Partial purification and characterization of the Ca2(+)-pumping ATPase of the liver plasma membrane.
A Ca2(+)-pumping ATPase has been characterized in rat hepatocyte plasma membranes. The enzyme has high Ca2+ affinity, and properties typical of a P-type ion pump. At variance with the Ca2+ pumps of other eukaryotic plasma membranes, it is not stimulated by calmodulin. The steady state concentration of the phosphoenzyme formed in the presence of ATP is increased by La3+. The enzyme cross-reacts with a monoclonal antibody (mAb-5F10) raised against the human erythrocyte Ca2+ pump. The enzyme has been purified using a mAb-5F10 antibody affinity column. CNBr digestion of the isolated protein has yielded two peptides which have been sequenced. One of them matches perfectly a sequence contained in the erythrocyte Ca2+ pump, the other is very homologous to another domain in the erythrocyte pump. In spite of the absence of calmodulin stimulation, 125I-calmodulin overlay experiments on the purified liver ATPase under denaturing conditions have revealed that the enzyme binds calmodulin even more strongly than the erythrocyte pump. Immunocytochemical experiments on liver slices using the mAb-5F10 antibody have shown that the enzyme is located predominantly in the blood sinusoidal domain of the hepatocyte plasma membrane.
Kessler
F
F
Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich.
Bennardini
F
F
Bachs
O
O
Serratosa
J
J
James
P
P
Caride
A J
AJ
Gazzotti
P
P
Penniston
J T
JT
Carafoli
E
E
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001693:Biological Transport, Active; D017301:Ca(2+) Mg(2+)-ATPase / Q000302:isolation & purification / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification* / Q000378:metabolism; D002147:Calmodulin / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D003488:Cyanogen Bromide; D005260:Female; D007124:Immunoenzyme Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D051381:Rats; D011919:Rats, Inbred Strains
D000911:Antibodies, Monoclonal; D002147:Calmodulin; D010446:Peptide Fragments; D000001:Calcimycin; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D003488:Cyanogen Bromide; D002118:Calcium