ATP‐dependent Ca2+ transport and Ca2+‐ATPase activities in an enriched plasma membrane fraction from gypsy moth larval midgut tissue

Abstract

AbstractA subcellular fraction enriched in plasma membranes was obtained from gypsy moth (Lymantria dispar) larval midgut tissue. Using [45Ca]2+ as a tracer, Ca2+ transport activity by membrane vesicles in the enriched fraction was measured and shown to be ATP‐dependent, with a very high affinity for Ca2+ (apparent Km for [Ca2+ free]Abbreviations used: [Ca2+free] = concentration of free (unbound) calcium ion;CaM = calmodulin; F = fraction; IOV = inside‐out membrane vesicles; W‐5 = N‐(6‐aminohexyl)‐1‐naphthalenesulfonamide; W‐7 = N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide. = 22 nM). Ca2+ transport was abolished upon addition of the calcium ionophore, A23187. Ca2+‐stimulated, Mg2+‐dependent ATPase activity peaked between 100 and 200 nM Ca2+free. Ca2+‐Mg2+‐ATPase activity was inhibited by vanadate, 2 phenothiazine drugs (trifluoperazine and chlorpromazine), and the naphthalene sulfonamide, W‐7; the related compound, W‐5, and ouabain had a negligible effect. These results suggest the presence of a high affinity plasma membrane Ca2+ pump in gypsy moth larval midgut cells and are discussed in light of earlier work involving calcium transport in isolated midguts of larval Hyalophora cecropia. Ionic and other conditions that characterize the midgut physiology of larval Lepidoptera (e.g., luminal pH; electrochemical gradient for Ca2+; effect of certain ions and inhibitors on Ca2+ transport) contrast significantly with those found in adult Diptera. The implications that these differences may have for calcium regulation are discussed. © 1992 Wiley‐Liss, Inc.