Abstract
We describe the results of a study designed to identify cDNAs encoding Ca2+-transporting ATPases and other cation-transporting ATPases of the aspartylphosphate class. Rat brain, kidney, and stomach cDNA libraries were screened with an oligonucleotide hybridization probe corresponding to a 23-amino acid sequence from part of the ATP-binding site of the sarcoplasmic reticulum Ca-ATPase. This procedure resulted in the isolation of cDNAs encoding (i) the plasma membrane Ca-ATPase, (ii) an apparent Ca-ATPase that exhibits high amino acid similarity to the sarcoplasmic reticulum Ca2+ pumps, (iii) a transport ATPase of unknown ion specificity and (iv) two Ca-ATPase isoforms encoded by the gene for the slow-twitch muscle sarcoplasmic reticulum Ca-ATPase. Several isoforms of the Na,K-ATPase and gastric H,K-ATPase that had been characterized previously were also identified. The complete nucleotide sequences have been determined for the two classes of cDNA derived from alternatively spliced transcripts of the slow-twitch muscle sarcoplasmic reticulum Ca-ATPase gene. One of these cDNAs, isolated from the stomach library, encodes a Ca-ATPase that is identical to the skeletal muscle enzyme. The second class of cDNA, found in brain, kidney, and stomach libraries, is identical to that of the slow-twitch isoform throughout much of its length but encodes an alternative C terminus and has a different 3'-untranslated sequence. Whereas the muscle isoform consists of 997 amino acids and terminates with the sequence Ala-Ile-Leu-Glu, the second isoform is 1043 amino acids in length due to the replacement of these last 4 amino acids with a 50-amino acid sequence that contains a potential transmembrane domain followed by a consensus sequence for an N-linked glycosylation site.
Highlights
A Novel Ca2+Pump Expressedin Brain,Kidney, and Stomach Is Encoded by an Alternative Transcript of the Slow-twitch Muscle Sarcoplasmic Reticulum Ca-ATPase Gene
We describe the resultosf a study designed to identify and endoplasmic reticulum [1].Ca2+-transporting ATPaseosf cDNAsencoding Ca2+-transportingATPases and other the SR’ have been purified [2] and characterized biochemication-transporting ATPases of the aspartylphosphate cally
Kidney, and stomach cDNA libraries etal muscle isoform [3] and two fast-twitch skeletal muscle were screened with an oligonucleotide hybridization isoforms which differ a t their C termini as a result of alterprobe corresponding toa 23-amino acid sequence from part of the ATP-binding site of the sarcoplasmic reticulum Ca-ATPase
Summary
Isolation of Brain, Kidney, andStomach cDNAs-When this study was initiated, there were no amino acid sequence data available for Ca-ATPases of the ER and plasma mem-. Isolation of cDNAs-The 20 replica filters of each library were prehybridizedovernight at 60 “Cin 6X SSC, 1X Denhardt’s solution, 0.1% SDS and100 pg of denatured salmon sperm DNA/ml (see Ref. ing to theanticodons for a 23-amino acid sequence (residues 698-720) of the SR Ca-ATPases [3, 4] was prepared (Fig. 1). Replica filters for each library were hybridized in 70 ml of the same solution for 6 h at 50 “C with 330ng of a 68-nucleotide probe derived from a 23amino acid sequence that is highly conserved in transport ATPases of the aspartylphosphate class (see Fig. 1and “Results” for a detailed description of the probe and screening strategy). The replica filters for the brain, kidney, and stomach cDNA libraries used in the present study had been screened previously using probes that identified Na,K-ATPase and H,KATPase cDNAs [20,21]. Na,K-ATPase (01. "3) Na.K-ATPase (a2) H.K-ATPase H-ATPases SR Ca-ATPases cDNA Cloning of Calcium-transportingATPases
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