ACTIVATION OF THE HUMAN RED-CELL CALCIUM-ATPASE BY CALCIUM PRETREATMENT

Some kinetic parameters of the human red cell Ca2+-ATPase were studied on calmodulin-free membrane fragments following preincubation at 37 d egrees C. After 30 min treatment with EGTA (1 mM) plus dithioerythrito l (1 mM), a V-max of about 0.4 mu mol P-i/mg x hr and a K-s of 0.3 mu M Ca2+ were found. When Mg2+ (10 mM) or Ca2+ (10 mu M) were also added during preincubation, V-max but not K-s was altered. Ca2+ was more ef fective than Mg2+, thus increasing V-max to about 1.3 mu mol P-i/mg X hr. The presence of both Ca2+ and Mg2+ during pretreatment decreased K -s to 0.15 mu M, while having no apparent effect on V-max. Conversely, addition of ATP (2 mM) with either Ca2+ or Ca2+ plus Mg2+ increased V -max without affecting K-s. Preincubation with Ca2+ for periods longer than 30 min further increased V-max and reduced K-s to levels as low as found with calmodulin treatment. The Ca2+ activation was not preven ted by adding proteinase inhibitors (iodoacetamide, 10 mM; leupeptin, 200 mu M; pepstatin A, 100 mu M; phenylmethanesulfonyl fluoride, 100 m u M). The electrophoretic pattern of membranes preincubated with or wi thout Mg2+, Ca2+ or Ca2+ plus Mg2+ did not differ significantly from e ach other. Moreover, immunodetection of Ca2+-ATPase by means of polycl onal antibodies revealed no mobility change after the various treatmen ts. The above stimulation was not altered by neomycin (200 mu M), wash ing with EGTA (5 mM) or by both incubating and washing with delipidize d serum albumin (1 mg/ml), or omitting dithioerythritol from the prein cubation medium. On the other hand, the activation elicited by Ca2+ pl us ATP in the presence of Mg2+ was reduced 25-30% by acridine orange ( 100 mu M), compound 48/80 (100 mu M) or leupeptin (200 mu M) but not b y dithio-bis-nitrobenzoic acid (1 mM). The fluorescence depolarization of 1,6-diphenyl- and 1-(4-trimethylammonium phenyl)-6-phenyl 1,3,5-he xatriene incorporated into membrane fragments was not affected after p reincubating under the different conditions. The results show that pro teolysis, fatty acid production, an increased phospholipid metabolism or alteration of membrane fluidity are not involved in the Ca2+ effect . Ca2+ preincubation may stimulate the Ca2+-ATPase activity by stabili zing or promoting the E(1) conformation.