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1.
cDNA cloning and amino acid sequence of bovine brain 2',3'-cyclic-nucleotide 3'-phosphodiesterase 总被引:4,自引:0,他引:4
2',3'-Cyclic-nucleotide 3'-phosphodiesterase (EC 3.1.4.37) has been widely used as a marker for myelin-oligodendrocytes in the central nervous system. Evidence has been provided that the enzyme is identical with one of the Wolfgram proteins of central nervous system myelin. The amino acid sequence of bovine 2',3'-cyclic-nucleotide 3'-phosphodiesterase was determined by both protein and cDNA sequence analyses. Protein sequence analysis was done on bovine elastase 2',3'-cyclic-nucleotide 3'-phosphodiesterase, a low molecular weight enzyme obtained by solubilization with pancreatic elastase (EC 3.4.21.36) (Nishizawa, Y., Kurihara, T., and Takahashi, Y. (1980) Biochem. J. 191, 71-82; Kurihara, T., Nishizawa, Y., Takahashi, Y., and Odani, S. (1981) Biochem. J. 195, 153-157). Based on the carboxyl-terminal sequence of bovine elastase 2',3'-cyclic-nucleotide 3'-phosphodiesterase, synthetic oligodeoxyribonucleotides were prepared and used as probes for screening a cDNA library of bovine brain. A cDNA of 2305 base pairs was obtained and sequenced, and the complete amino acid sequence of bovine 2',3'-cyclic-nucleotide 3'-phosphodiesterase was deduced. Bovine 2',3'-cyclic-nucleotide 3'-phosphodiesterase deduced contains 400 amino acids including initiation methionine and has a molecular weight of 44,850. Bovine elastase 2',3'-cyclic-nucleotide 3'-phosphodiesterase corresponds to the 236 amino acids of bovine 2',3'-cyclic-nucleotide 3'-phosphodiesterase. RNA blot analysis revealed a single-species mRNA of about 2600 bases. 相似文献
2.
Sakamoto Y Tanaka N Ichimiya T Kurihara T Nakamura KT 《Journal of molecular biology》2005,346(3):789-800
2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNP), a member of the 2H phosphoesterase superfamily, is firmly bound to brain white matter and found mainly in the central nervous system of vertebrates, and it catalyzes the hydrolysis of 2',3'-cyclic nucleotide to produce 2'-nucleotide. Recent studies on CNP-knockout mice have revealed that the absence of CNP causes axonal swelling and neuronal degeneration. Here, the crystal structure of the catalytic fragment (CF) of human CNP (hCNP-CF) is solved at 1.8A resolution. It is an alpha+beta type structure consisting of three alpha-helices and nine beta-strands. The structural core of the molecule is comprised of two topologically equivalent three-stranded antiparallel beta-sheets that are related by a pseudo 2-fold symmetry. Each beta-sheet contains an H-X-T-X motif, which is strictly conserved among members of the 2H phosphoesterase superfamily. The phosphate ion is bound to the side-chains of His and Thr from each of the two motifs. Structural comparison of hCNP-CF with plant 1',2'-cyclic nucleotide phosphodiesterase (CPDase) and bacterial 2'-5' RNA ligase reveals that the H-X-T-X motifs are structurally conserved among these enzymes, but the surface properties of the active site are quite different among the enzymes, reflecting the differences in their substrates. On the basis of the present crystal structure of the hCNP-CF/phosphate complex, the available structure of the CPDase/cyclic-nucleotide analogue complex, and the recent functional studies of rat CNP-CF, we propose a possible substrate-binding mode and catalytic mechanism of CNP, which employs the nucleophilic water molecule activated by His310. The proposed mechanism is basically equivalent to the second step of the well-accepted reaction mechanism of RNase A. Since the overall structure of hCNP-CF differs considerably from that of RNase A, it is likely that the similar active sites with two catalytic histidine residues in these enzymes arose through convergent evolution. 相似文献
3.
S Sato N Fujita T Kurihara R Kuwano K Sakimura Y Takahashi T Miyatake 《Biochemical and biophysical research communications》1989,163(3):1473-1480
cDNA clones of human myelin-associated glycoprotein were isolated and analyzed. The combination of the two overlapping cDNA clones covered the full coding region and the complete amino acid sequence was deduced. In rat and mouse, expression of the two forms of mRNA is developmentally regulated; the mRNA without exon 12 portion is expressed mainly in the actively myelinating stage of development. Although the cDNA library used here was prepared from adult human brain poly(A)+ RNA, all five clones obtained corresponded to the mRNA without exon 12 portion. 相似文献
4.
Ballestero RP Dybowski JA Levy G Agranoff BW Uhler MD 《Journal of neurochemistry》1999,72(4):1362-1371
We previously reported cloning of cDNAs encoding both components of a protein doublet induced during goldfish optic nerve regeneration. The predicted protein sequences showed significant homology with the mammalian 2',3'-cyclic-nucleotide 3'-phosphodiesterases (CNPases). CNPases are well-established markers of mammalian myelin; hence, the cDNAs were designated gRICH68 and gRICH70 (for goldfish Regeneration-Induced CNPase Homologues of 68 and 70 kDa). Homologous cDNAs have now been isolated from zebrafish encoding a highly related protein, which we have termed zRICH. RNase protection assays show that zRICH mRNA is induced significantly (fivefold) in optic nerve regenerating zebrafish retinas 7 days following nerve crush. Western blots show a single band in zebrafish brain and retina extracts, with immunoreactivity increasing three-fold in regenerating retinas 21 days postcrush. Immunohistochemical analysis indicated that this increase in zRICH protein expression is localized to the retinal ganglion cell layer in regenerating retina. We have characterized and evaluated the relevance of a conserved beta-ketoacyl synthase motif in zRICH to CNPase activity by means of site-directed mutagenesis. Two residues within the motif, H334 and T336, are critical for enzymatic activity. A cysteine residue within the motif, which corresponds to a critical residue for beta-ketoacyl synthase, does not appear to participate in the phosphodiesterase activity. 相似文献
5.
Molecular cloning of the myelin specific enzyme 2',3'-cyclic-nucleotide 3'-phosphohydrolase 总被引:2,自引:0,他引:2
We describe the isolation of cDNA clones for bovine brain 2',3'-cyclic-nucleotide 3'-phosphohydrolase (CNPase, EC 3.1.4.37), the third most abundant protein in central nervous system myelin. The cDNA encodes the complete protein (400 amino acids) and hybridizes to a major size species of mRNA in bovine brain tissue, approx. 2.7 kb in size. CNPase mRNA levels do not appear to be affected in quaking dysmyelinating mutant mice. The sequence reveals probable sites for CNPase phosphorylation by cAMP-dependent protein kinase and a region of homology with haemocyanin. 相似文献
6.
Lovato L Cianti R Gini B Marconi S Bianchi L Armini A Anghileri E Locatelli F Paoletti F Franciotta D Bini L Bonetti B 《Molecular & cellular proteomics : MCP》2008,7(12):2337-2349
The presence of autoantibodies in multiple sclerosis (MuS) is well known, but their target antigens have not been clearly identified. In the present study, IgG autoreactivity to neural antigens of normal human white matter separated by bidimensional electrophoresis was assessed in serum and cerebrospinal fluid of 18 MuS and 20 control patients. Broad IgG autoreactivity was detected by two-dimensional immunoblotting in all cases to neural antigens, most of which were identified by mass spectrometry. The comparative analysis of MuS and non-MuS reactive spots showed that a restricted number of neural protein isoforms were specifically recognized by MuS IgG. Almost all MuS patients had cerebrospinal fluid IgG directed to isoforms of one of the oligodendroglial molecules, transketolase, 2',3'-cyclic-nucleotide 3'-phosphodiesterase type I, collapsin response mediator protein 2, and tubulin beta 4. Interestingly 50% of MuS IgG recognized transketolase, which was mostly localized on oligodendrocytes in human white matter from normal and MuS samples. IgG autoreactivity to cytoskeletal proteins (radixin, sirtuin 2, and actin-interacting protein 1) was prevalent in secondary progressive MuS patients. Among the proteins recognized by serum IgG, almost all MuS patients specifically recognized a restricted number of neuronal/cytoskeletal proteins, whereas 2',3'-cyclic-nucleotide 3'-phosphodiesterase type I was the oligodendroglial antigen most frequently recognized (44%) by MuS seric IgG. Our immunomics approach shed new light on the autoimmune repertoire present in MuS patients revealing novel oligodendroglial and/or neuronal putative autoantigens with potential important pathogenic and diagnostic implications. 相似文献
7.
Complete amino acid sequence of canine cardiac calsequestrin deduced by cDNA cloning 总被引:19,自引:0,他引:19
B T Scott H K Simmerman J H Collins B Nadal-Ginard L R Jones 《The Journal of biological chemistry》1988,263(18):8958-8964
cDNA cloning was used to deduce the complete amino acid sequence of canine cardiac calsequestrin, the principal Ca2+-binding protein of cardiac junctional sarcoplasmic reticulum. Cardiac calsequestrin contains 391 amino acid residues plus a 19-residue amino-terminal signal sequence. The molecular weight of the mature protein, excluding carbohydrate, is 45,269. Cardiac calsequestrin is highly acidic, and a striking feature is the enrichment of acidic residues (60%) within the 63 carboxyl-terminal residues. No part of the sequence contains EF hand Ca2+-binding structures. The photo-affinity probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine was used to localize the Ca2+-regulated hydrophobic site to amino acid residues 192-223. The cardiac and skeletal muscle isoforms of calsequestrin (Fliegel, L., Ohnishi, M., Carpenter, M. R., Khanna, V. K., Reithmeier, R. A. F., and MacLennan, D. H. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1167-1171), although the products of different genes, are 65% identical, are acidic, and share one glycosylation site. However, cardiac calsequestrin has several unique features. First, it has a 31-amino acid extension at its carboxyl terminus (residues 361-391), which contains 71% acidic residues and a second glycosylation site. Second, its mRNA contains a second open reading frame with the capacity to code for a 111-amino acid protein. Third, contrary to the restricted expression of the fast skeletal isoform, cardiac calsequestrin mRNA is present in both cardiac and slow skeletal muscle, but not in fast skeletal muscle. We conclude that the deduced amino acid sequence of cardiac calsequestrin is consistent with its ability to bind large amounts of Ca2+ (40 mol of Ca2+/mol of calsequestrin). The protein probably binds Ca2+ by acting as a charged surface rather than by presenting multiple discrete Ca2+-binding sites. 相似文献
8.
The three major monomer hemoglobins from Glycera dibranchiata erythrocytes isolated in this laboratory were sequenced from their N-termini. A stretch of amino acid sequence identity was used to determine the sequence of a mixed oligodeoxynucleotide that would be complementary to all 12 possible mRNA sequences coding for the amino acids. A cDNA library was constructed by using poly(A+) RNA from G. dibranchiata erythrocytes, the library was probed with the oligonucleotide, and the longest positive inserts found were subcloned into a sequencing plasmid and then sequenced. The first one was 745 bases long, containing 85 bases of 5'-untranslated RNA, an open reading frame of 444 bases coding for 148 amino acids, and a 3'-untranslated region of 216 bases. The predicted amino acid sequence matches the first 25 amino acids of G. dibranchiata monomer globin component IV. The sequence contains an N-terminal methionine plus 18 other mostly conservative sequence changes compared to the published sequence of Imamura et al. (1972), which appears from our partial sequencing to be monomer globin component II. We confirm the presence of leucine in the E7 position, which is histidine in most myoglobins and hemoglobins. 相似文献
9.
Plasma cell membrane glycoprotein PC-1. cDNA cloning of the human molecule, amino acid sequence, and chromosomal location 总被引:10,自引:0,他引:10
M F Buckley K A Loveland W J McKinstry O M Garson J W Goding 《The Journal of biological chemistry》1990,265(29):17506-17511
The murine cell membrane glycoprotein PC-1 is a homodimer with restricted tissue distribution, being first characterized in plasma cells. We now describe the isolation of cDNA clones encoding the human homolog of the murine PC-1 protein, its complete amino acid sequence, and its chromosomal location. Overall, the amino acid sequence of the human protein is about 80% identical to the murine protein, although the extent of homology varies in different domains. It had not been possible to assign a definitive amino terminus to the murine protein. Comparison of the murine and human sequence necessitates reassignment of the amino terminus, resulting in a cytoplasmic tail of 24 amino acids rather than 58 amino acids as previously published for the mouse. The sequence of several independently obtained cDNA clones indicates that the 3' end of the mRNA is subject to alternative splicing. Southern blots suggest a single copy gene. In situ chromosomal hybridization localizes the gene for human PC-1 to chromosome 6q22-q23, a common site for deletions in human lymphoid neoplasia. 相似文献
10.
Simon W. Law Glenn Gray H.Bryan Brewer 《Biochemical and biophysical research communications》1983,112(1):257-264
ds-cDNA to human liver apoA-I mRNA has been cloned. Nucleic acid sequence analysis revealed that apoA-I mRNA codes for a precursor apolipoprotein, preproapoA-I, which contains 24 amino acids on the NH2-terminal end of the mature plasma apoA-I. Eighteen amino acids are contained within the hydrophobic prepeptide (Met-Lys-Ala-Ala-Val-Leu-Thr-Leu-Ala-Val-Leu-Phe-Leu-Thr-Gly-Ser-Gln-Ala) followed by a 6 amino acid propeptide (Arg-His-Phe-Top-Gln-Gln). Our results on human apoA-I are in agreement with the partial sequence of the precursor of rat apoA-I with respect to the length of the precursor sequence, location of the prepeptide, and the presence of an unusual propeptide sequence terminating in a neutral dipeptide Gln-Gln. A detailed analysis of the primary structure of normal human apoA-I mRNA will be essential to our ultimate understanding of the processing of human apoA-I and diseases characterized by molecular defects in apoA-I structure and function. 相似文献
11.
Fernández-Centeno E Heredia CF 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2000,125(2):161-167
We describe the properties of a 2',3'-cyclic nucleotide 2'-phosphodiesterase (EC 3.1.4.16), found in Fusarium culmorum, which hydrolyzes nucleoside 2',3'-cyclic monophosphates to nucleoside 3'-phosphates. In contrast with a similar enzyme found in bacteria, the Fusarium enzyme does not exhibit nucleotidase activity and does not show a requirement for metal ions, but is inhibited by micromolar concentrations of Cu++ and Zn++, and is very stable to heat. This cyclic phosphodiesterase hydrolyzes the four major nucleoside 2',3'-cyclic monophosphates and has greater affinity for purine (Kms for Ado-2',3'-P = 0.3 mM and for Guo-2',3'-P = 0.1 mM) than for pyrimidine nucleotides (Kms for Cyd-2',3'-P = 0.6 mM and for Urd-2',3'-P = 2 mM). The respective Vmax for Urd-2',3'-P; Cyd-2',3'-P; Ado-2',3'-P; and Guo-2',3' are 100:45:16:5. The efficacy of the phosphodiesterase to hydrolyze the four major 2',3' cyclic nucleotides (based on the relative values of Vmax/Km) is not significantly different. The Fusarium enzyme differs from a previously described 2',3' cyclic phosphodiesterase from Neurospora, in that it is inactive on 3',5'-nucleoside monophosphates and nucleoside 2' or 3' phosphates. 相似文献
12.
Molecular cloning of cDNA for human complement component C1s. The complete amino acid sequence 总被引:5,自引:0,他引:5
C M Mackinnon P E Carter S J Smyth B Dunbar J E Fothergill 《European journal of biochemistry》1987,169(3):547-553
The complete amino acid sequence (673 residues plus 15 residues of leader sequence) of human complement component C1s has been determined by nucleotide sequencing of cDNA clones from a human liver library probed with synthetic oligonucleotides. Much of the sequence is supported by independent amino acid sequence information. The cDNA sequence contains an anomalous "intron-like" sequence, including a stop codon, that can be discounted because of the amino acid sequence evidence. The N-terminal chain (422 residues) of C1s, like that of C1r with which it is broadly homologous, contains five domains: domains I and III are homologous to one another and to similar regions in C1r, domain II is homologous to the epidermal growth factor sequence found in C1r and several other proteins, and domains IV and V are homologous to one another and to the 60-residue repeating sequence found in C1r, C2, factor B, C4-binding protein and some apparently unrelated proteins. The sequence of the C-terminal chain (251 residues) agrees with that already established to be the "serine protease" domain of C1s. 相似文献
13.
Molecular cloning and sequencing of the human erythrocyte 2,3-bisphosphoglycerate mutase cDNA: revised amino acid sequence. 总被引:7,自引:0,他引:7
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V Joulin J Peduzzi P H Roméo R Rosa C Valentin A Dubart B Lapeyre Y Blouquit M C Garel M Goossens et al. 《The EMBO journal》1986,5(9):2275-2283
The human erythrocyte 2,3-bisphosphoglycerate mutase (BPGM) is a multifunctional enzyme which controls the metabolism of 2,3-diphosphoglycerate, the main allosteric effector of haemoglobin. Several cDNA banks were constructed from reticulocyte mRNA, either by conventional cloning methods in pBR322 and screening with specific mixed oligonucleotide probes, or in the expression vector lambda gt 11. The largest cDNA isolated contained 1673 bases [plus the poly(A) tail], which is slightly smaller than the size of the intact mRNA as estimated by Northern blot analysis (approximately 1800 bases). This cDNA encodes for a protein of 258 residues; the protein yielded 34 tryptic peptides which were subsequently isolated by h.p.l.c. Our nucleotide sequence data were entirely confirmed by the amino acid composition of these tryptic peptides and reveal several major differences from the published sequence; the revised amino acid sequence of human BPGM is presented. These findings represent the first step in the study of the expression and regulation of this enzyme as a specific marker of the erythroid cell line. 相似文献
14.
The diastereomers of adenosine and uridine 2',3'-cyclic phosphorothioates were tested as substrates for 2',3'-cyclic nucleotide 3'-phosphodiesterase from bovine brain. The enzyme cleaves the Sp (or exo) diastereomers efficiently, whereas the Rp (or endo) diastereomers are resistant to hydrolysis, even after long incubation. As the enzyme exhibits strong substrate inhibition the precise determination of kinetic parameters posed problems, particularly with phosphorothioates. The stereoselectivity of this enzyme is opposite to that of RNase T1 and RNase A and thus could be a useful complement in determination of the configuration of nucleoside 2',3'-cyclic phosphorothioates resulting from hydrolysis reactions of unknown stereochemical course. 相似文献
15.
A cDNA clone has been isolated from a pEX expression library that encodes alpha 1 acid glycoprotein. We present the complete nucleotide sequence encoding this protein and compare the derived amino acid sequence with pre-existing data. 相似文献
16.
Molecular cloning and sequence analysis of cDNA encoding human prostatic acid phosphatase 总被引:6,自引:0,他引:6
lambda gt11 clones encoding human prostatic acid phosphatase (PAP) (EC 3.1.3.2) were isolated from human prostatic cDNA libraries by immunoscreening with polyclonal antisera. Sequence data obtained from several overlapping clones indicated that the composite cDNAs contained the complete coding region for PAP, which encodes a 354-residue protein with a calculated molecular mass of 41,126 Da. In the 5'-end, the cDNA codes for a signal peptide of 32 amino acids. Direct protein sequencing of the amino-terminus of the mature protein and its proteolytic fragments confirmed the identity of the predicted protein sequence. PAP has no apparent sequence homology to other known proteins. However, both the cDNA clones coding for human placental alkaline phosphatase and PAP have an alu-type repetitive sequence about 900 nucleotides downstream from the coding region in the 3'-untranslated region. Two of our cDNA clones differed from others at the 5'-ends. RNA blot analysis indicated mRNA of 3.3 kb. We are continuing to study whether acid phosphatases form a gene family as do alkaline phosphatases. 相似文献
17.
18.
The ST2 gene is a member of the IL-1 receptor family and is hypothesized to be involved in helper T cell function, but its functional ligand and physiological role remain unknown. We have cloned the human ST2L cDNA that encodes a distinct type of membrane-bound ST2 protein. The predicted 556-amino-acid sequence showed 67% identity to the mouse ST2L protein. The human ST2 gene (IL1RL1) contains 13 exons and spans 40 kb in length. Its exon-intron organization was elucidated from a registered human genomic sequence derived from chromosome 2q, which contains three other genes belonging to the IL-1 receptor family in an approximately 202-kb genomic region. The tissue distribution of ST2 expression was examined by RT-PCR, and the soluble form (ST2, IL1RL1-a) and ST2L (IL1RL1-b) appear to be expressed differentially. We also established stable transfectants of a human glioblastoma cell line, T98G, that express human ST2L constitutively, and we confirmed cell-surface expression of human ST2L protein on the transfectants. 相似文献
19.
2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP) activity was studied in human peripheral blood lymphocytes. Incubation of human lymphocytes with optimum mitogenic concentrations of Robinia pseudoacacia lectin produced an increase in CNP activity. This increase was not detected before 24 h of incubation. Furthermore, whereas unfractionated lymphocytes exhibited activation of CNP, this was not observed in B and T cell subpopulations. 相似文献
20.
Purification and characterization of wheat germ 2',3'-cyclic nucleotide 3'-phosphodiesterase 总被引:1,自引:0,他引:1
The 2',3'-cyclic nucleotide 3'-phosphodiesterase which hydrolyzes nucleoside 2',3'-cyclic phosphates (N greater than p) to nucleoside 2'-phosphates has been purified 16,000-fold to near homogeneity from wheat germ. The purified enzyme is a single polypeptide with a molecular weight of 23,000-24,000. It has a pH optimum of 7.0. The apparent Km values for A greater than p, G greater than p, C greater than p, and U greater than p are 13.1, 9.2, 25.2, and 25.3 mM, respectively. Vmax values for A greater than p, G greater than p, C greater than p, and U greater than p are 2090, 280, 2140, and 600 mumol/min/mg of purified protein, respectively. Wheat germ 2',3'-cyclic nucleotide 3'-phosphodiesterase does not hydrolyze 2',3'-cyclic esters in cyclic phosphate-terminated oligoribonucleotides or in nucleoside 5'-phosphate, 2',3'-cyclic phosphate (pN greater than p). This is in contrast to the 3'-phosphodiesterase activity associated with a wheat germ RNA ligase which hydrolyzes cyclic phosphate-terminated oligonucleotides and pN greater than p substrates much more efficiently than nucleoside 2',3'-cyclic phosphates. The enzyme characterized in this work appears to be the only known 2',3'-cyclic nucleotide 3'-phosphodiesterase specific for 2',3'-cyclic mononucleotides. 相似文献