Purification and characterization of a calcium-dependent ATPase from Paramecium tetraurelia.
We report the purification of a CaATPase of high specific activity from Paramecium tetraurelia. The enzyme is preferentially released into solution upon deciliation of cells by a Ca2+ shock procedure. Purification by ion exchange and gel filtration chromatography yields major peptides of 68 and 53 kDa and a minor peptide of 58 kDa, as determined by electrophoresis on sodium dodecyl sulfate polyacrylamide gels. These three peptides yield similar proteolytic peptide maps. Rabbit antisera to the purified enzyme inhibit enzyme activity and specifically label 68- and 53-kDa bands on nitrocellulose blots of the deciliation supernatant from which the enzyme is isolated. Concanavalin A-Sepharose precipitates about 60% of ATPase activity; only the 53-kDa band binds concanavalin A on nitrocellulose blots. The purified enzyme has a specific activity of 620 +/- 70 mumol/min/mg with ATP as substrate in the presence of Ca2+, which is required for enzyme activity. As substrates, ATP and GTP are strongly preferred to UTP and CTP. The Km for ATP in the presence of 3 mM Ca2+ is approximately 20 microM. Enzyme activity is strongly inhibited by the calmodulin antagonists trifluoperazine, fluphenazine, W7, and calmidazolium. However, calmodulin is not associated with the purified enzyme, based on the enzyme's inability to bind anti-calmodulin antibodies or to stimulate brain phosphodiesterase. The intracellular origin of this ATPase, its possible function, and its relationship to several other ATPases of Paramecium are discussed.
Levin
A E
AE
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53706.
Travis
S M
SM
DeVito
L D
LD
Park
K A
KA
Nelson
D L
DL
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D002413:Cations, Divalent; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D008970:Molecular Weight; D009711:Nucleotides / Q000378:metabolism; D010247:Paramecium / Q000201:enzymology*; D013379:Substrate Specificity; D013449:Sulfonamides / Q000494:pharmacology
D002147:Calmodulin; D002413:Cations, Divalent; D009711:Nucleotides; D013449:Sulfonamides; C017967:W 7; C034700:N-(6-aminohexyl)-1-naphthalenesulfonamide; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases