Genetic Complexity, Structure, and Characterization of Highly Active Bovine Intestinal Alkaline Phosphatases
1998; Elsevier BV; Volume: 273; Issue: 36 Linguagem: Inglês
10.1074/jbc.273.36.23353
ISSN1083-351X
AutoresThomas D. Manes, Marc Hoylaerts, Rainer Müller, Friedrich Lottspeich, Werner Hölke, José Luís Millán,
Tópico(s)Folate and B Vitamins Research
ResumoMammalian alkaline phosphatases (APs) display 10–100-fold higher k cat values than do bacterial APs. To begin uncovering the critical residues that determine the catalytic efficiency of mammalian APs, we have compared the sequence of two bovine intestinal APs, i.e. a moderately active isozyme (bovine intestinal alkaline phosphatase, bIAP I, ∼3,000 units/mg) previously cloned in our laboratory, and a highly active isozyme (bIAP II, ∼8,000 units/mg) of hitherto unknown sequence. An unprecedented level of complexity was revealed for the bovine AP family of genes during our attempts to clone the bIAP II cDNA from cow intestinal RNAs. We cloned and characterized two novel full-length IAP cDNAs (bIAP III and bIAP IV) and obtained partial sequences for three other IAP cDNAs (bIAP V, VI, and VII). Moreover, we identified and partially cloned a gene coding for a second tissue nonspecific AP (TNAP-2). However, the cDNA for bIAP II, appeared unclonable. The sequence of the entire bIAP II isozyme was determined instead by a classical protein sequencing strategy using trypsin, carboxypeptidase, and endoproteinase Lys-C, Asp-N, and Glu-C digestions, as well as cyanogen bromide cleavage and NH2-terminal sequencing. A chimeric bIAP II cDNA was then constructed by ligating wild-type and mutagenized fragments of bIAP I, III, and IV to build a cDNA encoding the identified bIAP II sequence. Expression and enzymatic characterization of the recombinant bIAP I, II, III, and IV isozymes revealed averagek cat values of 1800, 5900, 4200, and 6100 s−1, respectively. Comparison of the bIAP I and bIAP II sequences identified 24 amino acid positions as likely candidates to explain differences in k cat. Site-directed mutagenesis and kinetic studies revealed that a G322D mutation in bIAP II reduced its k cat to 1300 s−1, while the converse mutation, i.e. D322G, in bIAP I increased its k cat to 5800 s−1. Other mutations in bIAP II had no effect on its kinetic properties. Our data clearly indicate that residue 322 is the major determinant of the high catalytic turnover in bovine IAPs. This residue is not directly involved in the mechanism of catalysis but is spatially sufficiently close to the active site to influence substrate positioning and hydrolysis of the phosphoenzyme complex. Mammalian alkaline phosphatases (APs) display 10–100-fold higher k cat values than do bacterial APs. To begin uncovering the critical residues that determine the catalytic efficiency of mammalian APs, we have compared the sequence of two bovine intestinal APs, i.e. a moderately active isozyme (bovine intestinal alkaline phosphatase, bIAP I, ∼3,000 units/mg) previously cloned in our laboratory, and a highly active isozyme (bIAP II, ∼8,000 units/mg) of hitherto unknown sequence. An unprecedented level of complexity was revealed for the bovine AP family of genes during our attempts to clone the bIAP II cDNA from cow intestinal RNAs. We cloned and characterized two novel full-length IAP cDNAs (bIAP III and bIAP IV) and obtained partial sequences for three other IAP cDNAs (bIAP V, VI, and VII). Moreover, we identified and partially cloned a gene coding for a second tissue nonspecific AP (TNAP-2). However, the cDNA for bIAP II, appeared unclonable. The sequence of the entire bIAP II isozyme was determined instead by a classical protein sequencing strategy using trypsin, carboxypeptidase, and endoproteinase Lys-C, Asp-N, and Glu-C digestions, as well as cyanogen bromide cleavage and NH2-terminal sequencing. A chimeric bIAP II cDNA was then constructed by ligating wild-type and mutagenized fragments of bIAP I, III, and IV to build a cDNA encoding the identified bIAP II sequence. Expression and enzymatic characterization of the recombinant bIAP I, II, III, and IV isozymes revealed averagek cat values of 1800, 5900, 4200, and 6100 s−1, respectively. Comparison of the bIAP I and bIAP II sequences identified 24 amino acid positions as likely candidates to explain differences in k cat. Site-directed mutagenesis and kinetic studies revealed that a G322D mutation in bIAP II reduced its k cat to 1300 s−1, while the converse mutation, i.e. D322G, in bIAP I increased its k cat to 5800 s−1. Other mutations in bIAP II had no effect on its kinetic properties. Our data clearly indicate that residue 322 is the major determinant of the high catalytic turnover in bovine IAPs. This residue is not directly involved in the mechanism of catalysis but is spatially sufficiently close to the active site to influence substrate positioning and hydrolysis of the phosphoenzyme complex. alkaline phosphatase intestinal alkaline phosphatase bovine intestinal alkaline phosphatase I–VII isozymes tissue nonspecific alkaline phosphatase HPLC, high performance liquid chromatography polymerase chain reaction p-nitrophenylphosphate glycosylphosphatidylinositol base pair(s). Alkaline phosphatases (APs)1 are dimeric, zinc-containing nonspecific phosphomonoesterases that exist throughout speciation, from Escherichia coli to man (1McComb R.B. Bowers G.N. Posen S. Alkaline Phosphatases. Plenum Press, New York1979Crossref Google Scholar). Cloning of AP cDNAs from a variety of species and comparison of their primary structures has revealed a high degree of sequence conservation and even a 25–30% similarity between E. coli and mammalian APs (2Millán J.L. Anticancer Res. 1988; 8: 995-1004PubMed Google Scholar,3Harris H. Clin. Chim. Acta. 1989; 186: 133-150Crossref Scopus (433) Google Scholar). The three-dimensional crystallographic structure of the E. coli AP homodimer has been solved (4Sowadski J.M. Handschumacher M.D. Murthy H.M. Foster B.A. Wyckoff H.W. J. Mol. Biol. 1985; 186: 417-433Crossref PubMed Scopus (201) Google Scholar, 5Kim E.E. Wyckoff H.W. J. Mol. Biol. 1991; 218: 449-464Crossref PubMed Scopus (937) Google Scholar) and the reaction mechanism inferred (6Coleman J.E. Ann. Rev. Biophys. Biomol. Struct. 1992; 21: 441-483Crossref PubMed Scopus (765) Google Scholar). Efforts to crystallize any of the mammalian APs have so far been unsuccessful. Conservation of the residues that comprise the catalytic zinc binding site, as well as the substrate binding residues, suggests that the reaction mechanism is conserved throughout evolution (5Kim E.E. Wyckoff H.W. J. Mol. Biol. 1991; 218: 449-464Crossref PubMed Scopus (937) Google Scholar). However, there are significant structural differences between the E. coli and mammalian APs including several loop regions that have undergone deletion and/or insertion. Mammalian APs display the unique kinetic property, not shared by their bacterial ancestors, of being inhibited stereospecifically byl-amino acids and peptides through an uncompetitive mechanism (7Ghosh N.K. Fishman W.H. J. Biol. Chem. 1966; 241: 2516-2522Abstract Full Text PDF PubMed Google Scholar, 8Fishman W.H. Sie H.-G. Enzymologia. 1971; 41: 140-167Google Scholar). Using human placental AP as a paradigm for mammalian APs, we and others have established that residues within a surface loop unique to mammalian APs are responsible for the differential uncompetitive inhibition by l-amino acids (9Hummer C. Millán J.L. Biochem. J. 1991; 274: 91-95Crossref PubMed Scopus (40) Google Scholar, 10Hoylaerts M.F. Millán J.L. Eur. J. Biochem. 1991; 202: 605-616Crossref PubMed Scopus (44) Google Scholar, 11Watanabe T. Wada N. Kim E.E. Wyckoff H.W. Chou J.Y. J. Biol. Chem. 1991; 266: 21174-21178Abstract Full Text PDF PubMed Google Scholar, 12Hoylaerts M.F. Manes T Millán J.L. Biochem. J. 1992; 286: 23-30Crossref PubMed Scopus (77) Google Scholar), the heat stability properties (13Bossi M. Hoylaerts M.F. Millán J.L. J. Biol. Chem. 1993; 268: 25409-25416Abstract Full Text PDF PubMed Google Scholar), and protein-protein interaction specificities exhibited by some mammalian APs (13Bossi M. Hoylaerts M.F. Millán J.L. J. Biol. Chem. 1993; 268: 25409-25416Abstract Full Text PDF PubMed Google Scholar). A major property of APs that remains to be explained in terms of structure is the large variability in catalytic activity displayed by mammalian APs, which have 10–100-fold higher k cat values thanE. coli AP (14Murphy J.E. Tibbitts T.T. Kantrowitz E.R. J. Mol. Biol. 1995; 253: 604-617Crossref PubMed Scopus (85) Google Scholar). Because among mammalian APs, the intestinal isozyme has the highest specific activity, the bovine intestinal APs (IAPs) represent a potentially useful system for addressing this question. Besman and Coleman (15Besman M. Coleman J.E. J. Biol. Chem. 1985; 260: 11190-11193Abstract Full Text PDF PubMed Google Scholar) demonstrated the existence of two IAP isozymes in the cow intestine, i.e. calf IAP and adult bovine IAP, by sequencing the amino termini of chromatographically purified AP fractions. We have previously reported the cloning and biochemical characterization of the recombinant adult bovine IAP, presently designated bIAP I (16Weissig H. Schildge A. Hoylaerts M.F. Iqbal M. Millán J.L. Biochem. J. 1993; 260: 503-508Crossref Scopus (47) Google Scholar). In this study we report the sequence and characterization of the calf IAP (bIAP II) and two novel bIAP isozymes (bIAP III and bIAP IV (GenBankTM accession nos.AF052226 and AF05227)) and present evidence for the existence of an unprecedented level of complexity in the cow AP gene family. Sequence comparisons and site-directed mutagenesis have unequivocally identified a Gly residue at position 322 as the crucial residue that determines the high specific activity of bIAP II. A λgt 11 cDNA library prepared from adult cow intestine (CLONTECH Laboratories, Palo Alto, CA) was screened using a 1,075-bp HindIII fragment from the 5′ end of the bIAP I cDNA (16Weissig H. Schildge A. Hoylaerts M.F. Iqbal M. Millán J.L. Biochem. J. 1993; 260: 503-508Crossref Scopus (47) Google Scholar) as a probe. Clones isolated from this library were used to screen an EMBL-3 SP6/T7 genomic library prepared from adult cow's liver (CLONTECH Laboratories). An unamplified λZAP II cDNA library was prepared from mRNA isolated, using the TrisolvTM reagent, from the small intestine of one adult cow using oligo(dT) as primer (Stratagene, San Diego, CA) and screened using the 1,075-bp HindIII fragment of the bIAP I cDNA as a probe. Probes were radiolabeled using a random primed DNA labeling kit (Boehringer Mannheim). Phage DNA was prepared as described previously (17Tsonis P.A. Manes T. BioTechniques. 1988; 6: 950-951PubMed Google Scholar) for λgt 11 and EMBL-3 SP6/T7 clones. In vivo excision of λZAP II clones was performed according to the manufacturer's protocol (Stratagene). Genomic clones were characterized by Southern blot analysis as described previously (18Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). EcoRI cDNA fragments from λgt 11 clones and various restriction fragments from clones derived from the other libraries were subcloned into the KS+ vector (Stratagene). Plasmid DNA was prepared by the alkaline lysis procedure (18Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Sequencing was performed using Sequenase according to the manufacturer's protocol (Amersham Pharmacia Biotech). Oligos used for sequencing bIAPs III and IV are as follows. 1s, GCC AAG AAT GTC ATC CTC; 1a, GAG GAT GAC ATT CTT GGC; 2s, GGT GTA AGT GCA GCC GC; 2a, GCG GCT GCA CTT AGA CC; 3s, AAT GTA CAT GTT TCC TG; 3a, CAG GAA ACA TGT ACA TT; 4s, CCA GGG CTT CTA CCT CTT; 4a, AAG AGG TAG AAG CCC TGG; 5s, ACC AGA GCT ACC ACC TCG; 5a, AAG CAG GAA ACC CCA AGA; and 6s, CTT CAG TGG CTT GGG ATT; 6a, AAT CCC AAG CCA CTG AAG. Nucleic acid sequences were analyzed using the MacVector sequence analysis program (International Biotechnologies, Inc., New Haven, CT). Approximately 500 μg of purified, highly active (approximately 7800 units/mg) cow intestinal AP was dissolved in 450 μl of 6 m guanidinium hydrochloride, 0.25 mTris, 1 mm EDTA, pH 8.5, followed by the addition of 30 μl of mercaptoethanol. After reduction for 30 min at 100 °C, cysteines were alkylated by addition of 35 μl of vinylpyridine, and the mixture was incubated for 45 min at room temperature in the dark. The reaction mixture was then immediately desalted by a short reversed phase HPLC on a Aquapore RP300 column (30 × 2.1 mm, Applied Biosystems, Weiterstadt) using a steep gradient of acetonitrile in 0.1% trifluoroacetic acid to elute bound enzyme. Protein-containing fractions were evaporated to dryness. In order to deglycosylate the enzyme, 125 μg of AP were dissolved in 15 μl of distilled water, 6 μl of incubation buffer (250 mmNa2HPO4, 50 mm EDTA, pH 7.2), and 15 units of endoglycosidase F/N-glycosidase F (Boehringer Mannheim, Penzberg). The mixture was left overnight at 37 °C and subsequently used for cleavages. Reduced and alkylated AP was enzymatically cleaved with different enzymes (i.e.endoproteinase Lys-C, endoproteinase Asp-N, endoproteinase Glu-C, and trypsin (Boehringer Mannheim, Penzberg) according to the instructions given on the data sheets for individual enzymes. Cyanogen bromide cleavage was performed with 10% (w/w) CNBr in 70% (v/v) formic acid for 8 h. After dilution with water the solution was reduced in volume on a SpeedVac concentrator (Savant) and applied onto reversed phase HPLC. Carboxypeptidase Y (8 ng/μl) digestion of the COOH-terminal tryptic peptide was performed for 4 min, and the released peptides were analyzed by matrix-assisted laser desorption/ionization mass spectrometry using a Bruker Reflex III instrument according to the instructions of the manufacturer. 2,5-Dihydroxybenzoic acid (10 mg/ml) in acetonitrile/water (50/50, v/v) was used as a matrix. Peptides derived from enzymatic or chemical cleavages were separated by reversed phase HPLC on a LiChrospher C18 sel B column, 125 × 2 mm (Merck, Darmstadt) using a 0.1% trifluoroacetic acid/acetonitrile solvent system. Flow rate was 300 μl/min. The effluent was monitored by UV at 206 nm, and the fractions were collected manually. Mass determination of the peptides was performed on an API III electrospray mass spectrometer (PE-Sciex, Langen) according to the instruction of the manufacturer. Amino acid sequencing was performed using a 492A protein sequencer (Applied Biosystems, Weiterstadt) according to the instructions of the manufacturer. To construct a cDNA coding for bIAP II, wild-type restriction fragments and site-directed mutagenized PCR fragments of cDNAs bIAP I, III, and IV were assembled, creating the L1N8 (three fragments) and INT 1 (nine fragments) intermediate cDNA constructs. INT 1 and bIAP III then served as the templates for site-directed mutagenesis and fragments from these were assembled into a complete INT 2 (eight fragments) cDNA. Restriction fragments from INT 2 and site-directed mutagenized fragments of INT 2 were then assembled into the INT 3 (five fragments) cDNA and finally the bIAP II (four fragments) cDNA. Site-directed mutagenesis was performed as described previously (19Tomic M. Sunjevaric I. Savtchenko E.S. Blumenberg M. Nucleic Acids Res. 1990; 18: 1656Crossref PubMed Scopus (50) Google Scholar) using BsaI as the restriction enzyme that cuts at a distance from its recognition site (GGTCTCN1/N5). All PCR products were sequenced to verify the absence of secondary mutations, and all constructs were verified by sequencing and restriction digestion. The sequence of the oligonucleotide primers used for amplifying the site-directed mutagenized fragments are as follows, with the name of the primer given first followed by the sequence (positions which denote the mutation are underlined): KS, CGA GGT CGA CGG TAT CG; 1L, GCA GGT CTC TCA GCT GGG ATG AGG GTG AGG; 8N, GCA GGT CTC AGC TGA GGA GGA AAA CCC CGC; 122, GCA GGT CTC TGT TGT GTC GCA CTG GTT; 1s, GCC AAG AAT GTC ATC CTC; M133I, GGT CTC TTT CTT GGC CCG GTTGAT CAC; S142A, GGT CTC AAG AAA GCA GGG AAG GCC GTC; 180, GGT CTC GTG CAT CAG CAG GCA GGT CGG C; M180K, GGT CTC ATG CAC AGA AGA ATG GCT GCC AG; K205M, GGT CTC AAA CAT GTACAT TCG GCC TCC ACC; V210E, GGT CTC CAT GTT TCC TGA GGG GAC CCC A; A236E, GGT CTC CTG CCA TTCCTG CAC CAG GTT; 236, GGT CTC TGG CAG GCC AAG CAC CAG GGA; 289, GGT CTC CAG GGT CGG GTC CTT GGT GTG; E289A, GGT CTC GAC CCT GGC GGA GAT GAC G; 330, GGT CTC CTC AGT CAG TGC CAT ATA; 330E, V332I, GGT CTC ACT GAG GCG ATC ATG TTT GAC; XIa, GTG CAC CAG GTG CGC CTG CGG GCC; N192Y, GCC GCA CAG CTG GTCTAC AAC ATG GAT; S380G, GCT GTC TAA GGC CTT GCCGGG GGC; N192Y, GCC GCA CAG CTG GTC TAC AAC ATG GAT; D411G, GGG GGT CTC GCT TGC TGC CAT TAA C; D416E, GTT AAT GGT CTC ACA AGC GAG GAA CCC TCG; S428A, CCC GTG GGT CTC GCT AGC CAG GGG CAC; D416E, GTT AAT GGT CTC ACA AGC GAG GAA CCC TCG; T480S, GAT GCT GGT CTC GGT GGA GGG GGC TGG CAG; 480, CTG CCA GGT CTC ACC ACC GCC ACC AGC ATC; SP6, CAT ACG ATT TAG GTG ACA CTA TAG; 236, GGT CTC TGG CAG GCC AAG CAC CAG GGA; Q304R−, GTA GAA GCC CCG GGG GTT CCT GCT; Q304+, AGC AGG AAC CCC CGG GGC TTC TAC; E321D, TGC CAT ATA AGC TTT GCCGTC ATG GTG. The different PCR reactions are numbered 1–16, the templates are either the wild-type cDNAs bIAP I, III, or IV, or chimeric constructs INT 1 or INT 2, and the oligonucleotide primers (indicated in parentheses) are those listed above. 1, bIAP IV (KS, 1L); 2, bIAP IV (8N, 122); 3, bIAP III (1S, M133I); 4, bIAP I (S142A, 180); 5, bIAP I (M180K, K205 m); 6, bIAP I (V210E, A236E); 7, bIAP I (236, 289); 8, bIAP IV (E289A, 330); 9, bIAP III (330E, V332I, XIa); 10, INT 1 (N192Y, S380G); 11, INT 1 (N192Y, D411G); 12, bIAPIII (D416E, S428A); 13, INT 1 (D416E, T480S); 14, INT 1 (480, SP6); 15, INT 2 (236, Q304R−); and 16, INT 2 (Q304R+, E321D). The following ligation reactions were performed using, in all cases, the pcDNA-3 (Invitrogen, San Diego, CA) expression vector. The fragments are numbered according to the PCR reaction number given above or by the name of the wild-type or chimeric cDNA, followed by the restriction enzymes used to create the cohesive termini of that fragment. L1N8 = pcDNA-3/ EcoRI-XbaI + 1/EcoRI-BsaI + 2/BsaI-BamHI + bIAP I/BamHI-XbaI. INT 1 = pcDNA-3/EcoRI-XbaI + L1N8/EcoRI-NcoI + 3/NcoI-BsaI + 4/BsaI + 5/BsaI + 6/BsaI + 7/BsaI + 8/BsaI + 9/BsaI-StuI + bIAP I/StuI-XbaI. INT 2 = pcDNA-3/EcoRI-NotI + INT 1/EcoRI-PstI + 10/PstI-StuI + 11/StuI-BsaI + 12/BsaI + 13/BsaI + 14/BsaI + bIAP I/BsaI-NotI. INT 3 = pcDNA-3/EcoRI-XbaI + INT 2/EcoRI-NcoI + INT 2/NcoI-PvuII + 10/PvuII-EagI + INT 2/EagI-HindIII + INT 2/HindIII-XbaI. bIAP II = pcDNA-3/EcoRI-XbaI + INT 3/EcoRI-EagI + 15/EagI-SmaI + 16/SmaI-HindIII + INT 3/HindIII-XbaI. Ten additional constructs were made to identify the residue(s) responsible for the differential kinetic properties of bIAPs I and II. All constructs were subcloned in pcDNA-3/EcoRI-XbaI. Five constructs were built by exchanging restriction fragments between LIN8 or bIAP I (I) and bIAP II (II). L1N8 EcoRI-PmlI and (II) PmlI-XbaI were ligated to produce the (N122K)bIAP II mutant cDNA. (II) EcoRI-BstEII, (I)BstEII-PvuII, (II) PvuIIXbaI were combined for the (K180M)bIAP II mutant cDNA. (II) EcoRI-EagI, (I)EagI-BstEII, (II)BstEII-XbaI were ligated for the (A289Q,A294V,Q297R,L299V)bIAP II mutant. (II)EcoRI-EagI, (II)EagI-BstEII, (I)BstEII-HindIII, (II)HindIII-XbaI for the (G322D)bIAP II mutant. (II)EcoRI-HindIII, (I)HindIII-SacI, (II)SacI-XbaI for the (I332G)bIAP II mutant. Five others positions required new site-directed mutagenesis, and the oligos used for these are as follows: I133M−, GGT CTC TTT CTT GGC CCG GTT CAT CAC; A142S−, TGG TCA CCA CTC CCA CGG ACT TCC CTG; M205K−, GGT CTC AAA CAT GTA TTT TCG GCC TCC ACC; E210V+, GGT CTC ATG TTT CCT GTG GGG ACC CCA GAC; E236A, GGT CTC CTG CCA TGC CTG CAC CAG GTT. Using these and previously listed oligos the following eight PCR reactions (a–h) were carried out using bIAP II as template: a, 1s, I133M−; b, S142A+,M205K−; c, 1s, A142S−; d, V210E+,330−; e, E210V+,330−; f, M180K+,E236A−; g, 236+,330−; h, S142A,K205M−. The products of these were subcloned and sequenced, and then fragments isolated for the following ligations: (II) EcoRI-NcoI, (a)NcoI-BsaI, (b) BsaI, PvuII, (II)PvuII-XbaI for I133M. (II)EcoRI-NcoI, (c)NcoI-BstEII, (II)BstEII-PvuII, (II)PvuII-XbaI for A142S. (II) EcoRI-BstEII, (b) BstEII-BsaI, (d)BsaI-HindIII, (II)HindIII-XbaI for M205K. (II)EcoRI-BstEII, (h)BstEII-BsaI, (e)BsaI-HindIII, (II)HindIII-XbaI for E210V. (II)EcoRI-NcoI, (II)NcoI-PvuII, (f) PvuII-BsaI, (g)BsaI-HindIII, (II)HindIII-XbaI for E236A. All cDNAs (bIAP I, bIAP II, bIAP III, bIAP IV, and corresponding mutants) were cloned into the pcDNA-3 expression vector (Invitrogen, San Diego, CA), transfected into Chinese hamster ovary cells and stable transfectants were selected by growing the cells in the presence of 500 μg/ml geneticin (Life Technologies, Inc.). Recombinant APs were extracted from the stably transfected Chinese hamster ovary cells as described previously (20Hoylaerts M.F. Manes T. Millán J.L. J. Biol. Chem. 1997; 272: 22781-22787Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). To measurek cat, microtiter plates coated with 0.1 μg/ml high affinity anti-bovine AP monoclonal antibody (Scottish Antibody Production Unit, Lanarkshire, Scotland) were incubated with increasing concentrations of enzyme and the activity of bound enzyme was measured as the change in absorbance at 405 nm over time at 20 °C upon addition of 30 mm p-nitrophenylphosphate (pNPP) as substrate in 1.0 m diethanolamine buffer (pH 9.8), 1 mm MgCl2, and 20 μmZnCl2. The p-nitrophenol concentration formed was calculated using an extinction coefficient of 10,080 liters mole−1 cm−1. Commercial preparations with known specific activities (Biozyme Laboratories, 7,822 units/mg, and Boehringer Mannheim, 3,073 units/mg) as well as purified bIAP II (8,600 units/mg) were used as standards. The concentration of enzyme at those dilutions that saturated the antibody (E 0) was calculated from a standard curve of activity versus known enzyme concentrations using identical assay conditions. The maximal rate of substrate conversion (V max) was then divided by E 0 to calculatek cat. To calculate K m, substrate concentration was varied between 0.25–2.0 mmpNPP, and the initial reaction rate was measured at 20 °C over a time interval of 10 min. Regression plots of [pNPP]/V versus [pNPP] (Hanes plots) to the x intercept equaled −K m. Dividing the standard error of the predicted y value for each x in the regression by the slope of the regression gave the standard error of theK m. V max ± S.E. was calculated by dividing K m ± S.E. with they intercept ± S.E., using the appropriate equations to obtain V max S.E. Heat stability curves were produced by incubating extracts at 45–75 °C in 5 °C increments, 10 min each as described previously (16Weissig H. Schildge A. Hoylaerts M.F. Iqbal M. Millán J.L. Biochem. J. 1993; 260: 503-508Crossref Scopus (47) Google Scholar). The activity of each sample was then determined as above, and residual activity calculated as the percentage remaining compared with the nonheated sample. The temperature at which 50% residual activity remained (T 50) was calculated from the residual activityversus temperature plots. We set out to determine the structure of the fetal intestinal AP (bIAP II) defined by Besman and Coleman (15Besman M. Coleman J.E. J. Biol. Chem. 1985; 260: 11190-11193Abstract Full Text PDF PubMed Google Scholar) as possessing a LIPAEEEN amino-terminal sequence since we knew that this amino-terminal sequence was found in purified high activity intestinal AP preparations (range 7,000 to 8,000 units/mg) available commercially (Biozyme Laboratories and Boehringer Mannheim GmbH). We screened a commercial 5′ Stretch λgt 11 bovine small intestinal cDNA library (CLONTECH Laboratories) with a 1,075-bp HindIII fragment of the bIAP I cDNA that contains sequences included in exons I through VIII of the bIAP I gene. Twelve cDNAs were isolated that represented different size spots and intensity of hybridization on the filters. The fragments were subcloned and completely sequenced. Four of these cDNAs were identical to the bIAP I sequence as previously published (16Weissig H. Schildge A. Hoylaerts M.F. Iqbal M. Millán J.L. Biochem. J. 1993; 260: 503-508Crossref Scopus (47) Google Scholar). Four clones represent a new tissue-specific AP isozyme gene homologous, but not identical, to bIAP I, although the clones were unspliced. The largest of these clones (2,561 bp) aligned to the bIAP I gene 570 bp 5′ of the start codon and extended to exon eight, and identifies the isozyme referred to here as bIAP VII (GenBankTM accession no. AF052230). Two cDNAs represented another variation of the bIAP I sequence and represented unspliced clones, the largest (783 bp) contained exon I to exon III sequences which define the bIAP V isozyme (GenBankTM accession no. AF052228). One cDNA (clone VIII) appeared to be yet another tissue-specific AP transcript. This clone is 1,642 bp long, is also unspliced, aligns with bIAP I from intron 2 to intron 8, and defines the bIAP VI isozyme GenBankTM accession no. AF052229). All these novel clones encode predicted amino-terminal sequences that are different from the expected LIPAEEEN sequence. Fig. 1 shows differences in the deduced first 80 amino-terminal amino acids of the newly identified isozymes bIAP III, bIAP IV, bIAP V, bIAP VI, bIAP VII in comparison with the corresponding residues of bIAP I (16Weissig H. Schildge A. Hoylaerts M.F. Iqbal M. Millán J.L. Biochem. J. 1993; 260: 503-508Crossref Scopus (47) Google Scholar) and of bIAP II as determined below. Still another cDNA was isolated that represents a new tissue nonspecific AP molecule. This partially spliced cDNA clone aligns with bovine kidney AP (21Garattini E. Hua J.C Udenfriend S. Gene (Amst.). 1987; 59: 41-46Crossref PubMed Scopus (33) Google Scholar) (starting at residue 8) in exon II and extends to exon IX. This appears to be a different TNAP (TNAP-2, GenBankTM accession no. AF052231) molecule expressed in the bovine intestine. Northern blot analysis was performed on RNA samples isolated from different portions of the cow intestine of a single animal, and the sample with the highest expression was chosen for the construction of a new cDNA library in λZAP II vector (Stratagene). Adjacent segments of the intestine were used for enzyme purification. The entire unamplified library (1.0 × 106 independent recombinant clones) was screened with the 1,075-bp HindIII bIAP I probe, and 65 clones were isolated and sequenced. All clones corresponded in sequence to one or the other of two novel bIAP cDNAs designated bIAP III and bIAP IV. The sequence of the 2,460-bp bIAP III cDNA is shown in Fig. 2 as well as the differences found in the coding region of the 2,536-bp bIAP IV cDNA. Neither of these full-length novel bIAP cDNAs coded for an amino-terminal LIPAEEEN sequence, while at the protein level it was clear that the LIPAEEEN sequence was the major component of the purified preparation from the same intestinal region. We had to conclude that the bIAP II sequence was either “toxic” to the bacterial cells used for library construction or “unclonable” for some other reason.Figure 2Complete nucleotide sequence of the 2,460-bp bIAP III cDNA and deduced amino acid sequence. Nucleotide differences found in the coding region of the bIAP IV cDNA are written above the nucleotide sequence and those mutations that translate into amino acid differences are spelled out under the deduced amino acid sequence of bIAP III. Nucleotide differences in the 5′- and 3′-untranslated regions of the bIAP III and bIAP IV cDNA are not shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Our previous work on the cloning of bIAP had revealed the structure of the bIAP I gene and also of a transcribed pseudogene (R201) (16Weissig H. Schildge A. Hoylaerts M.F. Iqbal M. Millán J.L. Biochem. J. 1993; 260: 503-508Crossref Scopus (47) Google Scholar). Southern blot analysis using the bIAP cDNA as a probe had revealed a complex pattern of bands and only two of them could be accounted for by the cloned bIAP I gene and the R201 pseudogene (16Weissig H. Schildge A. Hoylaerts M.F. Iqbal M. Millán J.L. Biochem. J. 1993; 260: 503-508Crossref Scopus (47) Google Scholar). Human and mouse APs are the two best characterized gene families, and both display the same degree of genetic complexity. Human APs are encoded by three tissue-specific AP loci, i.e. GCAP (22Millán J.L. Manes T. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3024-3028Crossref PubMed Scopus (132) Google Scholar), PLAP (23Knoll B.J. Rothblum K.N. Longley M. J. Biol. Chem. 1988; 263: 12020-12027Abstract Full Text PDF PubMed Google Scholar), and IAP (24Henthorn P.S. Raducha M. Kadesch T. Weiss M.J. Harris H. J. Biol. Chem. 1988; 263: 12011-12019Abstract Full Text PDF PubMed Google Scholar) and one TNAP locus (25Weiss M.J. Ray K. Henthorn P.S. Lamb B. Kadesch T. Harris H. J. Biol. Chem. 1988; 263: 12002-12010Abstract Full Text PDF PubMed Google Scholar), and the mouse AP genes include two active tissue-specific AP genes, i.e. embryonic and IAP (26Manes T. Glade K. Ziomek C.A. Millán J.L. Genomics. 1990; 8: 541-554Crossref PubMed Scopus (53) Google Scholar), one pseudogene (26Manes T. Glade K. Ziomek C.A. Millán J.L. Genomics. 1990; 8: 541-554Crossref PubMed Scopus (53) Google Scholar), and one TNAP gene (27Terao M. Studer M. Gianni M. Garattini E. Biochem. J. 1990; 268: 641-648Crossref PubMed Scopus (63) Google Scholar). Both the human and mouse tissue-specific genes are highly homologous and are each comprised of 11 exons contained in less than 5 kilobase pairs of DNA while the single TNAP gene in both species is compose
Referência(s)