CYTOCHEMISTRY OF PHOSPHATASES OF THE SARCOPLASMIC RETICULUM: I. Biochemical Studies (original) (raw)
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Kinetics and Regulation of Sarcoplasmic Reticulum ATPase
European Journal of Biochemistry, 1977
The measurement of ATP binding to the sarcoplasmic reticulum membrane reveals that the calcium pump possesses one high affinity (& = 2 -3 pM) site. Competition with substrate analogs show the high specificity of that site. At high ATP concentration another class of site can be detected with a much higher dissociation constant (Kd z 500 pM). This class of sites is of low specificity and ATP is easily displaced by other polyphosphates.
J Membrane Biol, 1978
Tryptic digestion of (Ca 2 § + Mg 2 +)-ATPase from sarcoplasmic reticulum of rabbit skeletal muscle has previously been shown to cleave the enzyme initially into a 55,000-dalton fragment and a 45,000-dalton fragment. In the present study the two fragments are solubilized in sodium dodecyl sulfate (SDS) and separated by preparative polyacrylamide gel electrophoresis. The 45,000-dalton fragment is found to be a relatively nonselective, divalent cation-dependent ionophore when incorporated into an oxidized cholesterol membrane (BLM). Ionophoric activity of this fragment is inhibited by low concentrations of LaC13, HgCI2, and various reducing agents. There appears to be one or two relatively inaccessible disulfide bonds in the 45,000-dalton fragment that are essential for transport. Addition of reducing agents inhibits the ionophoric activity of the succinylated undigested enzyme and the 45,000-dalton fragment, but has no effect on the 55,000-dalton fragment. These experiments imply that the 45,000-dalton fragment and the 55,000-dalton fragment are in a series arrangement in the membrane. By hydrolysis of ATP, the sarcoplasmic reticulum lowers the cytoplasmic calcium concentration in the region of the myofibrils to micromolar levels, pumping calcium against a 1000-to 3000-fold calcium gradient (Hasselbach & Makinose, 1962). This depletion of calcium leads to muscle relaxation. The main protein constituent of sarcoplasmic reticulum from rabbit white skeletal muscle is (
Biochimica et Biophysica Acta (BBA) - General Subjects, 1980
Sarcoplasmic reticulum from rabbit fast skeletal muscle contains intrinsic protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) and a substrate. The protein kinase activity was Mg 2+ dependent and could also phosphorylate exogenous protein substrates. Autophosphorylation of sarcoplasmic reticulum vesicles was not stimulated by cyclic AMP, neither was it inhibited by the heat-stable protein kinase inhibitor protein. The phosphorylated membranes had the characteristics of a protein with a phosphoester bond. An average of 73 pmol Pi/mg protein were incorporated in 10 min at 30°C. Addition of exogenous cyclic AMP-dependent protein kinase increased the endogenous level of phosphorylation by 25--100%. Sarcoplasmic reticulum membrane phosphorylation, mediated by either endogenous cyclic AMP-independent or exogenous cyclic AMP-dependent protein kinase, occurred on a 100 000 dalton protein and both enzyme activities resulted in enhanced calcium uptake and Ca2+-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3), in a manner similar to cardiac microsomal preparations.
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.
Effect of pH on the activity of the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum
Biochemical Journal, 1990
A kinetic model for the Ca2(+) + Mg2(+)-activated ATPase of sarcoplasmic reticulum was presented in a previous paper [Stefanova, Napier, East & Lee (1987) Biochem. J. 245, 723-730]. Here, that model is modified to account for the pH-dependence of ATPase activity and for the effects of Mg2+ on activity at high pH. It is shown that effects of Mg2+ on measurements of ATPase activity as a function of ATP concentration at pH 8.0 and pH 8.5 are consistent with binding of Mg2+ to the Ca2(+)-binding sites on the phosphorylated ATPase, such binding inhibiting dephosphorylation of the ATPase. It is also shown that slow dissociation of Ca2+ from the phosphorylated ATPase is consistent with the previously published model.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1990
The CaZ+-dependent adenosinetriphosphatase (CaZ+-ATPase) from the sarcoplasmic reticulum (SR) of rat skeletal muscles is pbosphorylated by inorganic phosphate (Pi) in the absence of Ca z +. The reaction can be described by the following simplified scheme: 1 2 E+ Pi~E-Pi~E-P+ H20 where E-P is a covalent, acid-stable and ADP-insensitive phosphoenzyme, and E. Pi is a noncovalent and acid-labile complex. The reaction is Mg2+-dependent. Membrane fragments deposited on Miilipore filters were successively pertused with two solutions, at constant flow. The effluent samples were analyzed. The pertused solutions were Ca z÷ free and always contained 40% dimethylsulfoxide (DMSO), plus other reactants. Following the successive perfusion of solutions without and with [3ZpIPi, 32p binding is only detected in the presence of Mg 2+, indicating the formation of the phosphoenzymes (E. Pi and E-P). Following pertusions of the phosphoenzymes with 5% trichluroacetic acid, 32p release indicates the mount of the acid-labile moiety (E. Pi). After pbosphorylations, the filters were washed with acid and unlabeled Pi, and the remaining radioactivity was measured to evaluate the acid-stable phosphoenzyme (E-P). The acid-labile and acid-stable phosphoenzymes amounted, respectively, 0.72 5: 0.12, and 1.48 + 0.10 nmol of Pi/mg of protein (+S.E., n = 5), after pbosphorylations with 20 pM Pi. The results indicate: (1) The method allowed the evaluation of the acid-labile intermediate of the SR Ca2+-ATPase cycle. K~=kz/k2, in the above scheme, approaches 2.0. (2) The substrate of the phosphorylation reaction, in the presence of DMSO, is likely to be the Mg • Pi complex, since Mg 2+ is necessary for step 1 in the above scheme.