AJP - Gastrointestinal and Liver Physiology, Vol 244, Issue 5 480-G490, Copyright © 1983 by American Physiological Society
Characterization of Mg-ATP-dependent Ca2+ transport in cat pancreatic microsomes
A. Kribben, T. Tyrakowski and I. Schulz
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.
