PH-induced changes in the reactions controlled by the low- and high-affinity Ca2+-binding sites in sarcoplasmic reticulum.
The effect of pH on the Ca2+-binding sites of high and low affinity, located respectively on the outer and inner surfaces of the sarcoplasmic reticulum membrane, was investigated using intact and leaky sarcoplasmic reticulum vesicles. With the use of intact vesicles, different pH profiles of membrane phosphorylation and rates of nucleoside triphosphate hydrolysis were obtained depending on the assay temperature, on the Ca2+ concentration, and on whether ATP or ITP was used as substrate. The different pH profiles were related to the amount of Ca2+ accumualted by the vesicles, i.e., to different degrees of saturation of the inner, low-affinity Ca2+-binding site. With the use of leaky vesicles, the saturation of the two Ca2+-binding sites could be controlled more precisely since the Ca2+ concentration on both sides of the membrane was equal to the Ca2+ concentration of the assay medium. Using leaky vesicles and measuring the rates of nucleotide hydrolysis, nucleotide-phosphate exchange and membrane phosphorylation by nucleotide as an indication of the degree of saturation of the Ca2+-binding sites, we observed that the affinity of both the high- and low-affinity sites increased three to four orders of magnitude when the pH of the assay medium was increased from 6.1 to 8.65.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002474:Cell-Free System; D006863:Hydrogen-Ion Concentration; D007292:Inosine Nucleotides / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D009132:Muscles / Q000378:metabolism; D010539:Permeability; D010710:Phosphates / Q000378:metabolism; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013696:Temperature
D007292:Inosine Nucleotides; D008565:Membrane Proteins; D010710:Phosphates; D000255:Adenosine Triphosphate; D008274:Magnesium; D002118:Calcium