The Mg2+-ATPase of rabbit skeletal-muscle transverse tubule is a highly glycosylated multiple-subunit enzyme

Kirley, Terence L.

doi:10.1042/bj2780375

Cited by 13 publications

(12 citation statements)

References 25 publications

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“…The results presented here and in other studies [5][6][7]11,21] indicate that the chicken muscle ecto-ATPase is an enzyme that can be finely regulated and that exhibits several unusual enzymic properties. With the sole exception of chicken brain enzyme [7], these uncommon properties of the avian muscle ecto-ATPase are not shared by ecto-NTPDases from other chicken tissues [8,21] or by the ecto-ATPases present in membrane preparations isolated from the skeletal muscle of other species [4,18,23]. Of the modulators that affect chicken ecto-ATPase, Con A is of particular interest because it was found to abolish its anomalous behaviour, converting the enzyme to Michaelis-Menten-type properties [5].…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2001

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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'-[beta,gamma-imido]triphosphate) and even in the presence of 3 mM P(i). 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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Section: Discussionmentioning

confidence: 99%

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

et al. 2001

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“…The tissues were excised and membranes prepared from these tissues as described previously (Stout & Kirley, 1994b). Rabbit skeletal muscle transverse tubule membranes (Kirley, 1991) and chicken gizzard membranes (Stout & Kirley, 1994a) were processed and prepared as described previously. Protein concentrations of all the resultant membranes were determined by the Bio-Rad dye binding assay, using the modification of Stoscheck (1990) and bovine serum albumin as the standard.…”

Section: Methodsmentioning

confidence: 99%

“…Cross-Linking/Mg-ATPase Assay/Western Analysis. Solubilized chicken gizzard or rabbit t-tubule protein [membrane protein was solubilized with digitonin as described previously (Stout & Kirley, 1994a)] or membrane-bound chicken gizzard protein was cross-linked with DTSSP, DSP, or BS 3 basically as described previously (Kirley, 1991). As a control for the effects of lysine modification in the absence of cross-linking, sulfo-NHS-acetate was employed as a monofunctional reagent very similar in structure and specificity to each half of the divalent DTSSP cross-linker.…”

Section: Methodsmentioning

confidence: 99%

Control of Cell Membrane Ecto-ATPase by Oligomerization State: Intermolecular Cross-Linking Modulates ATPase Activity

Stout

Kirley

1996

Biochemistry

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The extracellular ATPase (ecto-ATPase) is a divalent cation-dependent nucleoside triphosphatase with an unusually high specific activity. Monoclonal antibodies, described previously [Stout, J. G., Strobel, R. S., & Kirley, T. L. (1995) J. Biol. Chem. 270, 11845-11850], and newly generated polyclonal antibodies, both raised against the chicken gizzard ecto-ATPase, were evaluated for their ability to cross-react with mammalian ecto-ATPases and were used as specific immunochemical probes to identify non-cross-linked and cross-linked ecto-ATPase. Unlike previous results obtained with the rabbit skeletal muscle ecto-ATPase enzyme, cross-linking the chicken gizzard smooth muscle ecto-ATPase with 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP) and dithiobis(succinimidylpropionate) (DSP) increased the activity of the enzyme which corresponded to an increase in a approximately 130 kDa immunoreactive band, proposed to be a ecto-ATPase homodimer, and a concomitant decrease in a approximately 66 kDa immunoreactive band, the ecto-ATPase monomer. Ecto-ATPase was immunochemically identified in chicken, rat, mouse, rabbit, and pig. Interestingly, under nonreducing conditions, the ecto-ATPase activity in rat and pig (unlike chicken and rabbit) was evident on Western blots as an immunoreactive band at approximately 200 kDa, proposed to be an intermolecularly disulfide-linked ecto-ATPase homotrimer. Nonreducing Western blot analysis of various rat tissues with three different monoclonal antibodies that recognize the 66 kDa chicken gizzard ecto-ATPase monomer strengthened the hypothesis that this 200 kDa band indeed represents the trimeric ecto-ATPase. After reduction, ecto-ATPase monomers were found to be approximately 66 kDa in all species examined. The differences in ecto-ATPase quaternary structure stability may account for the observed species differences in ecto-ATPase enzymatic properties. Intermolecular disulfide bonds appear to be one of the species-specific ways to stabilize the native, active ecto-ATPase quaternary structure (the homotrimer). Based on the data obtained, as well as previous data from this and other laboratories, a hypothesis was developed to explain the modulation of ecto-ATPase activity by a variety of agents, including detergents, chemical cross-linkers, lectins, antibodies, and small molecule inhibitors. It is proposed that agents and conditions stabilizing ecto-ATPase oligomers stimulate enzyme activity, whereas agents and conditions destabilizing ecto-ATPase homooligomers would inhibit the ecto-ATPase.

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“…Gels were either 0.75 mm (analytical) or 1.5 mm (native) thick. The native gel consisted of a 6% Laemmli resolving gel containing 0.1% digitonin and no SDS as described previously (28). The gels were either stained with silver according to Ansorge (29) or electroblotted onto 0.2-f.Lm polyvinylidene fluoride membranes for 2 h at 33 V in 10 mM CAPS buffer, pH 11.0 (30).…”

Section: Methodsmentioning

confidence: 99%

Properties of and Proteins Associated with the Extracellular ATPase of Chicken Gizzard Smooth Muscle

Stout

Strobel

Kirley

1995

Journal of Biological Chemistry

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The chicken gizzard smooth muscle extracellular ATPase (ecto-ATPase) is a low abundance, high specific activity, divalent cation-dependent, nonspecific nucleotide triphosphatase (NTPase). The ATPase is a 66-kDa glycoprotein with a protein core of 53 kDa (Stout, J.G. and Kirley, T.L. (1994) J. Biochem. Biophys. Methods 29, 61-75). In this study we evaluated the characteristics of a bank of monoclonal antibodies raised against a partially purified chicken gizzard ecto-ATPase. 18 monoclonal antibodies identified by an ATPase capture assay were tested for effects on ATPase activity as well as for their Western blot and immunoprecipitation potential. The five most promising monoclonal antibodies were used to immunopurify the ecto-ATPase. The one-step immunoaffinity purification of solubilized chicken gizzard membranes with all five of these monoclonal antibodies isolated a 66-kDa protein whose identity was confirmed by N-terminal sequence analysis to be the ecto-ATPase. Several of these monoclonal antibodies stimulated ecto-ATPase activity similar to that observed previously with lectins. Western blot analysis revealed that three of the five monoclonal antibodies recognized a major immunoreactive band at 66 kDa (53-kDa core protein), consistent with previous purification results. The other two antibodies recognized proteins of approximately 90 and 160 kDa on Western blots. The 90-kDa co-immunopurifying (and presumably associated or related) protein was identified by N-terminal analysis as LEP100, a glycoprotein that shuttles between the plasma and lysosomal membranes. The approximately 160-kDa co-immunopurifying protein was identified by N-terminal analysis as integrin, a protein involved in extracellular contacts with adhesion molecules. Extended N-terminal sequence analysis of the immunopurified 66-kDa ecto-ATPase revealed some sequence homology with mouse lysosomal associated membrane protein. Tissue distribution of the ecto-ATPase showed that the highest levels of protein were expressed in muscle tissues (cardiac, skeletal, and smooth) and brain.

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The Mg2+-ATPase of rabbit skeletal-muscle transverse tubule is a highly glycosylated multiple-subunit enzyme

Cited by 13 publications

References 25 publications

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

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

Control of Cell Membrane Ecto-ATPase by Oligomerization State: Intermolecular Cross-Linking Modulates ATPase Activity

Properties of and Proteins Associated with the Extracellular ATPase of Chicken Gizzard Smooth Muscle

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