The N-terminal region of the plasma membrane Ca(2+) pump does not separate from the main catalytic fragments after proteolysis.
The purified plasma membrane Ca(2+) pump (PMCA) was digested with trypsin, and the proteolytic products were identified by immunoblotting with monoclonal antibodies JA9 or 5F10 directed against the extreme N-terminal segment and the central portion of the molecule, respectively. After a short treatment with low concentrations of the protease, JA9 reacted predominantly with a peptide of 35 kDa whereas 5F10 detected a peptide of 90 kDa. The trypsin cut leading to the production of these fragments had no effect on the maximal activity of the enzyme. At higher concentrations of trypsin, JA9 detected a main fragment of 33 kDa and smaller fragments of 19 and 15 kDa. The persistence of fragments reacting with JA9 indicates that the N-terminal region containing its epitope (residues 51-75) was not easily accessible to the protease in the native PMCA. However, the reactivity with JA9 was rapidly lost during proteolysis of the denatured protein. The passage of the mixture of PMCA fragments through a calmodulin-Sepharose column resulted in the retention of the N-terminal 35 kDa fragment together with that of 90 kDa, despite the fact that only the latter binds calmodulin. The ethylenediaminetetraacetic acid (EDTA) eluate, which contained about equal amounts of both fragments, had a Ca(2+) ATPase activity similar to that of the intact enzyme. The tight association between the two peptides was evidenced by the fact that concentrations of polyoxyethylene 10 lauryl ether (C(12)E(10)), sodium dodecyl sulfate (SDS) high enough for inactivating the enzyme and dissociate the pump from calmodulin were unable of breaking the interaction between the 35 and 90 kDa fragments. Altogether, these results show that after digestion with trypsin, the N-terminal portion of the PMCA, including the extreme N-terminal segment, remains part of a fully functional catalytic complex.
Adamo
H P
HP
Instituto de Química y Fisicoquímica Biológicas (IQUIFIB), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113, Buenos Aires, Argentina. hpadamo@mail.retina.ar
Grimaldi
M E
ME
Bredeston
L M
LM
D000906:Antibodies / Q000276:immunology; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000276:immunology; D002147:Calmodulin / Q000737:chemistry; D002462:Cell Membrane / Q000201:enzymology*; D002846:Chromatography, Affinity; D003902:Detergents; D004492:Edetic Acid; D015151:Immunoblotting; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D014357:Trypsin
D000906:Antibodies; D002147:Calmodulin; D003902:Detergents; D010446:Peptide Fragments; D004492:Edetic Acid; D014357:Trypsin; D000252:Calcium-Transporting ATPases