Transverse tubule Mg(2+)-ATPase of skeletal muscle. Evidence for extracellular orientation of the chicken and rabbit enzymes.

Saborido, Ana; Moro, G; Megías, Alicia

doi:10.1016/s0021-9258(18)54524-5

Cited by 34 publications

(4 citation statements)

References 42 publications

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“…While the importance of H59 is consistent with a previous report implicating a histidine residue in hydrolysis (35,36), the finding that H59 affects primarily the ATP rather than ADP hydrolysis rate is counterintuitive in light of the putative role of ACR1 as the β-phosphate binding domain (25). The crystal structures of actin with ATP and with ADP (26), as well as the proposed mechanism for hsp70 ATPase activity (37) based on the hsp70 (38) and actin structures (26), suggest that ACR4, the putative γ-phosphate binding domain, contains the residues involved in ATP hydrolysis and that ACR1 plays a more direct role in ADP hydrolysis.…”

Section: Discussionsupporting

confidence: 91%

Substitution of His59 Converts CD39 Apyrase into an ADPase in a Quaternary Structure Dependent Manner

Grinthal

Guidotti

1999

Biochemistry

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The two transmembrane domains of CD39 ecto-apyrase regulate the formation of fully active homotetramers. We show that mutations in apyrase conserved region 1 (ACR1) have two dramatically different sets of effects determined by whether they occur in intact tetramers or in disrupted tetramers or monomers. In intact tetramers, substitution of H59 in the rat brain CD39 ACR1 with G or S abolishes more than 90% of the ATPase activity but less than 50% of the ADPase activity, converting the enzyme into an ADPase with relative ADP:ATP hydrolysis rates of 6:1 or 8:1, respectively. In contrast, the same substitutions in tetramers lacking either transmembrane domain, in monomers lacking both transmembrane domains, or in detergent-solubilized full-length monomers have no effect on ATPase activity and increase ADPase activity approximately 2-fold, resulting in equal ATPase and ADPase activities. N61R substitution has a much smaller effect on the ADPase:ATPase ratio in both cases. While the data for truncated and monomeric constructs are consistent with the proposed role of ACR1 as the beta-phosphate binding domain by analogy with the actin/hsp70/hexokinase superfamily, the finding that H59 substitutions in full-length CD39 primarily diminish the ATP hydrolysis rate suggests that ACR1 may play a different role in intact tetramers. We propose that CD39 uses different ATPase and ADPase mechanisms in different quaternary structure contexts, and that H59 in ACR1 plays a central role specifically in ATP hydrolysis in intact tetramers.

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Section: Discussionsupporting

confidence: 91%

Substitution of His59 Converts CD39 Apyrase into an ADPase in a Quaternary Structure Dependent Manner

Grinthal

Guidotti

1999

Biochemistry

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“…The 43 kDa protein was detected in almost every tissue and species examined. Experiments designed to examine the possibility that it could be a proteolytic fragment of the ecto-ATPase were negative, in agreement with the consistent finding that the ecto-ATPase is very resistant to proteolysis (Kirley, 1988;Saborido et al, 1991). It is unlikely that this immunoreactivity was due to an impurity in the antigen used to generate the polyclonal antisera, since the antigen was size selected after immunoaffinity purification, and the antisera was affinity purified using size selected, immunoaffinity pure 66 kDa chicken gizzard ecto-ATPase.…”

Section: Ecto-atpase Modulation By Oligomerizationsupporting

confidence: 73%

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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“…The ecto-nucleoside triphosphate diphosphohydrolase (eNTPDase) family of enzymes (), previously called E-type ATPases (), hydrolyze a variety of nucleoside 5‘-triphosphates and 5‘-diphosphates in the extracellular space, and the relative hydrolysis rates for nucleoside triphosphates versus diphosphates vary considerably between enzymes in this family ( , ). The enzymes in this family consist of membrane-associated and soluble forms ( , ), and are expressed on a variety of cell types including endothelial cells (), activated lymphocytes (), skeletal and smooth muscle ( − ), and several types of tumors ( − ). Enzymatic activities of all eNTPDases are dependent on divalent cations such as calcium and magnesium.…”

mentioning

confidence: 99%

Site-Directed Mutagenesis of Human Nucleoside Triphosphate Diphosphohydrolase 3: The Importance of Residues in the Apyrase Conserved Regions

et al. 2001

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Ecto-nucleoside triphosphate diphosphohydrolase 3 (eNTPDase-3, also known as HB6 and CD39L3) is a membrane-associated ecto-apyrase. Only a few functionally significant residues have been elucidated for this enzyme, as well as for the whole family of eNTPDase enzymes. Four highly conserved regions (apyrase conserved regions, ACRs) have been identified in all the members of eNTPDase family, suggesting their importance for biological activity. In an effort to identify those amino acids important for the catalytic activity of the eNTPDase family, as well as those residues mediating substrate specificity, 11 point mutations of 7 amino acid residues in ACR1-4 of eNTPDase-3 were constructed by site-directed mutagenesis. Mutagenesis of asparagine 191 to alanine (N191A), glutamine 226 to alanine (Q226A), and arginine 67 to glycine (R67G) resulted in an increase in the rates of hydrolysis of nucleoside diphosphates relative to triphosphates. Mutagenesis of arginine 146 to proline (R146P) essentially converted the eNTPDase-3 ecto-apyrase to an ecto-ATPase (eNTPDase-2), mainly by decreasing the hydrolysis rates for nucleoside diphosphates. The Q226A mutant exhibited a change in the divalent cation requirement for nucleotidase activity relative to the wild-type and the other mutants. Mutation of glutamate 182 to aspartate (E182D) or glutamine (E182Q), and mutation of serine 224 to alanine (S224A) completely abolished enzymatic activity. We conclude that the residues corresponding to eNTPDase-3 glutamate 182 in ACR3 and serine 224 in ACR4 are essential for the enzymatic activity of eNTPDases in general, and that arginine 67, arginine 146, asparagine 191, and glutamine 226 are important for determining substrate specificity for human ecto-nucleoside triphosphate diphosphohydrolase 3.

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Transverse tubule Mg(2+)-ATPase of skeletal muscle. Evidence for extracellular orientation of the chicken and rabbit enzymes.

Cited by 34 publications

References 42 publications

Substitution of His59 Converts CD39 Apyrase into an ADPase in a Quaternary Structure Dependent Manner

Substitution of His59 Converts CD39 Apyrase into an ADPase in a Quaternary Structure Dependent Manner

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

Site-Directed Mutagenesis of Human Nucleoside Triphosphate Diphosphohydrolase 3: The Importance of Residues in the Apyrase Conserved Regions

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