Molecular and Cellular Biochemistry 126: 87-91, 1993. 9 1993 Kluwer Academic Publishers. Printed in the Netherlands. Effect of pH on stability of sarcoplasmic reticulum calcium pump in rabbit heart A. Xu, 1 N. Narayanan, 1 S.E. Samson 2 and A.K. Grover 2 1 Department of Physiology, University of Western Ontario, London, Ontario, Canada; e Department of Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada Received i April 1993; accepted 19 July 1993 Abstract The sarcoplasmic reticulum (SR) membranes isolated from rabbit heart were preincubated at pH 6.8 or 7.8 and their Ca 2+ pump properties were compared at pH 6.8. The ATP-dependent azide insensitive oxalate-stimulated Ca 2+ uptake was reduced more rapidly from the membranes preincubated at 37~ C at pH 7.8 than from those preincubated at pH 6.8. The Ca2+-MgZ+-ATPase,and the CaZ+-dependent formation of 110 kDa acylphosphate were also inhibited by the preincubation at the higher pH. Including 1 mM DTT in the preincubation medium reduced the inactivation. The preincubation at 37 ~ C in the presence or absence of DTT caused membranes to become more leaky as the loss of Ca 2+ uptake was more rapid than that of ATPase or the aeylphosphate formation. The loss of these activities was not accom- panied by a breakdown of the protein as monitored in Western blots. It is hypothesized that the SR Ca 2+ pump inactiva- tion involves a key-SH group and that the lower pH provides a compensatory protective mechanism for the SR during acidosis. (Mol Cell Biochem 126: 87-91, 1993) Key words: ischemia, acidosis, oxygen, free radicals, myocardium ATPase Introduction The regulation of intracellular [Ca 2+] is key to the con- tractility of all muscle cells (Carafoli 1987, Grover and Khan 1992, Inesi 1990). Several mechanisms involving Ca > channels and Na+-Ca2+-exchange and Ca 2+ pumps play a role in the Ca 2§ regulation. The sarcoplasmic retic- ulum (SR) in the myocardium contains a CaZ+-Mg2+-AT- Pase which is a 110 kDa protein encoded by the splice SERCA2a of the gene SERCA2 (Lytton et aL 1989, Ma- cLennan et al. 1985). During relaxation of cardiac myo- cytes the SR CaZ+-Mg2+-ATPase plays a key role by se- questering cytoplasmic Ca 2+ into the SR from where Ca 2+ may be released through Ca 2+ channels for the next contraction. Ischemic tissues not only accumulate a large amount of reactive oxygen, especially, hydrogen peroxide, su- peroxide and perhydroxy radicals but also have a large amount of hydrogen ions (Downey 1990). There are sev- eral reports of damage to the Ca2+ pumps and other en- zymes by reactive oxygen in various tissues (Grover and Samson 1989, Grover et al. 1992, Hebbel et al. 1986, Ka- neko et al. 1989, Kukreja et al. 1988, Tien et al. 1982, Vles- sis et al. 1991). The pH optimum of the SR and the PM Ca > pumps has also been previously reported (Grover Address for offprints: A.K. Grover, Department of Biomedical Sciences, McMaster University, HSC-4N41,1200 Main Street West, Hamilton, Ont- ario L8N 3Z5, Canada