Characterization of (Mg,Ca)-ATPase Activity in Rat Pancreatic Plasma Membranes

Abstract

Pancreatic plasma membranes containing a high adenylate cyclase activity and a low contamination by cytochrome c oxidase were isolated from the rat by sucrose density centrifugation. The preparation contained an (Mg,Ca)‐ATPase of high activity with the following characteristics. The ATPase activity was shown to have two apparent Km values for Mg‐ATP (0.24 ± 0.09 mM and 1.15 ± 0.21 mM) and two apparent Km values for Ca‐ATP (0.14 ± 0.09 mM and 0.68 ± 0.10 mM). Mg‐GTP and Ca‐GTP were also hydrolysed by the preparation. The phase transition temperature was 19.3 ± 1.0°C for the Mg‐ATPase and 22.6 ± 1.1°C for the Ca‐ATPase activities. Three lines of evidence suggest that Mg‐ATP and Ca‐ATP were substrates for the same enzyme: Mg‐dependent and Ca‐dependent activities were not additive; the two activities showed the same pH optimum at 8.0; and the nonionic detergents Triton X‐100, Triton X‐305, Triton N‐101, Lubrol P 12 A, and digitonin, produced a parallel solubilization of the two activities. Enzyme activities were insensitive to potassium, sodium, ouabain, pancreozymin, carbamoylcholine, secretin, concanavalin A, wheat germ agglutinin, and soybean lectin.

Mechanism of action of magnesium on acetylcholine‐evoked secretory responses in isolated rat pancreas

“…Other workers have shown that the initial Ca 2+ rise in stimulated acini is attributed to a release of Ca2+ from intracellular stores Ansah et al 1986). During secretion there is an increase in net extracellular Ca2+ taken up (Williams, 1980;Argent, Case & Hirst, 1982;Case, 1984;Putney, 1986 One such enzyme is the Mg2"-dependent Ca2+-ATPase which is located at the plasma membrane and actively extrudes intracellular Ca2+ against an inwardly directed electrochemical gradient (Lambert & Christophe, 1978;Williams, 1980;Putney, 1988). This enzyme should increase Ca2" extrusion in the presence of high Mg2" levels.…”

“…However, Mg2+ is found in abundant quantities in cells and is involved in protein synthesis and membrane stabilization and is a co-factor for numerous enzymes (Wacker, 1968;Case & Clausen, 1973;Flatman, 1984;Streb, Heslop, Irvine, Schulz & Berridge, 1985;Squire & Petersen, 1987). More specifically, Mg2" has been shown to be the co-factor for a membrane-bound Ca2+-ATPase directly acting to bring intracellular Ca2+ levels to a point of stasis following stimulation by the secretagogue ACh (Wacker, 1968;Lambert & Christophe, 1978;Lucas, Schmid, Komas & Loeffler, 1978;Galvan & Lucas, 1987). Protein kinase C is thought to play an important role in secretagogue-evoked secretion and its activity is known to be subject to modulation by mono-and divalent cations, particularly Ca2t and Mg2+ (Trudell, Costa & Csernansky, 1989).…”

Calcium uptake into acini from rat pancreas: Evidence for intracellular ATP-dependent calcium sequestration

“…A Ca 2 +-stimulated Mg 2 +-dependent ATPase has been described in microsomes from rat pancreas [42,44]. On the other hand, the presence of a Ca 2 +-stimulated Mg 2 +-independent ATPase has been demonstrated in pancreatic membranes, too [39,48]. We therefore tested if the presence of Mg 2 + was required for the ATP-and ADP-promoted 45Ca2 + uptake in our system.…”

Relation of ATPases in rat renal brush-border membranes to ATP-driven H+ secretion

“…However, despite numerous investigations it is not clear which ATPase activity in proximal tubules and in isolated brush-border membrane vesicles reflects the H+-ATPase. The "Mg2+-ATPase '' measured in cortex homogenates [45], microdissected nephrons [32], and isolated brush-border membranes [5,20,31,44] is stimulated by Ca ~-+ and other nucleotides besides ATP [13,31,44,45], This pattern is typical for ecto-ATPases found in other cells [14,18,28,29,40,42] rather than for an ATPase involved in H + transport. The "bicarbonate-stimulated ATPase" in a rat kidney brush-border membrane preparation was related to ATP-driven H+se -cretion [35,36,41,43], but was later shown to be of mitochondrial origin [13,31,38].…”