Cytosolic and cell-wall-bound acid invertases from leaves of Urtica dioica L.: a comparison

Two different forms of acid invertase (EC 3.2.1.26) were extracted from expanding leaves of the stinging nettle (Urtica dioica L.). One form was soluble and could be localized within the cytosol, whereas the other was ionically bound to the cell-wall and could not be detected in protoplasts. Both forms were purified, the latter to homogeneity. Western blotting with antibodies against the pure enzyme from cell walls was positive with the cell-wall enzyme but negative with the soluble form of acid invertase. Both forms are glycoproteins with identical molecular weights of 58 kDa. The K_m values for sucrose (raffinose) are 5 mM (4.8 mM) for the soluble and 1.2 mM (3.6 mM) for the cell-wall-bound enzyme. The pH optimum of the latter is slightly more acidic (4.5) than that of the soluble invertase (5.5). Both forms could easily be distinguished by their isoelectric points which were determined at pH 4.6 for the soluble and pH 9.3 for the wall-bound enzyme. When extraction and purification were carried out in the absence of protease inhibitors, both acid invertases showed microheterogeneity (multiple forms). However, with benzamidine and phenylmethylsulfonylfluoride as protease inhibitors each invertase produced only one protein band upon isoelectric focusing and gel electrophoresis, respectively.Abbreviations B benzamidine - Con A concanavalin A - FPLC fast protein liquid chromatography - IEF isoelectric focusing - kDa kilodalton - pI isoelectric point - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonylfluoride - SDS sodium dodecyl sulfate This work was supported by the Deutsche Forschungsgemeinschaft within the scope of the Sonderforschungsbereich 137.     

Biochemical studies on sulfate-reducing bacteria. XV. Separation and comparison of two forms of desulfoviridin.

Desulfoviridin from Desulfovibrio vulgaris was separated into two forms by DEAE-Sephadex column chromatography. The major form had a pI of 4.4 and the minor form one of 4.5-4.6. Both forms produced mainly trithionate, besides thiosulfate and sulfide, in methylviologen-linked sulfite reduction. The specific activities of sulfite reduction, as well as of hydroxylamine reduction, were virtually identical in both forms. There were no great differences in their absorption spectra, CD spectra, molecular weights, subunit compositions, labile sulfide, and iron contents, and amino acid compositions. The N-terminal amino acid was alanine in both forms.     

Characterization of multiple forms of prostatropin (prostate epithelial cell growth factor) from bovine brain

Two molecular forms of prostatropin distributed among five chromatographic peaks have been isolated from bovine brain by heparin-Sepharose affinity and reverse phase high performance liquid chromatography. One form has an apparent molecular weight of 16000 and an amino terminal sequence of asn-tyr-lys-lys-pro-lys-leu-leu-tyr-x-ser-asn-gly. The other form has an apparent molecular weight of 18000 and a blocked amino terminus. Both forms are similar in amino acid composition. The sequence of a proteolytic fragment from the blocked form overlaps the NH2-terminal sequence of the unblocked form, therefore, the smaller form may be derived from the larger form through proteolytic processing. Both forms contain regions identical in sequence to brain-derived, heparin-binding growth factors that have been isolated on the basis of mitogenic activity for fibroblasts and endothelial cells. Both forms of prostatropin exhibit potent mitogenic activity for normal and tumor prostate epithelial cells.     

The Role of beta-Galactosidases in the Modification of Cell Wall Components during Muskmelon Fruit Ripening

The changes in activities of soluble β-galactosidase and two forms of wall-bound β-galactosidases extracted with NaCl and EDTA were investigated throughout the development of muskmelon (Cucumis melo L. cv Prince) fruits. DEAE-cellulose ion-exchange chromatography of soluble β-galactosidase revealed the presence of two isoforms. Soluble isoform I was detected in all stages throughout the fruit development, whereas soluble isoform II appeared around 34 d after anthesis when fruit ripening initiated. Both NaCl- and EDTA-released β-galactosidase activities also increased as ripening proceeded. The soluble and wall-bound forms behaved differently upon ion-exchange chromatography. Enzymological properties such as optimum pH, optimum temperature, K_m values for p-nitrophenyl β-d-galactopyranoside, and inhibition by metal ions were nearly similar in all forms. Molecular sizes of pectic polymers and hemicelluloses extracted from fruit mesocarp cell walls were shifted from larger to smaller polymers during ripening, as determined by gel filtration profiles. NaCl-released β-galactosidase from cell walls of ripe fruits had the ability to degrade in vitro the pectin extracted from preripe fruit cell walls to smaller sizes of pectin similar to those that were observed in ripe cell walls in situ. Both soluble isoform I and II were able to degrade in vitro the 5% KOH-extractable hemicellulose from preripe fruit cell walls to sizes of molecules similar to those that were observed in ripe cell walls in situ. Soluble isoform I and the NaCl-released form from ripe fruits were able to modify in vitro 24% KOH-extractable hemicellulose from preripe cell walls to sizes of molecules similar to those that were observed in ripe fruits in situ.     

Comparison of the Soluble and Membrane-Bound Forms of the Puromycin-Sensitive Enkephalin-Degrading Aminopeptidases from Rat

Enkephalin degradation in brain has been shown to be catalyzed, in part, by a membrane-bound puromycin-sensitive aminopeptidase. A cytosolic puromycin-sensitive aminopeptidase with similar properties also has been described. The relationship between the soluble and membrane forms of the rat brain enzyme is investigated here. Both of these aminopeptidase forms were purified from rat brain and an antiserum was generated to the soluble enzyme. Each of the aminopeptidases is composed of a single polypeptide of molecular mass 100 kilodaltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography. The antisoluble aminopeptidase antiserum reacts with both enzyme forms on immunoblots and inhibits both with nearly identical inhibition curves. The isoelectric points (pI = 5.0) of both forms were shown to be identical. N-terminal sequencing yielded a common sequence (P-E-K-R-P-F-E-R-L-P-T-E-V-S-P-I-N-Y) for both enzyme forms, and peptide mapping yielded 26 peptides that also appeared identical between the two enzyme forms. Studies on the nature of the association of the membrane enzyme form with the cell membrane suggest that this enzyme form does not represent the soluble form trapped during the enzyme preparation. It is suggested that the membrane form of the puromycin-sensitive aminopeptidase is identical to the soluble enzyme and that it associates with the membrane by interactions with other integral membrane proteins.     

Identification, cDNA cloning, and gene expression of soluble starch synthase in rice (Oryza sativa L.) immature seeds.

Three forms of soluble starch synthase were resolved by anion-exchange chromatography of soluble extracts from immature rice (Oryza sativa L.) seeds, and each of these forms was further purified by affinity chromatograph. The 55-, 57-, and 57-kD proteins in the three preparations were identified as candidates for soluble starch synthase by western blot analysis using an antiserum against rice granule-bound starch synthase. It is interesting that the amino-terminal amino acid sequence was identical among the three proteins, except that the 55-kD protein lacked eight amino acids at the amino terminus. Thus, these three proteins are products of the same gene. The cDNA clones coding for this protein have been isolated from an immature rice seed library in lambda gt11 using synthetic oligonucleotides as probes. The deduced amino acid sequence of this protein contains a lysine-X-glycine-glycine consensus sequence for the ADP-glucose-binding site of starch and glycogen synthases. Therefore, we conclude that this protein corresponds to a form of soluble starch synthase in immature rice seeds. The precursor of the enzyme contains 626 amino acids, including a 113-residue transit peptide at the amino terminus. The mature form of soluble starch synthase shares a significant but low sequence identity with rice granule-bound starch synthase and Escherichia coli glycogen synthase. However, several regions, including the substrate-binding site, are highly conserved among these three enzymes. Blot hybridization analysis demonstrates that the gene encoding soluble starch synthase is a single-copy gene in the rice genome and is expressed in both leaves and immature seeds. These results suggest that soluble and granule-bound starch synthases play distinct roles in starch biosynthesis of plant.     

Amino acid sequence analysis of two mouse calbindin-D9k isoforms by tandem mass spectrometry. Protein modification by internal insertion of a single amino acid

Two forms of calbindin-D9k have sometimes been observed within a single tissue. Sequencing of these proteins has been complicated by the presence of blocked amino termini. Tandem mass spectrometry is a powerful tool for comparing related proteins, and its use does not depend upon an unblocked amino terminus. In the present studies, calbindin-D9k was purified from the intestines of mice (270 animals per purification) by use of gel permeation chromatography and two preparative electrophoresis steps in the presence and absence of EDTA. The purified protein appeared to be homogeneous following electrophoresis under nondenaturing conditions, but two components were identified by sodium dodecyl sulfate-gel electrophoresis and immunoblotting. Two forms of the protein were isolated by reverse-phase high performance liquid chromatography. In each of three preparations, the average ratio of the major:minor isoforms was 2:1. The major form contained 77 amino acids and lacked the amino-terminal serine found in 78-amino acid calbindins from rat and pig. The amino acid sequence was identical with the deduced sequence reported for rat intestinal calbindin-D9k in 73 of 77 positions. In the minor form, a glutamine was found in a location between Lys-43 and Ala-44 of the major form and between the two calcium binding sites of the protein. The minor form was otherwise identical with the major form, including the presence of a blocked amino terminus. The inserted glutamine was located at the site of an intron in the rat calbindin gene, suggesting the possibility that alternative splicing produced the two forms of calbindin-D9k. The functional significance of an inserted amino acid between the two calcium binding sites remains to be explored.     

Purification and characterization of a low and a high molecular weight form of epidermal growth factor from rat urine

Two forms of epidermal growth factor (EGF) have been purified to homogeneity from rat urine by immunoaffinity chromatography and gel filtration. For one of the purified peptides the molecular mass has been determined to be 5891 by mass spectrometry. This peptide consists of 51 amino acid residues. The sequence of the first 48 amino acid residues is identical to the previously published sequence for submandibular rat EGF. The C-terminal three residues (49-51) are Trp-Trp-Lys. The other purified peptide has a molecular mass of 45 kDa as determined by SDS-polyacrylamide gel electrophoresis. The N-terminal sequence is Asn-Tyr-Lys-Asp-(Cys)-Gly-Pro-Gly-Gly-(Cys)-Gly-Ser-His-Ala. Both the high and the low molecular mass form of urinary rat EGF are able to bind to the human placenta receptor for EGF.     

Purification and partial sequencing of human placental alkaline phosphatase

Two forms of human placental alkaline phosphatase have been purified to homogeneity utilizing high performance liquid chromatography. Both have the same amino acid composition but they differ in their carbohydrate substituents. Sequence data indicate that the two forms are identical for the first forty two residues from the amino terminus are presented.     

Amino terminal amino acid sequences and carbohydrate of the two major forms of rabbit plasminogen

Two major forms of plasminogen exist in the plasma of many animal species and are distinguished by their affinities for certain antifibrinolytic amino acids. Quantitative end group analysis demonstrated that each isolated form of rabbit plasminogen possessed a single amino terminal residue of glutamic acid. Amino acid sequence analysis indicated that at least the first twelve amino terminal amino acids were identical in the two forms. The unique amino terminal sequence obtained for each form was NH₂-glu-pro-leu-asp-asp-tyr-val-asn-thr-gln-gly-ala-. Analysis of the carbohydrate content of each major plasminogen form revealed some striking differences. The first major form of rabbit plasminogen isolated from affinity chromatography columns contained 1.5–1.7 percent neutral carbohydrate and 3.0–3.3 moles of sialic acid per mole of protein. The second major form of rabbit plasminogen isolated from affinity chromatography columns contained 0.6–0.8 percent neutral carbohydrate and 1.8–2.2 moles of sialic acid per mole of protein.     

Isolation of four forms of acetone-induced cytochrome P-450 in chicken liver by h.p.l.c. and their enzymic characterization.

The purpose of this study was to purify and characterize the forms of cytochrome P-450 induced in chicken liver by acetone or ethanol. Using high performance liquid ion-exchange chromatography, we were able to isolate at least four different forms of cytochrome P-450 which were induced by acetone in chicken liver. All four forms of cytochrome P-450 proved to be distinct proteins, as indicated by their N-terminal amino acid sequences and their reconstituted catalytic activities. Two of these forms, also induced by glutethimide in chicken embryo liver, appeared to be cytochromes P450IIH1 and P450IIH2. Both of these cytochromes P-450 have identical catalytic activities towards benzphetamine demethylation. However, they differ in their abilities to hydroxylate p-nitrophenol and to convert acetaminophen into a metabolite that forms a covalent adduct with glutathione at the 3-position. Another form of cytochrome P-450 induced by acetone is highly active in the hydroxylation of p-nitrophenol and in the conversion of acetaminophen to a reactive metabolite, similar to reactions catalysed by mammalian cytochrome P450IIE. Yet the N-terminal amino acid sequence of this form has only 30-33% similarity with cytochrome P450IIE purified from rat, rabbit and human livers. A fourth form of cytochrome P-450 was identified whose N-terminal amino acid sequence and enzymic activities do not correspond to any mammalian cytochromes P-450 reported to be induced by acetone or ethanol.     

Characterization of the glycosyl-phosphatidylinositol-anchored human renal dipeptidase reveals that it is more extensively glycosylated than the pig enzyme.

Renal dipeptidase (EC 3.4.13.11) has been purified from human kidney cortex by affinity chromatography on cilastatin-Sepharose following solubilization with either n-octyl-beta-D-glucopyranoside or bacterial phosphatidylinositol-specific phospholipase C (PI-PLC). Phase separation in Triton X-114 revealed that the detergent-solubilized form was amphipathic and retained the glycosyl-phosphatidylinositol membrane anchor whereas the phospholipase solubilized form was hydrophilic. Both forms of the enzyme existed as a disulphide-linked dimer of two identical subunits of Mr 59,000 each. The glycosyl-phosphatidylinositol anchor of purified human renal dipeptidase was hydrolysed by a range of bacterial PI-PLCs and by a plasma phospholipase D. Mild acid treatment and nitrous acid deamination of the hydrophilic form revealed that the cross-reacting determinant, characteristic of the glycosyl-phosphatidylinositol anchor, was due exclusively to the inositol 1,2-cyclic phosphate ring epitope. The N-terminal amino acid sequences of the amphipathic and hydrophilic forms were identical, locating the membrane anchor at the C-terminus. The N-terminal sequence of human renal dipeptidase showed a high degree of similarity with that of the pig enzyme, and enzymic deglycosylation revealed that the difference in size of renal dipeptidase between these two species is due almost entirely to differences in the extent of N-linked glycosylation.     

Soluble and membrane-bound neutral alpha-glucosidase from the human kidney

In human kidney cortex neutral alpha-glucosidases 1 and 2 are represented by two forms, soluble (cytosolic) and membrane-bound (brush border) ones. It has been shown that the soluble enzyme preexists in human kidney but does not derive from the membrane-bound form. Similar to the membrane-bound enzyme the soluble form is a glycoprotein. Both enzyme forms possess identical electrophoretic mobility, pH-optimum, heat sensibility and Km values for maltose (0.7 mM) and 4-methylumbelliferyl-alpha-D-glucopyranoside (0.57 mM), but differ by molecular weights as determined by gel filtration chromatography. The molecular weights of the soluble neutral alpha-glucosidases 1 and 2 are lower than those of the comparable brush border enzymes (470 000, 360 000, 520 000 and 440 000, correspondingly). Neutral membrane-bound alpha-glucosidase 1 is a sialylated enzyme with a pI of 4.10 +/- 0.02. The soluble enzyme contains no or only traces of neuraminic acid and has a pI 4.40 +/- 0.03. The soluble and membrane-bound neutral alpha-glucosidases are apparently independent forms of the enzyme, differing by the degree of sialylation and by the presence of an "anchor" in the membrane-bound enzyme. The synthesis of both forms is presumably coded by the same structural gene.     

Chemical identification of lipid components in the membranous form of rat liver alkaline phosphatase

Membranous and soluble forms of rat liver alkaline phosphatase were selectively prepared by extracting microsomes with n-butanol at pH 8.5 and 5.5, respectively, and purified in homogeneous forms by the method previously established (Miki et al. (1986) Eur. J. Biochem. 160, 41-48). When subjected to polyacrylamide gel electrophoresis, the two forms migrated to the same position in the presence of sodium dodecyl sulfate, while the membranous form remained at the top of gels in the absence of the detergent. Treatment of the membranous form with phosphatidylinositol-specific phospholipase C resulted in its conversion to a soluble form with the same electrophoretic mobility even in the absence of the detergent as that of the soluble form extracted at pH 5.5. Automated Edman degradation analysis showed that the two forms have the same N-terminal amino acid sequence up to the 30th residue determined. Chemical analyses of hydrolysates of the two forms by gas-liquid chromatography demonstrated that the membranous form contains palmitic acid, stearic acid, and inositol, while the soluble form contains inositol but is devoid of the fatty acids. Taken together, these results suggest that rat liver alkaline phosphatase is covalently attached to phosphatidylinositol acylated with palmitic acid and stearic acid, which functions as the membrane-anchoring domain of the enzyme molecule.     

Assessments of growth conditions on the production of cyanophycin by recombinant Escherichia coli strains expressing cyanophycin synthetase gene

The synthesis of cyanophycin, a biodegradable polymer, is directed by cyanophycin synthetase. Polymerase chain reaction (PCR) cloned the gene cphA coding for cyanophycin synthetase from Synechocystis sp. PCC 6803 into pET-21b followed by transformation into two Escherichia coli hosts. The culture conditions for cyanophycin production were investigated, and the molecular weight and compositions of purified cyanophycin were analyzed. The results showed that E. coli BL21-CodonPlus(DE3)-RIL could produce 120 mg cyanophycin per gram dry cell weight in terrific medium. The purified cyanophycin consisted of insoluble and soluble forms at pH 7. The insoluble form had a higher molecular weight (20-32 kDa) than the soluble form (14-25 kDa). Both forms are composed of three major amino acids, aspartic acid, arginine, and lysine, and the insoluble form showed a higher arginine/lysine molar ratio (4.61 ± 0.31) than the soluble form (0.89 ± 0.05). In addition, the nitrogen sources could affect the yields of insoluble and soluble forms of cyanophycin. The medium containing additional lysine could enhance the proportion of the soluble form, but had little effect on the lysine and arginine percentages of both soluble and insoluble forms. The medium containing additional arginine slightly decreased the proportion of soluble form and altered its amino acid composition, with a minimal effect on the lysine and arginine percentages in the insoluble form.     

Isolation and partial characterization of rabbit plasma alpha1-antitrypsin.

Alpha1-Antitrypsin was isolated from rabbit plasma by salting out with (NH4)2SO4 followed by ion-exchange chromatography either on DEAE-Sephadex or DEAE-cellulose (each at pH8.8 and 6.5), and affinity chromatography on Sepharose-Cibacron Blue and Sepharose-concanavalin A. The protein thus obtained was homogeneous during crossed immunoelectrophoresis by using an antiserum to whole rabbit plasma, but it migrated as two broad bands when electrophoresed in alkaline polyacrylamide gels. Under optimal loading conditions, two or three subcomponents could be distinguished in each band. The two major forms of rabbit alpha1-antitrypsin, designated components F and S, were separated by preparative polyacrylamide-gel electrophoresis, and some of their physico-chemical properties were established. Both forms reacted with trypsin at a molar ratio of 1:1. Their elution volumes from a Sephadex G-200 column were identical, corresponding to a mol.wt. of 58000; however, some heterogeneity was observed after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Isoelectric focusing in polyacrylamide gel in a pH 4-6 gradient revealed a multiple-band pattern for each form in the range of pH4.4-4.9. The two forms of rabbit alpha1-antitrypsin possessed the same N-terminal amino acid (glutamic acid) and had very similar amino acid and carbohydrate compositions.     

Chemical and biological characterization of a truncated form of acidic fibroblast growth factor from bovine brain

Two forms of acidic fibroblast growth factor were isolated from bovine brain by a combination of ammonium sulfate precipitation, cation-exchange chromatography, heparin-Sepharose affinity chromatography, and reverse-phase high-performance liquid chromatography. Amino acid analysis, polyacrylamide gel electrophoresis and amino-terminal sequence analysis showed that one form corresponds to a protein with a molecular mass of 16 kDa and the amino-terminal sequence Phe-Asn-Leu-Pro-Leu-Gly-Asn-Tyr-Lys-Lys-Pro-Lys-Leu-Leu-Tyr- and thus represents the acidic fibroblast growth factor as previously characterized [B?hlen et al. (1985) EMBO J. 5, 1951-1956]. The second mitogen form has a molecular mass of 15.5 kDa. The amino-terminal sequence was established as Asn-Tyr-Lys-Lys-Pro-Lys-Leu-Leu-Tyr-. This evidence indicates that the latter form represents an amino-terminally truncated acidic fibroblast growth factor, lacking the first six amino acid residues. Both forms of the protein are biologically active and equipotent with respect to stimulation of the proliferation in vitro of mesodermal cells such as vascular endothelial and adrenal cortex cells.     

Properties and biosynthesis of cell wall-bound acid phosphatase in Aspergillus nigerx

Acid phosphatase (EC 3.1.3.2 [EC] ) of Aspergillus niger myceliumwas distributed exclusively in the cell wall and soluble fractions,whereas alkaline phosphatase was distributed in the solubleand particulate fractions but only slightly in the cell wallfraction. Cell wall-bound acid phosphatase was released by fungal-walllytic enzymes such as snail gut juice. Cell wall-bound, released,and soluble acid phosphatases showed very similar enzymaticproperties except that the bound enzyme was more stable to heatand detergents. By DEAE-cellulose chromatography, the releasedacid phosphatase was found to correspond to acid phosphatasesI A, IB and II in the soluble fraction. When phosphate in the medium was consumed, the acid phosphataseactivity of the soluble fraction increased more rapidly thanthat of the cell wall fraction. When phosphate was added tothe derepressed culture, the acid phosphatase activity of thesoluble fraction decreased after a short lag period, while thatof the cell wall fraction continued to increase. When labeledamino acid was added to the derepressed culture, it was incorporatedinto the soluble acid phosphatase without a lag period, whileit was incorporated into the cell wall phosphatase after a lagperiod. From these observations, acid phosphatase was consideredto be synthesized first as the soluble form and then integratedinto the cell wall. ¹ The present experiments were carried out, for the most part,at the Institute of Applied Microbiology of the University ofTokyo. (Received January 19, 1976; )     

Purification and properties of a protamine kinase and a type II casein kinase from bovine kidney mitochondria

Bovine kidney mitochondrial extracts contain an inactive protamine kinase and an inactive casein kinase. The protamine kinase was activated by chromatography on poly(L-lysine)-agarose. Two forms of this soluble mitochondrial protamine kinase were separated by chromatography on protamine-agarose. Both forms were purified about 80,000-fold to apparent homogeneity. Both forms of the protamine kinase consist of a single polypeptide chain with an apparent Mr approximately 45,000. Both enzyme forms underwent autophosphorylation without significant effect on activity, and both forms exhibited identical substrate specificities. The protamine kinase showed little activity toward branched-chain alpha-keto acid dehydrogenase (less than 3%), and it was essentially inactive (less than 0.1%) with pyruvate dehydrogenase, casein, and ovalbumin. The enzyme was active with histone H1 and with bovine serum albumin. Protamine kinase activity was unaffected by heparin (up to 100 micrograms/ml), by the protein inhibitor of cyclic AMP-dependent protein kinase, by Ca2+ and calmodulin, and by monoclonal antibody to the catalytic domain of protein kinase C from rat brain. The casein kinase was activated in the presence of spermine or by chromatography of the extract on DEAE-cellulose or poly(L-lysine)-agarose. The enzyme was purified about 80,000-fold to apparent homogeneity. It exhibited an apparent Mr 130,000 as determined by gel-permeation chromatography on Sephacryl S-300 in the presence of 0.5 M NaCl. Two subunits, with apparent Mr's 36,000 (alpha) and 28,000 (beta) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The kinase underwent autophosphorylation of its beta-subunit, without significant effect on activity. Casein kinase activity was inhibited 50% by 1.5 micrograms/ml of heparin. Spermine (1.0 mM) stimulated activity of the purified kinase two- to three-fold at 1.5 mM Mg2+. Half-maximal stimulation occurred at 0.1 mM spermine. The kinase utilized both ATP and GTP as substrates. The casein kinase showed little activity (less than 1%) toward pyruvate dehydrogenase and branched-chain alpha-keto acid dehydrogenase from kidney mitochondria, and the kinase was essentially inactive with glycogen synthase a. The properties of this soluble mitochondrial kinase indicate that it is a type II casein kinase.     

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.