concentrations on acetylcholinesterase (AChE) enzyme activity in different tissues of Anabas testudineus. The fishes were exposed to varying concentrations of naphthalene (4.2 mgL–1, 4.4 mgL–1, 4.6 mgL–1, 4.8 mgL–1 and 5 mgL–1) for a period of 72 hours. Acetylcholinesterase enzyme activity was found to be significantly inhibited, in a dose-response manner. The inhibition percentage of AChE activity varied from 9.34–43.95% in brain tissue, 2.56–35.81% in liver tissue, 5.94–34.15% in muscle tissue and 3.92–33.75% in gills in comparison to the tissues of the control group. Maximum inhibition of acetylcholinesterase enzyme activity in treated fish was observed in the brain followed by liver, muscles, and gills. This study highlights the significance and role of acetylcholinesterase as a potential stress biomarker of naphthalene toxicity.

Vitamin E and ATPases: Protection of ATPase Activities by Vitamin E Supplementation in Various Tissues of Hypercholesterolemic Rats

2000 ◽  
Vol 70 (1) ◽  
pp. 3-7 ◽  
Author(s):  
Evin Ademoglu ◽  
Cahide Gökkusu ◽  
Sükrü Palandüz
Keyword(s):  
Vitamin E ◽  
Plasma Membranes ◽  
Cholesterol Content ◽  
Kidney Cortex ◽  
Hypercholesterolemic Diet ◽  
Membrane Atpase ◽  
Membrane Bound ◽  
Vitamin E Supplementation

It has been shown that the lipid composition of plasma membrane can be modified in vivo by dietary fat. It has also been observed that an increase in the cholesterol content of plasma membranes results in decreased activities of ATPases. In the present study, we evaluated the changes in the activities of ATPases from erythrocytes, hepatocytes, and kidney cortex caused by cholesterol-rich diet in rats and subsequently examined the role of vitamin E administration on the cholesterol-induced effects in these tissues. Administration of hypercholesterolemic diet to the rats for 4.5 months, significantly decreased membrane Na+-K+-ATPase and Ca+2-ATPase activities in comparison to the controls in all tissues studied. Vitamin E supplementation to the hypercholesterolemic rats led to a recovery in membrane ATPase activities. In conclusion, vitamin E supplementation to the rats provided protection against hypercholesterolemic diet-induced impairment of membrane-bound ATPases.

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