Characterization of Mg-ATP-dependent Ca2+ transport in cat pancreatic microsomes

Abstract

Mg-ATP-dependent 45Ca2+ uptake and Ca2+-ATPase activity have been examined in isolated microsomes obtained by differential centrifugation and in purified subcellular fractions obtained by Ficoll-sucrose density centrifugation in the presence of mitochondrial inhibitors. Mg-ATP-dependent 45Ca2+ uptake increased with increasing EGTA-buffered free [Ca2+], reaching a maximum of 2 nmol 45Ca2+ X 15 min-1 X mg prot-1 at 2 mumol/1 [Ca2+] in the incubation medium. Half-maximal 45Ca2+ uptake was at approximately 0.2 mumol/1 [Ca2+]. Maximal Ca2+ -Mg2+ -ATPase activity was 130 nmol X 15 min-1 X mg prot-1 at 2 mumol/l [Ca2+], with an apparent Km of approximately 0.3 mumol/l [Ca2+]. The Ca2+ ionophore A23187 (10(-6) mol/l), the mercurial compounds mersalyl (10(-5) mol/l) and CH3ClHg (10(-3) mol/l), as well as La3+ (10(-4) mol/l), vanadate (10(-4) mol/l), and saponin (50 micrograms/mg prot), abolished Mg-ATP-promoted 45Ca2+ uptake. In the absence of Mg2+, ATP did not provoke 45Ca2+ uptake. Using the purified smooth membrane fraction (F1) from the Ficoll-sucrose density gradient (enrichment of Na+-K+-ATPase specific activity by ninefold and of NADH-cytochrome c reductase by threefold as compared with total tissue homogenate), Mg-ATP-dependent 45Ca2+ uptake correlated better with Na+-K+-ATPase (r = 0.97) than with the smooth endoplasmic marker NADH-cytochrome c reductase (r = 0.52). No correlation was found with RNA, the marker for rough endoplasmic reticulum. We conclude that pancreatic plasma membranes contain a Ca2+-Mg2+-ATPase that represents the Ca2+ extrusion system from acinar cells. It is also possible that vesicular membrane structures associated with the plasma membrane, or endocytotic plasma membrane vesicles, take up Ca2+ and represent an intracellular Ca2+ pool.

Characterization of calcium uptake into rough endoplamic reticulum of rat pancreas

“…However, the evidence that this Ca 2+ uptake occurred into endoplasmic reticulum was rather weak and was mainly based on the stimulatory effect of oxalate [44]. Ca 2+ uptake into inside-out plasma membrane vesicles as previously described [30] could not be excluded.…”

“…This is a characteristic feature of the endoplasmic reticulum, in contrast to calcium uptake into plasma membrane vesicles [30,62], due to the permeability of the endoplasmic reticulum to oxalate. With increasing concentrations of calcium inside the vesicles during uptake, oxalate forms insoluble precipitates with calcium which keeps the intravesicular free calcium concentration low and thus leads to a constant calcium uptake for a longer period of time.…”

“…At least three structures are considered to influence the cytosolic calcium concentration: plasma membrane [30,43,55], mitochondria [5,6,10,12] and one or more not yet clearly identified nonmitochondrial structures [12,33,44,46]. Attempts to determine these nonmitochondrial structures in leaky acinar cells [33,57,61], as well as in isolated microsomal fractions of pancreatic acinar cells [34,44,46], suggest that the endoplasmic reticulum is involved in the regulation of the cytosolic free calcium concentration.…”

The effects of Na+ replacement on intracellular pH and [Ca2+] in rabbit salivary gland acinar cells.

“…Some evidence does exist to suggest that the plasma membrane Ca2+-ATPase may be sensitive to pH (Kraus-Friedmann, Biber, Murer & Carafoli, 1982;Kribben, Tyrakowski & Schulz, 1983), although Muallem et al (1989) found that changes in pHi did not alter the rate of Ca2+ efflux across the plasmalemma in pancreatic acinar cells. A final possibility is that interaction between H+ and Ca2+ may occur at the level of mitochondrial Ca2+ transport.…”

Isolation, purification and protein content of pancreatic endoplasmic reticulum

“…For integration into an earlier scheme of cell fractionation used for guinea pig pancreas see Tartakoff and Jamieson (19). Purification of cat pancreatic microsomes and characterization by enzymatic markers is described by Milutinovic et al (15) and Kribben et al (13). Preparation of canine microsomes and their use for protein translation studies is detailed in Walter and Blobel (21).…”