http://www.jbc.org/content/266/26/17067.full.pdf

Thapsigargin inhibits the sarcoplasmic or endoplasmic reticulum Ca-ATPase family of calcium pumps.

Functional comparisons between isoforms of the sarcoplasmic or endoplasmic reticulum family of calcium pumps.

Ecto-ATPases are ubiquitous in eukaryotic cells. They hydrolyze extracellular nucleoside tri- and/or diphosphates, and, when isolated, they exhibit E-type ATPase activity, (that is, the activity is dependent on Ca2+ or Mg2+, and it is insensitive to specific inhibitors of P-type, F-type, and V-type ATPases; in addition, several nucleotide tri- and/or diphosphates are hydrolysed, but nucleoside monophosphates and nonnucleoside phosphates are not substrates). Ecto-ATPases are glycoproteins; they do not form a phosphorylated intermediate during the catalytic cycle; they seem to have an extremely high turnover number; and they present specific experimental problems during solubilization and purification. The T-tubule Mg2+-ATPase belongs to this group of enzymes, which may serve at least two major roles: they terminate ATP/ADP-induced signal transduction and participate in adenosine recycling. Several other functions have been discussed and identity to certain cell adhesion molecules and the bile acid transport protein was suggested on the basis of cDNA clone isolation and immunological work.

Ecto-ATPases: identities and functions.

The trehalose myth revisited: introduction to a symposium on stabilization of cells in the dry state.

The primary active transport of Na +, K +, Ca 2T and H + in eukaryotic cells is driven by ATP-powered cation pumps, the Na,K pump, Ca pump and H,K pump. The intermediary steps of the pump reactions and their relationship to cation translocation have been examined in detail, particularly for the Na,K-pump and Ca-pump proteins. A common feature of these pumps is a protein of Mr close to 110,000 that binds ATP and accepts its y-phosphate in a covalent Asp-P bond. There is evidence that both phosphoand dephospho-forms of the protein exist in two major conformational states, E1 and E2, with different affinities and orientation of cation binding sites, and ATP-driven cation pumping is blocked by micromolar concentrations of vanadate [39, 68, 95, 96, 116, 132]. A wealth of structural information about the cation pump proteins appeared recently after identification of mRNA and sequencing of cloned cDNA. Several isoforms of the genes of o~-subunit of Na,K pump were identified in the human and rat genomes [147, 182] and the sequences ofa-subunit [110, 111,145, I46, 178,181] and B-subunit [22, 112, 140, 146, 179] were deduced from cDNA of mRNA from a variety of tissues in piscine and mammalian species. A similar intensity of work in adjacent fields provided the sequences of slow and fast twitch Ca pump from sarcoplasmic reticulum [19, 121] and H,K pump from stomach mucosa [180]. In addition the gene sequences are available for K pumps [82, 185] and H pumps [1, 177] from microorganisms. As paradigm for interpretation of this information the combined X-ray crystallographic structure at high resolution and

Structural basis for E1-E2 conformational transitions in Na,K-pump and Ca-pump proteins.

Substrate regulation of the sarcoplasmic reticulum ATPase. Transient kinetic studies.

The stoichiometry of phosphorylation (catalytic) sites in sarcoplasmic reticulum vesicles ( SRV ) and SR ATPase purified by differential solubilization with deoxycholate was found to be 4.77 +/- 0.4 and 6.05 +/- 0.18 nmol/mg of protein, respectively, when phosphorylation was carried out under conditions permitting 32P labeling of nearly all sites. Assuming that each site corresponds to a single 115K ATPase chain, the observed site stoichiometry accounts only for 55% and 70% of the total protein. Failure to obtain higher phosphorylation levels was due to the presence of nonspecific protein contaminants in SRV or to the presence of inactive aggregates in the ATPase purified with deoxycholate. This was demonstrated by dissolving SRV and purified ATPase with lithium dodecyl sulfate, subjecting them to molecular sieve HPLC, and collecting the elution fractions for determination of protein, measurement of 32P-labeled sites, and electrophoretic analysis. In fact, in the specific elution peak containing the 115K ATPase chains, phosphorylation levels were 6.62 +/- 0.33 and 7.03 +/- 0.18 in SRV and purified ATPase, corresponding to 68% and 86% of the protein in the specific elution peak. An alternate purification method was then developed, based on solubilization of SRV with dodecyl octaethylene glycol monoether ( C12E8 ), separation of delipidated ATPase by anion-exchange chromatography, and enzyme reactivation with phosphatidylcholine. This preparation yields 7.3 +/- 0.44 nmol of phosphorylation site/mg of protein of the SRV fraction before HPLC.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosinetriphosphatase site stoichiometry in sarcoplasmic reticulum vesicles and purified enzyme.

Yeast cells have had to develop mechanisms in order to protect themselves from chemical and physical agents of the environment to which they are exposed. One of these physical agents is thermal variation. Some yeast cells are known to accumulate high concentrations of trehalose when submitted to heat shock. In this work, we have studied the effect of trehalose on the protection against thermal inactivation of purified plasma membrane H+-ATPase from Schizosaccharomyces pombe, in the solubilized and in the reconstituted state. We observed that after 1 min of incubation at 51 degrees C in the presence of 1 M trehalose, about 50% of soluble enzyme remains active. In the same conditions, but in the absence of trehalose, the activity was completely abolished. The t0.5 for the enzyme inactivation increased from 10 to 50 s after reconstitution into asolectin liposomes. Curiously, in the presence of 1 M trehalose, the t0.5 for inactivation of the reconstituted enzyme was further increased to higher than 300 s, regardless of whether trehalose was added inside or outside the liposome. Additionally, the concentration that confers 50% for the protection by trehalose (K0.5) decreased from 0.5 M, in the solubilized state, to 0.04 M in the reconstituted state, suggesting a synergetic effect between sugar and lipids. Gel electrophoresis revealed that the pattern of H+-ATPase cleavage by trypsin changed when 1 M trehalose was present in the buffer. It is suggested that both in a soluble and in a phospholipid environment, accumulation of trehalose leads to a more heat-stable conformation of the enzyme, probably an E2-like form.

Helena M. Scofano

Papers

Substrate regulation of the sarcoplasmic reticulum ATPase. Transient kinetic studies.

Adenosinetriphosphatase site stoichiometry in sarcoplasmic reticulum vesicles and purified enzyme.

Protection against thermal denaturation by trehalose on the plasma membrane H+‐ATPase from yeast

Gill microsomal (Na+,K+)-ATPase from the blue crab Callinectes danae: Interactions at cationic sites.

Transient state kinetic studies of phosphorylation by ATP and Pi of the calcium-dependent ATPase from sarcoplasmic reticulum.