THE ISOLATION AND CHARACTERIZATION OF PORCINE ERYTHROCYTE Ca2+ - ADENOCINE TRIPHOSPHATASE

Abstract

The Ca2+- pumping adenosine triphosphatase, Ca2+-ATPase, (ATP phosphohydrolase, EC 3.6.1.3), was purified from porcine erythrocyte membranes by calmodulin affinity chromatography in the presence of phosphatidylcholine and characterized. The membrane-bound enzyme in situ has a specific activity of 3.12 ± 0.08 µmoles/mg protein/hour and a Vmax of 3.47 ± 0.21 µmoles/mg protein/hour in the absence of calmodulin. Corresponding values for the purified enzyme are 101.79 ± 5.82 and 112.92 ± 3.87 respectively. Both forms of the enzyme showed biphasic (high and low affinity) Ca2+ activation kinetics, and were stimulated by calmodulin about 7- fold.. Calmodulin increased both the Ca2+ affinity and the Vmax of the enzyme.. The enzyme is also highly sensitive to inhibition by orthovanadate (Ki = 1.6 ± 0.2 µM). While these properties are consistent with those of the human erythrocyte Ca2+ -ATPase, the specific activity of the enzyme in intact porcine erythrocyte membranes is one order of magnitude higher than in human erythrocytes. The amount of enzyme routinely isolated from porcine erythrocytes is also about 5 times higher than that isolated from human erythrocyte ghosts. The purified enzyme was reconstituted into asolectin liposomes where it accumulated Ca2+ into the vesicles with a Vmax of 1.43 ± 0.11µmoles Ca2+ per mg. protein per minute. The ATPase activity of the reconstituted enzyme was stimulated 4 - fold by the addition of the Ca2+ - ionophore A 23187. This indicates a tight coupling between ATP hydrolysis and Ca2+ transport. After electrophoresis 1n sodium dodecyl sulphate (SDS) polyacrylamide gels, the purified enzyme appeared as a single polypeptide with a molecular weight of about 140,000. However, on high resolution gels, two minor bands (2% and 8% of the total proteins respectively) are seen at positions corresponding to 124,000 and 90,000. These are either sub-units or proteolytic degradation products of the intact enzyme. In addition, upon freezing the purified enzyme at -80°C., 1t usually forms aggregates, probably dimers, with a molecular weight of about 250,000 on SDS gels. After incubation with 125I-ca1modulin, all these bands became labeled, suggesting that they are active forms of the purified ATPase. The enzyme was also subjected to a controlled proteolytic treatment with trypsin and chymotrypsin in the presence of different effectors of its activity. This led to the fragmentation of the ATPase molecule into a number of transient and limit polypeptides. These fragments were separated by electrophoresis on SDS gels and the calmodul1n-blnding polypeptides were detected by the125I-calmodu1in overlay assay. The porcine erythrocyte Ca 2+ - ATPase appears to be more resistant to proteolysis than the human enzyme, as revealed by the time-course of the proteolytic digestion. It also showed slightly different proteolytic patterns from those established for the human erythrocyte Ca2+ -ATPase. Antibodies were raised in rabbits against the purified porcine erythrocyte Ca2+ -ATPase and used to study immunological cross-reactivity between the human and porcine enzyme. These antibodies cross-reacted with the enzyme from the two species, suggesting that there are immunological, and probably amino acid sequence, similarities between the two proteins. These findings indicate that (i) the properties of the human and porcine erythrocyte Ca2+ -ATPase are similar, and (ii) porcine erythrocytes might be a good source of the enzyme for molecular studies.