cDNA cloning, functional expression, and mRNA tissue distribution of a third organellar Ca2+ pump.
We describe the characterization of a rat kidney cDNA that encodes a novel Ca2+-transporting ATPase. The cDNA, termed RK 8-13, was isolated previously using an oligonucleotide hybridization probe corresponding to part of the ATP binding site of the sarcoplasmic reticulum Ca-ATPases (Gunteski-Hamblin, A.-M., Greeb, J., and Shull, G. E. (1988) J. Biol. Chem. 263, 15032-15040). The complete nucleotide sequence of the 4.5-kilobase cDNA has been determined, and the primary structure of the protein has been deduced. The enzyme consists of 999 amino acids, has an Mr of 109,223, and contains all of the conserved domains found in transport ATPases of the E1-E2 class. It exhibits 75-77% amino acid identity with the fast-twitch and slow-twitch/cardiac isoforms of the sarcoplasmic reticulum Ca-ATPase, and the hydropathy plots of the three enzymes are virtually identical. High levels of ATP-dependent Ca2+ uptake were demonstrated in microsomes of COS-1 cells that had been transfected with a construct consisting of the entire coding sequence of the cDNA in the expression vector p91023(B). Northern blot analyses of poly(A)+ RNA revealed that the mRNA for this protein is expressed in heart, skeletal muscle, uterus, brain, lung, liver, kidney, testes, small intestine, large intestine, and pancreas. These data show that the enzyme is a Ca2+-transporting ATPase and that its mRNA is expressed in a broad variety of both muscle and non-muscle tissues.
Burk
S E
SE
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524.
Lytton
J
J
MacLennan
D H
DH
Shull
G E
GE
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels; D000252:Calcium-Transporting ATPases / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D005260:Female; D015870:Gene Expression; D007668:Kidney / Q000032:analysis; D008297:Male; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D051381:Rats; D015183:Restriction Mapping; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D014018:Tissue Distribution; D014162:Transfection
D015220:Calcium Channels; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D000255:Adenosine Triphosphate; D004247:DNA; D000252:Calcium-Transporting ATPases; D002118:Calcium