Characteristics of a Ca2+-ATPase activity measured in islet homogenates.
Ca2+-ATPase activity was measured in rat islet homogenates, in a medium of low ionic strength containing a low concentration of Ca2+ and Mg2+ and devoid of K+. The enzyme activity was highly sensitive to inhibition by compound 48/80 (a calmodulin inhibitor), stimulated by 120 nM calmodulin and slightly affected by 10 mM NaN3. The addition of Mg2+ to the assay medium promotes the disappearance of apparent Ca2+-ATPase activity. Ouabain (0.1 mM) did not modify this ATPase activity. The enzyme showed two kinetic components for Ca2+ as well as for ATP: one with high apparent affinity and low maximum velocity and the other with low apparent affinity and high maximum velocity. Incubation of islet homogenates in this assay medium with [gamma-32P]ATP in the presence of proteolytic inhibitors, results in the appearance of a single labelled band of 130 kDa, identified by gel electrophoresis. The incorporation of 32P into this band was similar in the presence of either 2.8 or 50 microM Ca2+ and susceptible to hydroxylamine attack. The results indicate that, under the conditions described above, the Ca2+-ATPase activity evidenced in the islet homogenates had characteristics resembling those of the enzyme which catalyzes the outward Ca2+ transport. On the other hand, the method could provide a useful tool to test the effect of different agents which affect insulin secretion upon the islet plasma membrane Ca2+-ATPase activity.
Rossi
J P
JP
IQUIFIB - Instituto de Química y Fisicoquímica Biológicas (UBA-CONICET), Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina.
Gronda
C M
CM
Fernandez
H N
HN
Gagliardino
J J
JJ
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007515:Islets of Langerhans / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D009994:Osmolar Concentration; D010766:Phosphorylation; D051381:Rats; D011919:Rats, Inbred Strains; D003189:p-Methoxy-N-methylphenethylamine / Q000494:pharmacology
D003189:p-Methoxy-N-methylphenethylamine; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium