RESUMO
Two proctolin-binding proteins solubilized from 1600 cockroach hindgut membranes were purified 1000-fold using five chromatography steps. Twenty-five micrograms of protein were recovered from the final size-exclusion chromatography as a single peak eluting at 74 kDa, whereas two major bands at 80 and 76 kDa were identified after silver staining of electrophoresis gels. The fragments, sequenced by tandem mass spectrometry and the Edman method, revealed a high homology with rat liver dipeptidyl aminopeptidase (DPP) III and a significant homology between the cockroach-purified proteins. From analysis of the Drosophila genome sequence database, it was possible to identify a putative DPP sharing high homology with the sequences obtained from the cockroach purified proteins and with the rat DPP III. Anti-(rat liver DPP III) Ig reacted specifically with both cockroach-purified proteins in Western blot analysis. The purified proteins removed the N-terminal dipeptide from the insect myotropic neuropeptide proctolin (Arg-Tyr-Leu-Pro-Thr) with a Km value of 3.8 +/- 1.1 microM. The specific DPP III inhibitor tynorphin prevented the degradation of proctolin by the purified insect DPP (IC50 = 0.68 microM). These results provide strong evidence that the cockroach-purified proteins represent an insect membrane DPP, presumably present in Drosophila, and that it is closely related to vertebrate DPP III.
Assuntos
Baratas/enzimologia , Dipeptidil Peptidases e Tripeptidil Peptidases/isolamento & purificação , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Neuropeptídeos , Oligopeptídeos/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Baratas/citologia , Baratas/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Etiquetas de Sequências Expressas , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Ratos , Análise de Sequência de ProteínaRESUMO
Dipeptidyl peptidase III (DPP III) (EC 3.4.14.4), which has a HELLGH-E (residues 450-455, 508) motif as the zinc binding site, is classified as a zinc metallopeptidase. The zinc dissociation constants of the wild type, Leu(453)-deleted, and E508D mutant of DPP III at pH 7.4 were 4.5 (+/-0.7) x 10(-13), 5.8 (+/-0.7) x 10(-12), and 3.2 (+/-0.9) x 10(-10) M, respectively. The recoveries of the enzyme activities by the addition of various metal ions to apo-DPP III were also measured, and Co(2+), Ni(2+), and Cu(2+) ions completely recovered the enzyme activities as did Zn(2+). The dissociation constants of Co(2+), Ni(2+), and Cu(2+) ions for apo-DPP III at pH 7.4 were 8.2 (+/-0.9) x 10(-13), 2.7 (+/-0.3) x 10(-12), and 1.1 (+/-0.1) x 10(-14) M, respectively. The shape of the absorption spectrum of Co(2+)-DPP III was very similar to that of Co(2+)-carboxypeptidase A or Co(2+)-thermolysin, in which the Co(2+) is bound to two histidyl nitrogens, a water molecule, and a glutamate residue. The absorption spectrum of Cu(2+)-DPP III is also very similar to that of Cu(2+)-thermolysin. The EPR spectrum and the EPR parameters of Cu(2+)-DPP III were very similar to those of Cu(2+)-thermolysin but slightly different from those of Cu(2+)-carboxypeptidase A. The five lines of the superfine structure in the perpendicular region of the EPR spectrum in Cu(2+)-DPP III suggest that nitrogen atoms should coordinate to the cupric ion in Cu(2+)-DPP III. All of these data suggest that the donor set and the coordination geometry of the metal ions in DPP III, which has the HExxxH motif as the metal binding site, are very similar to those of the metal ions in thermolysin, which has the HExxH motif.
Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Fígado/enzimologia , Zinco/metabolismo , Animais , Espectroscopia de Ressonância de Spin Eletrônica , Cinética , Ratos , Proteínas Recombinantes/metabolismoRESUMO
Dipeptidyl peptidase II (DPP II; EC 3.4.14.2) from rat kidney was purified to a specific activity of 65.4 micromol/min per mg of protein for Lys-Ala-beta-naphthylamide. The N-terminal and partial amino acid sequences of the enzyme were determined. The peptide sequences were used to identify expressed sequence tag (EST) clones. By using the cDNA fragment of one of the EST clones as a probe, we isolated a cDNA clone with 1710 bp encoding DPP II from a rat kidney cDNA library. The cDNA of rat DPP II contained an open reading frame of 1500 bp, coding for a protein of 500 amino acids. The first 10 residues of the purified enzyme matched the deduced protein sequence starting with residue 37, suggesting the presence of a signal peptide. The mature enzyme (464 residues) had a calculated molecular mass of 51400 Da, which was lower than the value (about 60000 Da) determined by SDS/PAGE; and the deduced amino acid sequence showed six potential N-glycosylation sites. The deduced amino acid sequence of rat DPP II shared high similarity with quiescent-cell proline dipeptidase (78% identity) and prolyl carboxypeptidase (38% identity) and bore the putative catalytic triad (Ser, Asp, His) conserved in serine peptidase families. We transiently transfected COS-7 cells with pcDNA3.1 containing the cloned cDNA and obtained the overexpression of an immunoreactive protein (of about 60000 Da). The transfected cells showed Lys-Ala-methylcoumarinamide-hydrolysing activity that was 50 times higher than the control cells.
Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Rim/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Clonagem Molecular , DNA Complementar/química , DNA Complementar/isolamento & purificação , DNA Complementar/metabolismo , Dipeptidases/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/isolamento & purificação , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Feminino , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , RNA Mensageiro/química , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Alinhamento de Sequência , TransfecçãoRESUMO
The role of the HELLGH (residues 450-455) motif in the sequence of rat dipeptidyl peptidase III (EC 3.4.14.4) was investigated by replacing Glu451 with an alanine or an aspartic acid residue and by replacing His450 and His455 with a tyrosine residue by site-directed mutagenesis. Mutated cDNAs were expressed three or four times in Escherichia coli, and the resulting proteins were purified to apparent homogeneity. None of the expressed mutated proteins exhibited DPP III activity. The mutants of Glu451 contained 1 mol of zinc per mole of protein, but mutants His450 and His455 did not contain significant amounts of zinc as determined by atomic absorption spectrometry. The Leu453-deleted enzyme (having the zinc aminopeptidase motif HExxH-18-E) had almost the same order of binding affinity (for Arg-Arg-2-naphthylamide) as the wild-type enzyme, but the specificity constant was about 10%. These results provide evidence that the suitable number of amino acids included between Glu451 and His455 is three residues for the enzyme activity and confirm that residues His450, His455, and Glu451 are involved in zinc coordination and catalytic activity.
Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/fisiologia , Fígado/enzimologia , Fragmentos de Peptídeos/fisiologia , Zinco/metabolismo , Sequência de Aminoácidos , Animais , Catálise , DNA Complementar/isolamento & purificação , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/isolamento & purificação , Ativação Enzimática/genética , Escherichia coli/genética , Ácido Glutâmico/genética , Histidina/genética , Humanos , Leucina/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Zinco/químicaRESUMO
Aminopeptidase B (Ap B; EC 3.4.11.6) is a zinc-binding protein that contains the consensus sequence HEXXHX18E (324-347), conserved among the M1 family of metallopeptidases. To determine if these putative zinc-binding residues (His324, His328 and Glu347) and the active-site Glu325 are essential for the enzyme activity, we replaced the histidines with tyrosines and the glutamic acid residues with alanines using site-directed mutagenesis. The cDNAs were expressed in Escherichia coli, and the resulting recombinant proteins, named H324Y, E325A, H328Y and E347A, were purified to apparent homogeneity. None of the expressed mutated proteins showed aminopeptidase activity. The E325A enzyme contained 1 mol of zinc per mol of protein, and the other three mutants, H324Y, H328Y and E347A, did not contain significant amounts of zinc, as determined by atomic absorption spectrometry. From sequence-homology searches, Ap B is known to be closely related to leukotriene (LT)-A4 hydrolase (EC 3.3.2.6). We examined human placental Ap B and recombinant rat Ap B, both of which had been purified previously [Fukasawa, Fukasawa, Kanai, Fujii and Harada (1996) J. Biol. Chem. 271, 30731-30735], to determine whether or not they had epoxide hydrolase activities. However, neither enzyme hydrolysed LTA4 into LTB4. We then replaced some amino acids in the domain of the rat enzyme similar to the LTA4-binding site of LTA4 hydrolase. However, these mutants, Y408F, N409S and NE409-410SS also did not possess any epoxide hydrolase activity. We concluded that Ap B is an M1-family zinc metallopeptidase without epoxide hydrolase activity.
Assuntos
Aminopeptidases/química , Aminopeptidases/metabolismo , Epóxido Hidrolases/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Aminopeptidases/genética , Aminopeptidases/isolamento & purificação , Animais , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Epóxido Hidrolases/metabolismo , Escherichia coli/genética , Humanos , Hidrólise , Cinética , Leucotrieno A4/metabolismo , Leucotrieno B4/metabolismo , Metaloendopeptidases/química , Metaloendopeptidases/genética , Metaloendopeptidases/isolamento & purificação , Metaloendopeptidases/metabolismo , Dados de Sequência Molecular , Placenta/enzimologia , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Espectrofotometria Atômica , Zinco/análiseRESUMO
We have purified dipeptidyl peptidase III (EC 3.4.14.4) from human placenta. It had a pH optimum of 8.8 and readily hydrolysed Arg-Arg-beta-naphthylamide. Monoamino acid-, Gly-Phe-, Gly-Pro- and Bz-Arg-beta-naphthylamides were not hydrolysed at all. The enzyme was inhibited by p-chloromercuriphenylsulphonic acid, metal chelators and 3,4-dichloroisocoumarin and contained 1 mol of zinc per mol of enzyme. The zinc dissociation constant was 250 fM at pH 7. 4 as determined by the zinc binding study. We isolated, by immunological screening of a Uni-ZAP XR cDNA library constructed from rat liver mRNA species, a cDNA clone with 2633 bp encoding the rat enzyme. The longest open reading frame encodes a 827-residue protein with a theoretical molecular mass of 92790 Da. Escherichia coli SOLR cells were infected with the pBluescript phagemid containing the cloned cDNA and established the overexpression of a protein that hydrolysed Arg-Arg-beta-naphthylamide. The recombinant protein was purified and the amino acid sequence of the protein was confirmed. We presumed that the putative zinc-binding domain involved in catalysis was present in the recombinant enzyme. It was a novel zinc-binding motif in that one amino acid residue was inserted into the conserved HEXXH motif characteristic of the metalloproteinases.
Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Feminino , Humanos , Dados de Sequência Molecular , Placenta/enzimologia , Ligação Proteica , Ratos , Ratos Wistar , Homologia de Sequência de Aminoácidos , Zinco/metabolismoRESUMO
We isolated, by immunological screening of a Uni-ZAP XR cDNA library constructed from rat liver mRNAs, a cDNA clone with 2212 base pairs encoding aminopeptidase B (EC 3.4.11.6). The open reading frame encodes a 649-amino acid protein with a theoretical molecular mass of 72,545 Da and bears the consensus sequence of the zinc metalloexopeptidases, indicating that the enzyme belongs to this family, which includes aminopeptidase A, aminopeptidase N, and leukotriene-A4 hydrolase. Escherichia coli SOLR cells infected with the pBluescript phagemid excised from the Uni-ZAP XR vector containing the aminopeptidase B cDNA had a high L-arginyl-beta-naphthylamidase activity. The recombinant protein was purified to homogeneity from the recombinant E. coli extracts. The enzyme had Cl--dependent aminopeptidase activity specifically restricted to the Arg and Lys derivatives and contained 1 mol of zinc per mol of the enzyme.
Assuntos
Aminopeptidases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Epóxido Hidrolases/química , Expressão Gênica , Humanos , Fígado/enzimologia , Metaloproteínas/genética , Dados de Sequência Molecular , Ratos , Alinhamento de Sequência , ZincoRESUMO
The complete sequence of 12,851 nucleotides of the mouse lactate dehydrogenase-A (LDH-A) gene has been determined. It includes eight exons, seven introns, promoter and regulatory regions. The B1 repetitive elements present in intron III and VI are oriented in opposite orientation, and they share 72% sequence homology. The exon-intron organization of mouse LDH-A gene is compared with the organizations of other dehydrogenase genes, and the molecular evolution of the nicotinamide adenine dinucleotide binding domains is discussed.
Assuntos
Genes , L-Lactato Desidrogenase/genética , Animais , Sequência de Bases , Enzimas de Restrição do DNA , Éxons , Humanos , Íntrons , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Ratos , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
The presence of at least ten mouse LDH-A pseudogenes was demonstrated in the genomic blot analysis, and four different processed pseudogenes have thus far been isolated and characterized. In this report, the nucleotide sequences to two different mouse lactate dehydrogenase-A processed pseudogenes, M11 and M14, were determined and compared with the protein-coding sequences of the mouse and rat LDH-A functional genes. In the pseudogene M11, the sequence of 64 nucleotides from codon no. 257 to 278 was tandemly duplicated. In the pseudogene M14, the sequence of 22 nucleotides from codon no. 68 to 75 was replaced by an inserted repetitive sequence of 242 nucleotides homologous to a mouse truncated R element. The pattern of nucleotide substitutions accumulated in mouse LDH-A pseudogenes M11 and M14, as well as that of pseudogene M10 identified previously, was analyzed, and the substitution frequencies of the C or G at the CG dinucleotide were found to be high.
Assuntos
Genes , L-Lactato Desidrogenase/genética , Mutação , Animais , Sequência de Bases , Evolução Biológica , Clonagem Molecular , DNA/genética , Isoenzimas , Camundongos , Sequências Repetitivas de Ácido NucleicoRESUMO
A mouse genomic clone containing a lactate dehydrogenase-A (LDH-A) processed pseudogene and a B1 repetitive element was isolated, and a nucleotide sequence of approximately 3 kb was determined. The pseudogene and B1 element are flanked by perfect 13-bp repeats, and the B1 sequence starts at 14 nucleotides 3' to the presumptive polyadenylation signal of the pseudogene. The nucleotide sequences of the LDH-A genes and processed pseudogenes from mouse, rat, and human were compared, and a phylogenetic tree was constructed. The rate and pattern of nucleotide substitutions in the LDH-A pseudogenes are similar to previously reported results (Li et al. 1984). The average rate of nucleotide substitutions in the LDH-A pseudogenes is 4.3 X 10(-9)/site/year. The substitutions of C----T and G----A are most frequent, and A----G substitutions are relatively high. The rate of synonymous substitutions in the LDH-A genes is 5.3 X 10(-9), which is not significantly higher than the average rate of 4.7 X 10(-9) for 35 mammalian genes. The rate of nonsynonymous substitutions in the LDH-A genes is 0.20 X 10(-9), which is considerably lower than the average rate of 0.88 X 10(-9) for 35 mammalian genes. Thus, the mammalian LDH-A gene appears to be highly conserved in evolution.
Assuntos
L-Lactato Desidrogenase/genética , Camundongos/genética , Pseudogenes , Animais , Sequência de Bases , Evolução Biológica , Humanos , Dados de Sequência Molecular , Filogenia , Sequências Repetitivas de Ácido NucleicoRESUMO
The nucleotide sequence of approx. 3 kilobases including the regulatory region, a non-coding exon and the first protein-coding exon from mouse lactate dehydrogenase-A (LDH-A) gene has been determined. The putative initiation sites of transcription and translation were deduced by comparing the nucleotide sequence of mouse LDH-A gene with those of a mouse LDH-A processed pseudogene and the LDH-A full-length cDNAs from rat and human. The tentative TATA and CAAT boxes, and the hexanucleotides CCGCCC have been identified. The sequence of AAATCTTGCTCAA of mouse LDH-A gene has also been found to show striking homology to the cyclic AMP-responsive sequences of eukaryotic genes regulated by cyclic AMP. It has been reported previously that the protein-coding sequence of mouse LDH-A gene is interrupted by six introns and the 3' untranslated sequence of 485 nucleotides is not interrupted [Li, Tiano, Fukasawa, Yagi, Shimiza, Sharief, Nakashima & Pan (1985) Eur. J. Biochem. 149, 215-225]. An additional intron of 1653 base-pairs was found in the 5' untranslated sequence of 101 nucleotides at 24 nucleotides upstream to the translation start site. Thus, mouse LDH-A gene containing seven introns spans approx. 11 kilobases and its length of mature mRNA is 1582 nucleotides, excluding the poly(A) tail.
Assuntos
Genes Reguladores , L-Lactato Desidrogenase/genética , Animais , Sequência de Bases , Sítios de Ligação , AMP Cíclico/genética , Isoenzimas , Camundongos , Camundongos Endogâmicos C57BL , Biossíntese de Proteínas , Transcrição GênicaRESUMO
The complete covalent structure of the 331 amino acids of mouse lactate dehydrogenase (LDH) A4 isozyme has been determined by sequence analyses of both the protein and the genomic DNA. The mouse LDH-A gene spans a length of at least 7000 bases from the translation initiation codon ATG to the end of the 3' untranslated region, and it contains six introns that interrupt the protein-coding sequence. The relationships between the exon-intron organization of LDH-A gene and the structural-functional domains of the protein are discussed.
Assuntos
Genes , L-Lactato Desidrogenase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Isoenzimas , Camundongos , Camundongos Endogâmicos DBA , Fragmentos de Peptídeos/análise , Biossíntese de Proteínas , Conformação Proteica , Especificidade da Espécie , SuínosRESUMO
Inactivation of pig kidney dipeptidyl peptidase IV (EC 3.4.14.5) by photosensitization in the presence of methylene blue at pH 7.5 was observed to have pseudo-first-order kinetics. During the process, until over 95% inactivation was achieved, the histidine and tryptophan residues were decreased from 14.0 to 2.7 and 12.6 to 7.1, respectively, per 94,000-Da subunit, without any detectable changes in other photosensitive amino acids. Modification of four histidine residues per subunit using diethylpyrocarbonate resulted in only 30% inactivation of the enzyme, while N-bromosuccinimide almost completely inactivated the enzyme with the modification of only one tryptophan residue per subunit, as determined by absorption spectrophotometry at 280 nm. The protective action of the substrate and inhibitors such as Ala-Pro-Ala and Pro-Pro against the modification of tryptophan residues with N-bromosuccinimide was observed both fluorometrically and by measurement of activity. On the basis of these results it is suggested that one of the tryptophan residues in the enzyme subunit is essential for the functioning of the substrate binding site of pig kidney dipeptidyl peptidase IV.
Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/antagonistas & inibidores , Histidina , Inibidores de Proteases , Triptofano , Animais , Sítios de Ligação , Bromosuccinimida/farmacologia , Dietil Pirocarbonato/farmacologia , Dipeptidil Peptidase 4 , Dipeptidil Peptidases e Tripeptidil Peptidases/efeitos da radiação , Histidina/efeitos da radiação , Cinética , Luz , Fotoquímica , Espectrometria de Fluorescência , Espectrofotometria , Suínos , Triptofano/efeitos da radiaçãoRESUMO
Specific antibody against purified alkaline phosphatase from bovine dental pulp was raised in a rabbit. Immunological analyses by Ouchterlony double-gel diffusion and quantitative immunoprecipitation showed that there was neither a detectable antigenic difference between liver and pulp alkaline phosphatases nor a detectable cross-reaction with the intestinal enzyme.
Assuntos
Fosfatase Alcalina/imunologia , Polpa Dentária/enzimologia , Fígado/enzimologia , Fosfatase Alcalina/metabolismo , Animais , Bovinos , Precipitação Química , Imunodifusão , Intestinos/enzimologia , Pirofosfatases/metabolismo , Coelhos , Especificidade da EspécieRESUMO
Soluble dipeptidyl peptidase IV (EC 3.4.14.5) was purified from the 100,000 X g supernatant fraction of pig liver homogenate. The purified enzyme had the same properties as, and immunological identity with, the membrane-bound enzyme which was described previously. However, the purified enzyme had a pattern of molecular heterogeneity different from the membrane-bound enzyme; this was shown by isoelectric focusing. Carbohydrate analysis revealed that the soluble enzyme contained glucose, which is not found in the membrane-bound one, and less fucose, mannose, and sialic acid than the latter. From these results, we conclude that the soluble form of dipeptidyl peptidase IV in pig liver is closely related to the membrane-bound enzyme, but is not simply a proteolytically solubilized product of it.