A fast passive Ca2+ efflux mediated by the (Ca2+ + Mg2+)-ATPase in reconstituted vesicles (original) (raw)
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Biochemical Journal, 1987
Preincubation of sarcoplasmic reticulum with 1 mM-ATP completely inhibits Ca2l accumulation and stimulates ATPase activity by over 2-fold. This effect of ATP is obtained only when the preincubation is carried out in the presence of Pi, but not with arsenate, chloride or sulphate. The inhibition by ATP of Ca2' accumulation is pH-dependent, increasing as the pH is increased above 7.5. Inhibition of Ca21 accumulation is observed on preincubation with ATP, but not with CTP, UTP, GTP, ADP, adenosine 5'-[fly-methylene]triphosphate or adenosine 5'-[/ly-imido]triphosphate. The presence of Ca2", but not Mg2", during the preincubation, prevents the effect of ATP + Pi on Ca2`accumulation. The ATP + Pi inhibition of Ca2+ accumulation is not due to modification of the ATPase catalytic cycle, but rather to stimulation of a rapid Ca2' efflux from actively or passively loaded vesicles. This Ca2+ effilux is inhibited by dicyclohexylcarbodi-imide. Photoaffinity labelling of sarcoplasmic-reticulum membranes with 8-azido-[a-32P]ATP resulted in specific labelling of two proteins, of approx. 160 and 44 kDa. These proteins were labelled in the presence of Pi, but not other anions. EXPERIMENTAL Materials ATP, ADP, p[NH]ppA, p[CH2]ppA, CTP, UTP, GTP, EGTA and Tricine were obtained from Sigma Chemical Co. 45CaC12 was from The Radiochemical Centre (Amersham, Bucks., U.K.), and [82P]P1 from Nuclear Research Center, Negev, Israel. 8-N3-[X-32P]ATP (6.6 Ci/mmol) was obtained from ICN. [_y32p]_ ATP was synthesized from [32P]P, and ADP by photophosphorylation with lettuce chloroplasts (Avron, 1960) and was purified on Dowex 1-X8 with NaCl as the eluent. Membrane preparation Sarcoplasmic-reticulum vesicles were prepared from rabbit fast-twitch skeletal muscle as described by Camp-Vol. 247 497 Abbreviations used: p[CHJppA, adenosine 5'-[fly-methylene]triphosphate; p[NH]ppA, adenosine 5'-[fly-imido]triphosphate; DDCD, dicyclohexylcarbodi-imide; 8-N3-ATP, 8-azido-ATP; FITC, fluorescein isothiocyanate.
Uptake at Ca2+ mediated by the (Ca2+ + Mg2+)-ATPase in reconstituted vesicles
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1987
The (Ca2+ + MgZ+)-ATPase was purified from skeletal muscle sarcoplasmic reticulum and reconstituted into sealed phospholipid vesicles by solution in cholate and deoxycholate followed by detergent removal on a column of Seplmdex G-50. The level of Ca 2+ accumulated by these vesicles, either in the presence or absence of phosphate within the vesicles, increased with increasing content of phosphatidylethanolamine in the phospholipid mixture used for the reconstitution. The levels of Ca 2+ accumulated in the absence of phosphate were very low for vesicles reconstituted with egg yolk phosphatidyicholine alone at pH 7.4, but increased markedly with decreasing pH to 6.0. Uptake was also relatively low for vesicles recoltstituted with dimyristoleoyl-or dinervonylphosphatidylcholine, and addition of cholesterol had little effect. The level of Ca 2+ accumulated increased with increasing external K + concentration, and was also increased by the ionophores FCCP and valinomycin. Vesicle sizes changed little with changing phosphatidylethanolamine content, and the sidedness of insertion of the ATPase was close to random at all phosphatidylethanolamine contents. It is suggested that the effect of phosphatidylethanolamine on the level of Ca 2+ accumulation follows from an effect on the rate of Ca 2+ efflux mediated by the ATPase.
Annals of the New York Academy of Sciences, 1978
The early phase of ATP-dependent Ca2+ uptake by sarcoplasmic reticulum (SR) vesicles was first monitored by optical methods involving the use of metallochromic indicators' and stopped flow mixing.2 Alternatively, the initial Ca'+ uptake can be measured directly with the aid o f a radioactive tracer in rapid quench experiments. This method requires the use of a forced flow device equipped with two mixing chambers for starting and quenching the reaction by sequential additions of ATP and EGTA.:'a4
Biochemistry, 1986
Time-resolved filtration measurements using radioactive calcium were conducted to investigate with leaky preparations the kinetic features of the dissociation of transported calcium to the luminal side of the sarcoplasmic reticulum calcium pump, which occurs concomitantly with isomerization of the phosphorylated ATPase. A t p H 6 and 20 OC, Ca2+ dissociation was moderately fast in the absence of potassium (3-5 s-l a t 0.05 m M ATP), implying that the dephosphorylation step (about 1.5 s-l) was the main contributor to rate limitation under these conditions. Potassium slowed down Ca2+ release but stimulated dephosphorylation, so that in its presence Ca2+-releasing isomerization did contribute to rate limitation, especially at neutral pH. At p H 6 in the absence of potassium and in the presence of magnesium, millimolar concentrations of A T P doubled the rate of Ca2+ dissociation, as also shown by dual-wavelength detection of fast changes in the absorbance of the Ca2+-sensitive dye Antipyrylazo 111. Under the same conditions, low-affinity binding of A T P to phosphoenzyme was demonstrated. It is suggested that this low-affinity acceleration by A T P of the crucial step leading to dissociation of transported Ca2+ is the specific interaction responsible for the low-affinity acceleration of overall ATPase activity generally observed in the presence of potassium a t neutral pH. Hydrolysis of the Ca2+-deprived phosphoenzyme was accelerated by A T P in the absence but not in the presence of Mg2+ in the dephosphorylation medium. W e suggest that metal-free A T P is a more potent activator than Mg-ATP for transitions involving phosphoenzyme. 'A preliminary communication has been presented at the 30th Biophysical Society Meeting in San Francisco (Champeil & Guillain, 1986). vesicles. Later, the covalent acyl-phosphate bond in the phosphoenzyme is hydrolyzed, and the ATPase conformation reverts to the initial one to which cytoplasmic calcium may
Effect of calcium on the interactions between Ca2+-ATPase molecules in sarcoplasmic reticulum
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1989
The interaction between CaZ+-ATPase molecules in the native sarcoplasmic reticulum membrane and in detergent solutions was analyzed by chemical crosslinking, high performance liquid chromatography (HPLC), and by the polarization of fluorescence of fluorescein 5'-isothiocyanate (FITC) covalcotly attached to the CaZ+-ATPase. Reaction of sarcoplasmic reticulum vesicles with glutaraldehyde causes the crnsslinking of Ca2÷-ATPnse molecules with the formation of dimers, tetramers and higher uligomers. At moderate concentrations of glutaraldehyde solubilization of sarc~plasmic retieulum by CnE a or Brij 36T (---d mg/mg protein) decreased the formation of higher oligo~ters without significant interference with the appearance of crosslinked ATPase dimers. These observations are consistent with the existence of CaZ+-ATPase dimers in detergent-solubilized sarcoplasmic reticulum. Ca 2+ (2-20 mM) and glycerol (10-20%) increased the degree of crnsslinking at pH 6.0 both in vesicular and in sulubilized sareoplnsmic reticulum, presumably by prmuoting interactions between ATPase molecules; at pH 7.5 the effect of Ca z+ was less pronounced. In agreement with these observations, high performance liquid chromatography of sarcoplasmic retiealum proteins solubilized by B~j 36T or CIzEto revealed the presence of components with the expected elution characteristics of CaZ+-ATPase oligomers. The polarization of fluorescence of FITC covalently attached to the CaZ+-ATPnse is low in the native sarcoplasmic reticulum due to energy transfer, consistent with the existence of ATPase oligomers (Highsmith, S. a,,d Cohen, J.A. (!q~lT) Biochemistry 26,154-161); upon solnbilization of the sarcoplasmie retieulum by detergents, the polarization of fluorescence increased due to dissociation of ATPase oligomers. Based on its effects on the fluorescence of FITC-ATPase, Ca z+ promoted the interaction between ATPase molecules, both in the native membrane and in detergent solutions. the field. In spite of intense effort at its solution [4], there is still disagreement about the size of the putative ATPas~; oligomers, their association constants, and functional significance.
Calcium Transport by Sarcoplasmic Reticulum Ca2+-ATPase
Journal of Biological Chemistry, 2002
After treatment of sarcoplasmic reticulum Ca 2؉-ATPase with proteinase K (PK) in the presence of Ca 2؉ and a protecting non-phosphorylated ligand (e.g. adenosine 5-(,␥-methylenetriphosphate), we were able to prepare in high yield an ATPase species that only differs from intact ATPase because of excision of the MAATE 243 sequence from the loop linking the A domain with the third transmembrane segment. The PK-treated ATPase was unable to transport Ca 2؉ and to catalyze ATP hydrolysis, but it could bind two calcium ions with high affinity and react with ATP to form a classical ADPsensitive phosphoenzyme, Ca 2 E1P, with occluded Ca 2؉. The ability of Ca 2 E1P to become converted to the Ca 2؉free ADP-insensitive form, E2P, was strongly reduced, as was the ability of PK-treated ATPase to react with orthovanadate or to form an E2P intermediate from inorganic phosphate in the absence of Ca 2؉. PK-treated ATPase also reacted with thapsigargin to form a complex with altered properties, and the tryptic cleavage "T2" site in the A domain was no longer protected in the absence of Ca 2؉. It is probable that disrupting the C-terminal link of the A domain with the transmembrane region severely compromises reorientation of A and P domains and the functionally critical cross-talk of these domains with the membrane-bound Ca 2؉ ions.