Abstract
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.
Highlights
Extracts of soluble proteins from bovine retina con-for viability of yeast (Lundin et al, 1991) and bacteria (Chen tain multiplespeciesofinorganicpyrophosphatase et al, 1990)
RNA from humanc, anine, and bovineretinas revealed The highest level of cytoplasmic proposed that its activity maybe (PPase) activity detected so that each containedsinagle major band o1f.4kilobases far is that of retinal rod outer segments, where it probably that hybridized strongwlyith a pyrophosphatase cDNArequired due to thehigh levels of cyclincucleotide metabolism probe
Rified the PPasefrom rod outer segments and have found that the multiple forms separated by chromatographmy ay it is a homodimer of subunits with an apparent molecular be of derived from a common very similarsize
Summary
0 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Vol 267, No 34, Issue of December 5,pp. 24641-24647,1992 Printed in U.S.A. Thebuffer for electroelution was 0.1 M panesulfonic acid PAGE, polyacrylamide gel electrophoresis; bp, base NaCl, 1.5 g/liter (12.4 mM) Tris base, and 7.2 g/liter (96 mM) glycine pair(s); kb and kbp, kilobases and kilobase pairs; PCR, polymerase (no pHadjustment).Other buffers used for protein workwere as chain reaction; HPLC, high pressure liquid chromatography; ROS, described in a companion study (Yang and Wensel, 1992b) (Sambrook et al, 1989).Digestion with restriction endonucleases was While a very broad peak of activity was eluted from the first carried out according to the manufacturers' instructions, digested DNA was separated on a 0.7% agarose gel, and Southern analysis was carried out using capillary transfer (Sambrook et al, 1989).The final high stringency wash wascarried out twice using 0.1 X SSC and 0.5% SDS at 68 "C for 40 min. Ing amino acids deduced from the cDNA sequence are listed underneath in all cases where they differ
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