JBC, Vol. 252, Issue 3, 994-1001, Feb, 1977
Proton inactivation of Ca2+ transport by sarcoplasmic reticulum
M. C. Berman, D. B. McIntosh and J. E. Kench
The effects of acid on fragmented sarcoplasmic reticulum from rabbit white
skeletal muscle have been studied. Brief exposure of sarcoplasmic reticulum
membranes to pH values in the range 5.5 to 6.0 at 37 degrees caused rapid
inactivation of calcium accumulation measured at 25 degrees in the presence
of oxalate (calcium uptake) while (Ca2+, Mg2+)-ATPase (EC 3.6.1.3) activity
was enhanced by 75%. ATPase activity, measured at 37 degrees in the absence
of oxalate and in the calcium steady state, was unaltered when calcium
uptake was inactivated. Calcium efflux from sarcoplasmic reticulum
vesicles, previously loaded passibely with 45CaCl2, was only slightly
increased when calcium uptake was abolished. At still lower pH values, 5.0
to 5.5, (Ca2+, Mg2+)-ATPase was inactivated while Mg2+ ATPase was more
acid-resistant. Acid inactivation of calcium uptake followed simple first
order kinetics for at least 80% of the time course. The rate constant, k,
increased from 0.043 min-1 to 1.63 min-1 between pH 6.50 and pH 5.35. At pH
4.65, Ea, the energy of activation, was 31 kcal mol-1 between 24 degrees
and 43 degrees. Inactivation, once initiated, was irreversible. Aged
suspensions of sarcoplasmic reticulum were more sensitive to acid
inactivation. Ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic
acid enhanced inactivation, and calcium specifically protected against
inactivation with half-maximal effect at 1 to 2 mM. The sulfhydryl reagent,
dithiothreitol (1 mM), caused significantly increased rates of
inactivation. Calcium binding was studied by dual wavelength
spectrophotometry and stopped flow analysis. Acid inactivation
distinguished two ATP-induced binding sites, previously described (Entman,
M. L., Snow, T. R., Freed, D., and Schwartz, A. (1973) J. Biol. Chem. 248,
7762-7772) as a superficial Mg2+-independent Site A which binds and
releases calcium rapidly and a deeper Mg2+-dependent Site B which binds and
releases calcium more slowly. Rates of binding to both sites were decreased
by acid inactivation. Binding of calcium to Site A increased, however, from
4.6 to 6.4 nmol mg of protein-1 whereas that to Site B decreased from 17.0
to 6.9 nmol mg of protein-1. Passive binding of calcium to sites of medium
affinity (K = 7 X 10(4) M-1) was unaffected by acid inactivation of calcium
uptake. Temperature dependence of (Ca2+, Mg2+)-ATPase was unchanged in the
range 9-34 degrees. Above 34 degrees, the higher activation energy process
(Ealpha = 33.7 kcal mol-1) observed in control sarcoplasmic reticulum and
thought to arise from a conformational change in the ATPase (Inesi, G.,
Millman, M., and Eletr, S. (1973) J. Mol. Biol. 81, 483-504) was diminished
by acid inactivation (Ealpha = 8.2 kcal mol-1) in a manner suggesting that
it is related to active calcium transport. The ATP in equilibrium 32Pi
exchange reaction was diminished by acid, but 25% of the activity remained
when calcium uptake was completely abolished...
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