Molecular cloning and functional expression of cDNA encoding a mammalian inorganic pyrophosphatase.
Extracts of soluble proteins from bovine retina contain multiple species of inorganic pyrophosphatase (PPase) that can be resolved by hydroxylapatite or ion exchange chromatography. We have purified one of these isoforms by a combination of chromatography and electrophoresis under denaturing conditions and have partially sequenced four peptides generated from it by CNBr digestion. This sequence information was used to clone PPase cDNA from a retinal cDNA library. Of five cDNA inserts, three were 1.3 kilobase pairs in length and two of these contained a complete open reading frame that was 867 base pairs long and encoded a 289-amino acid protein of 33 kDa. The deduced amino acid sequence is 49.5% identical to that of PPase from Saccharomyces cerevisiae, and contains identical amino acid residues at all of the positions previously identified as essential for catalytic activity in that enzyme. When the bovine PPase cDNA was expressed in Escherichia coli, catalytically active PPase was produced that comigrated with bovine retinal PPase in a nondenaturing gel and was clearly distinguishable from the host PPase. Northern analysis of poly(A)+ RNA from human, canine, and bovine retinas revealed that each contained a single major band of 1.4 kilobases that hybridized strongly with a pyrophosphatase cDNA probe. Southern analysis of bovine genomic DNA was consistent with the existence of one PPase gene. Thus, the multiple forms separated by chromatography may be derived from a common precursor or from mRNAs of very similar size.
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Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002417:Cattle; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004285:Dogs; D017886:Durapatite; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D006882:Hydroxyapatites; D043564:Inorganic Pyrophosphatase; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D011755:Pyrophosphatases / Q000235:genetics* / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping; D012160:Retina / Q000201:enzymology*; D012374:Rod Cell Outer Segment / Q000201:enzymology; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D006882:Hydroxyapatites; D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D012333:RNA, Messenger; D004247:DNA; D017886:Durapatite; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase