Biochemical properties of isolated transverse tubular membranes

Sabbadini, Roger A.; Dahms, A. Stephen

doi:10.1007/bf00812068

Cited by 51 publications

(46 citation statements)

References 191 publications

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“…One possible effect of detergents is the dissociation of protein oligomers. The proposal that the oligomeric state of the ecto-ATPase affects its enzymatic activity was made previously (33,34). To answer the question about the relationship of the transmembrane domains to the oligomeric state of the protein and its sensitivity to detergents, we made variants of the ectoapyrase with one or no transmembrane domains.…”

Section: Resultsmentioning

confidence: 99%

“…Second, sucrose density gradient sedimentation profiles of both the expressed ectoapyrase in COS-7 cells and the native ectoapyrases from LG2 cells (data not shown) are identical: ectoapyrase behaved as a tetramer after solubilization with digitonin, a detergent that has little effect on the enzymatic activity, and became monomeric after solubilization with Triton X-100, a detergent that inhibits the activity. It has been suggested that detergents may inactivate ectoapyrase by destabilizing an oligomeric state of the protein (33,34), and it has been reported that chicken gizzard muscle ecto-ATPase (34) and rat lung ectoapyrase (39) are disulfide-linked homotrimers and homodimers, respectively. However, this work is the first demonstration that the ectoapyrase is a noncovalent tetramer.…”

Section: Fig 4 Expression Of Wild Type and Mutant Ectoapyrases In Cmentioning

confidence: 99%

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The Transmembrane Domains of Ectoapyrase (CD39) Affect Its Enzymatic Activity and Quaternary Structure

Wang

Guidotti

1998

Journal of Biological Chemistry

140

193

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Mammalian ectoapyrase (CD39) is an integral membrane protein with two transmembrane domains and a large extracellular region. The enzymatic activity of ectoapyrase is inhibited by most detergents used for membrane protein solubilization. In contrast, the enzymatic activities of soluble E-type ATPases, including potato tuber (Solanum tuberosum) apyrase and parasite ectoATPase, are not affected by detergents. Here we show that ectoapyrase is a tetramer and that detergents that reduce the activity of the enzyme promote dissociation of the tetramer to monomers. We expressed a secreted form of the ectoapyrase in COS-7 cells by fusing the signal peptide of murine CD4 with the extracellular domain of the ectoapyrase. The soluble ectoapyrase is catalytically active and its activity is not affected by detergents. Mutants of the ectoapyrase with only the NH 2 -or the COOH-terminal transmembrane domain are membrane-bound, and their activity is no longer affected by detergents. The enzymatic activity of all of the mutant proteins is less than that of the native enzyme. These results suggest that the proper contacts between the transmembrane domains of the monomers in the tetramer are necessary for full enzymatic activity.Ectoapyrases, or ATP diphosphohydrolases, hydrolyze extracellular nucleotide tri-and diphosphates in the presence of Ca 2ϩ or Mg 2ϩ (1). The rate of hydrolysis of nucleotide diphosphates is ϳ50% of that of the triphosphates. Mammalian ectoapyrases play important roles in many biological processes, including the modulation of neural cell activities (for a review, see Ref.2), prevention of intravascular thrombosis (3, 4), and regulation of immune responses (5). The enzymatic activity of ectoapyrases suggests that their functions might be to regulate purinergic signaling systems (6).The amino acid sequence of potato apyrase (7) revealed regions of similarity between the potato apyrase and several other proteins: garden pea nucleotide triphosphatase (8), Toxoplasma gondii nucleotide triphosphatase (9), yeast guanosine diphosphatase (10), and the lymphocyte cell activating antigen CD39 (11). We showed that CD39 has ectoapyrase activity (5) and that it is likely that the gene for CD39 is the only ectoapyrase gene in the mammalian genome (12). Subsequently, it was shown that CD39 also has ecto-ADPase activity in endothelial cells and is responsible for inhibition of ADP-induced platelet aggregation (3, 4). The ecto-ATPases in chicken gizzard (13), humans (CD39L1 (14)), and rats (15) are also encoded by genes similar to that for CD39. These latter enzymes hydrolyze nucleoside diphosphates at less than 10% of the rate of ATP hydrolysis (15).Mammalian ectoapyrases (CD39) are integral membrane proteins with two transmembrane domains (one at each end of the protein), small cytoplasmic NH 2 -and COOH-terminal segments, and a large extracellular domain (11) with enzymatic activity (5). This arrangement is unusual for ecto-enzymes, since these are usually attached to the membrane by a single protein or lipid link (16, 17)...

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Section: Resultsmentioning

confidence: 99%

Section: Fig 4 Expression Of Wild Type and Mutant Ectoapyrases In Cmentioning

confidence: 99%

The Transmembrane Domains of Ectoapyrase (CD39) Affect Its Enzymatic Activity and Quaternary Structure

Wang

Guidotti

1998

Journal of Biological Chemistry

140

193

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“…Diabetes-induced hyperlipidemia and the alterations in membrane phospholipids and fatty acids have been shown to depress membrane-bound enzyme activity (Kuwahara et al, 1997 (Sabbadini & Dahms, 1989). Thus, the pumps are essential for the regulation of cell volume, uptake of nutrients, and cell growth and differentiation, and are also critical for normal functioning of excitable and non-excitable tissues.…”

Section: Discussionmentioning

confidence: 99%

Influence of kaempferol, a flavonoid compound, on membrane-bound ATPases in streptozotocin-induced diabetic rats

Al‐Numair

Veeramani

Alsaif

et al. 2015

Pharmaceutical Biology

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Context: Kaempferol is a flavonoid found in many edible plants (e.g. tea, cabbage, beans, tomato, strawberries, and grapes) and in plants or botanical products commonly used in traditional medicine. Numerous preclinical studies have shown that kaempferol have a wide range of pharmacological activities, including antioxidant, anti-inflammatory, anticancer, cardioprotective, neuroprotective, and antidiabetic activities. Objective: The present study investigates the effect of kaempferol on membrane-bound ATPases in erythrocytes and in liver, kidney, and heart of streptozotocin (STZ)-induced diabetic rats. Materials and methods: Diabetes was induced into adult male albino rats of the Wistar strain, by intraperitoneal administration of STZ (40 mg/kg body weight (BW)). Kaempferol (100 mg/kg BW) or glibenclamide (600 mg/kg BW) was administered orally once daily for 45 d to normal and STZinduced diabetic rats. The effects of kaempferol on membrane-bound ATPases (total ATPase, Na + /K + -ATPase, Ca 2+ -ATPase, and Mg 2+ -ATPase) activity in erythrocytes and in liver, kidney, and heart were determined. Results: In our study, diabetic rats had significantly (p50.05) decreased activities of total ATPases, Na + /K + -ATPase, Ca 2+ -ATPase, and Mg 2+ -ATPase in erythrocytes and tissues. Oral administration of kaempferol (100 mg/kg BW) or glibenclamide (600 mg/kg BW) for a period of 45 d resulted in significant (p50.05) reversal of these enzymes' activities to near normal in erythrocytes and tissues when compared with diabetic control rats. Discussion and conclusion: Thus, obtained results indicate that administration of kaempferol has the potential to restore deranged activity of membrane-bound ATPases in STZ-induced diabetic rats. Further detailed investigation is necessary to discover kaempferol's action mechanism.

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“…Because T-tubule membranes are particularly enriched in cholesterol [3] and it has been reported that the oxidation of membrane cholesterol increases membrane fluidity [19], we studied the effects of cholesterol oxidase treatment on ecto-ATPase activity. Figure 4 shows a complete loss of enzymic activity after a 30 min treatment of chicken T-tubules with cholesterol oxidase.…”

Section: Figure 4 Effect Of Treatment With Cholesterol Oxidase On Ectmentioning

confidence: 99%

“…In skeletal muscle, a very efficient E-type ATPase activity, first designated as Mg# + -ATPase or ' basic '-ATPase, is associated with transverse tubule (T-tubule) membranes [3]. This activity was extensively characterized in rabbit and chicken skeletal muscle T-tubule vesicles [4][5][6].…”

Section: Introductionmentioning

confidence: 99%

Regulation of transverse tubule ecto-ATPase activity in chicken skeletal muscle

Megías

Martinez-Senac

Delgado

et al. 2001

Biochemical Journal

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Transverse tubule (T-tubule) ecto-ATPase from chicken skeletal muscle is an integral membrane glycoprotein that seems to exist as a homodimer and exhibits unusual properties. Treatment of T-tubule membranes with concanavalin A (Con A) did not significantly affect the thermal variation of the fluorescence anisotropy of vesicles labelled with 1,6-diphenyl-1,3,5-hexatriene or trimethylammonium-1,6-diphenyl-1,3,5-hexatriene. Cross-linking of membrane components with glutaraldehyde elicited effects on ecto-ATPase activity very similar to those of Con A treatment: a severalfold increase in activity, a decrease in Triton X-100 sensitivity and a requirement to be present before ATP to exert its action. In addition, glutaraldehyde and Con A normalized the temperature dependence and the kinetic behaviour of the enzyme. Membrane-perturbing agents (detergents, alcohols and cholesterol oxidase), with the sole exception of digitonin, caused a marked decrease in ecto-ATPase activity; the prior presence of Con A prevented this inhibition, whereas when the lectin was added after the membrane perturbing agent, recovery of the activity was not always possible. The addition of nucleotides before Con A led to a suppression of ecto-ATPase stimulation; it occurred when the nucleotide was hydrolysed (ATP or UTP) and when it was not (adenosine 5′-[β,γ-imido]triphosphate) and even in the presence of 3mM Pi. A model is proposed for the complex regulatory mechanisms of chicken T-tubule ecto-ATPase that involves the occurrence of two different catalytic states in an equilibrium modulated by lectins and cross-linking agents, by the structure of the membrane and by the presence of ligands for a regulatory site.

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Biochemical properties of isolated transverse tubular membranes

Cited by 51 publications

References 191 publications

The Transmembrane Domains of Ectoapyrase (CD39) Affect Its Enzymatic Activity and Quaternary Structure

The Transmembrane Domains of Ectoapyrase (CD39) Affect Its Enzymatic Activity and Quaternary Structure

Influence of kaempferol, a flavonoid compound, on membrane-bound ATPases in streptozotocin-induced diabetic rats

Regulation of transverse tubule ecto-ATPase activity in chicken skeletal muscle

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