共查询到20条相似文献,搜索用时 31 毫秒
1.
Yubisui T Takabayashi T Takahashi F Fujiwara S Kawamura K 《Journal of biochemistry》2004,135(2):231-236
A cDNA clone for cytochrome b(5) was isolated from a cDNA library of an ascidian, Ciona savignyi, by a plaque hybridization method using a digoxigenin-labeled cDNA for the soluble form of human cytochrome b(5). The cDNA is composed of 5'- and 3'-noncoding sequences, and a 396-base pair coding sequence. The 3'-noncoding sequence contains polyadenylation signal sequences. The amino acid sequence of 132 residues deduced from the nucleotide sequence of the cDNA showed 61% identity and 82% similarity to the cytochrome b(5) of another ascidian species, Polyandrocarpa misakiensis, which we previously cloned. The amino-terminal hydrophilic domain of 98 residues contains well-conserved structures around two histidine residues for heme binding. A cDNA expression system was constructed to prepare a putative soluble form of Ciona cytochrome b(5). The recombinant soluble cytochrome b(5) showed an asymmetrical absorption spectrum at 560 nm as is shown by mammalian cytochromes b(5) upon reduction with NADH and NADH-cytochrome b(5) reductase. The recombinant Ciona cytochrome b(5) is reduced by NADH-cytochrome b(5) reductase with an apparent K(m) value of 3.3 microM. This value is similar to that of the cytochrome b(5) of Polyandrocarpa misakiensis. The expression of Ciona cytochrome b(5) mRNA during development was examined by an in situ hybridization method and ubiquitous expression in embryonic tissues was observed. The results indicate that cytochrome b(5) plays important roles in various metabolic processes during development. 相似文献
2.
Ikegami T Kameyama E Yamamoto SY Minami Y Yubisui T 《Bioscience, biotechnology, and biochemistry》2007,71(3):783-790
A cDNA for NADH-cytochrome b(5) reductase of Physarum polycephalum was cloned from a cDNA library, and the nucleotide sequence of the cDNA was determined (accession no. AB259870). The DNA of 943 base pairs contains 5'- and 3'-noncoding sequences, including a polyadenylation sequence, and a coding sequence of 843 base pairs. The amino acid sequence (281 residues) deduced from the nucleotide sequence was 25 residues shorter than those of vertebrate enzymes. Nevertheless, the recombinant Physarum enzyme showed enzyme activity comparable to that of the human enzyme. The recombinant Physarum enzyme showed a pH optimum of around 6.0, and apparent K(m) values of 2 microM and 14 microM for NADH and cytochrome b(5) respectively. The purified recombinant enzyme showed a typical FAD-derived absorption peak of cytochrome b(5) reductase at around 460 nm, with a shoulder at 480 nm. These results suggest that the Physarum enzyme plays an important role in the organism. 相似文献
3.
Information on the amino acid sequences of the internal peptide fragments of cytochrome b5 from Mortierella hygrophila was used to prepare synthetic oligonucleotides as primers for the polymerase chain reaction. A 100-base DNA fragment was thus amplified, by using a genomic gene from Mortierella alpina 1S-4 as a template, which produced polyunsaturated fatty acids such as arachidonic acid. The amplified DNA fragment was used as the probe to clone both a 523-base cDNA fragment and a 2.1-kilobase SalI-NruI genomic fragment coding for the whole M. alpina 1S-4 cytochrome b5. On the basis of nucleotide sequences of both cytochrome b5 genomic gene and cDNA, the genomic cytochrome b5 gene was found to consist of four exons and three introns. A novel type of RNA editing, in which the cDNA included either guanine insertion or adenine-->guanine substitution at one base upstream of poly(A), was interestingly observed. The deduced amino acid sequence of M. alpina 1S-4 cytochrome b5 showed significant similarities with those of cytochrome b5s from other organisms such as rat, chicken, and yeast. The soluble form of the cytochrome b5 gene was expressed to 16% of the total soluble protein in Escherichia coli. The holo-cytochrome b5 accounted for 8% of the total cytochrome b5 in the transformants. The purified cytochrome b5 showed the oxidized and reduced absorbance spectra characteristic of fungal microsomal cytochrome b5. 相似文献
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6.
Soluble and membrane-bound forms of cytochrome b5 are the products of a single gene in chicken 总被引:1,自引:0,他引:1
In order to determine the relationship of the soluble cytochrome b5 found in erythrocytes to the membrane-bound form found in other tissues, a cDNA clone encoding cytochrome b5 in chicken erythrocytes was isolated by using mixed oligonucleotides based on a partial amino acid sequence of the protein. Complete nucleotide sequence identity between the erythrocyte cDNA and the sequence of a cDNA clone of the liver protein suggests that they are transcribed from the same gene. The isolation and structural analysis of genomic clones was also consistent with the presence of only one cytochrome b5 gene in chicken. These results suggest that the formation of soluble erythrocyte cytochrome b5 occurs by proteolytic processing of the membrane-bound form. Thus, previous reports indicating that the carboxyl terminal amino acid residue of the erythrocyte form differs from the corresponding residue of the membrane-bound form may suggest the existence of a novel post-translational modification. 相似文献
7.
S Ishihara K Morohashi H Sadano S Kawabata O Gotoh T Omura 《Journal of biochemistry》1990,108(6):899-902
cDNA clones coding for hemoprotein H-450 were isolated from a rat liver cDNA library using anti-H-450 antibody. The molecular weight calculated from the deduced amino acid sequence comprising 547 amino acid residues was 60,085. The N-terminal sequence and a partial internal amino acid sequence of purified H-450, which were determined chemically, were both found in the amino acid sequence of H-450 deduced from the nucleotide sequence. H-450 mRNA is expressed in liver, kidney, and brain. A homology search of amino acid sequences indicated that H-450 shows no homology with cytochrome P-450, but shows significant homology with bacterial O-acetylserine (thiol)-lyases. However, H-450 has no O-acetylserine (thiol)-lyase activity. 相似文献
8.
H Takematsu Y Kozutsumi A Suzuki T Kawasaki 《Biochemical and biophysical research communications》1992,185(3):845-851
The rabbit genomic segments for the soluble cytochrome b5 (b5) and microsomal b5 were amplified and isolated, respectively, by means of the polymerase chain reaction using primers corresponding to various portions of the open reading frame of microsomal b5 cDNA. The DNA sequence analysis revealed that the soluble b5 gene has an extra 24 nucleotide long insert which encodes a C-terminal amino acid and a termination codon which are specific to the soluble b5. Except for the insert, the sequences of the soluble and microsomal b5 genes are identical with each other from the 5' end to the 3' end of the open reading frame of the microsomal b5 cDNA. Comparison of the genomic sequences with the cDNA sequences suggested that the soluble and microsomal genes are intronless within their open reading frames. These data indicate that rabbit soluble and microsomal b5 mRNAs are encoded by two highly conserved but separate genes. 相似文献
9.
Human cDNAs coding for angiogenin, a human tumor derived angiogenesis factor, were isolated from a cDNA library prepared from human liver poly(A) mRNA employing a synthetic oligonucleotide as a hybridization probe. The largest cDNA insert (697 base pairs) contained a short 5'-noncoding sequence followed by a sequence coding for a signal peptide of 24 (or 22) amino acids, 369 nucleotides coding for the mature protein of 123 amino acids, a stop codon, a 3'-noncoding sequence of 175 nucleotides, and a poly(A) tail. The gene coding for human angiogenin was then isolated from a genomic lambda Charon 4A bacteriophage library employing the cDNA as a probe. The nucleotide sequence of the gene and the adjacent 5'- and 3'-flanking regions (4688 base pairs) was then determined. The coding and 3'-noncoding regions of the gene for human angiogenin were found to be free of introns, and the DNA sequence for the gene agreed well with that of the cDNA. The gene contained a potential TATA box in the 5' end in addition to two Alu repetitive sequences immediately flanking the 5' and 3' ends of the gene. The third Alu sequence was also found about 500 nucleotides downstream from the Alu sequence at the 3' end of the gene. The amino acid sequence of human angiogenin as predicted from the gene sequence was in complete agreement with that determined by amino acid sequence analysis. It is about 35% homologous with human pancreatic ribonuclease, and the amino acid residues that are essential for the activity of ribonuclease are also conserved in angiogenin. This provocative finding is thought to have important physiological implications. 相似文献
10.
We have isolated from rabbit liver three cDNA clones of 1400-1800 base pairs that hybridize selectively to RNA from animals treated with phenobarbital. The nucleotide sequences of the cDNAs have been determined. In the protein coding region the nucleotide sequences of two of the cDNAs are 88% homologous, and the third cDNA is about 72-74% homologous to the other two. All three are 55-60% homologous to rat liver cytochrome P-450b cDNA. The amino acid sequences derived from the cDNA sequences are about 50% homologous to those of rat liver cytochrome P-450b and rabbit liver cytochrome P-450 (form 2). The degree of homology differs substantially in different regions of the protein. The hydrophobicity profiles of these five mammalian cytochromes P-450 are very similar and contain up to eight regions of hydrophobicity that are long enough to span a membrane. These results indicate that these three cDNAs code for rabbit liver cytochromes P-450 which are different from any rabbit liver cytochrome P-450 for which amino acid sequence information is published. These cDNAs are part of a family of genes that are related to rabbit liver cytochrome P-450 (form 2) and rat liver cytochrome P-450b which are the major phenobarbital-inducible forms. The divergence of amino acid sequence between the rat and rabbit forms and the divergence of nucleotide sequences of silent sites in the two most closely related rabbit forms suggest that cytochromes P-450 have a relatively high rate of amino acid divergence compared to many other vertebrate proteins. 相似文献
11.
The isolation and characterization of a human apolipoprotein B 100-specific cDNA clone (lambda gt-B1) containing a 1321 base pairs (bp) spanning insert is described. It encodes the 3'-nontranslated 281 bp long region up to the polyadenylation site and 1040 bp of the C-terminal coding region of 345 amino-acid residues of human apo B 100 and the stop codon. The lambda gt-B1 cDNA clone has been isolated from a human hepatoma cDNA expression library by immunoscreening using affinity-purified polyclonal anti apo B 100 antibodies. The nucleotide sequence of the apo B 100 insert has been determined. A part of the polypeptide sequence derived from this nucleotide sequence was identical with the amino-acid sequence obtained by protein sequencing of a purified cyanogen bromide fragment of apo B 100. The fusion protein consisting of beta-galactosidase and the 345 amino-acid residue long C-terminus of apo B 100 had an apparent molecular mass of 148 kDa in NaDodSO4 polyacrylamide gel electrophoresis. In Northern blot hybridization analysis the insert of the apo B 100-cDNA clone hybridized to a 20 to 22 kb mRNA from adult human liver. 相似文献
12.
Total mRNA was isolated from the pituitary glands of bullfrog (Rana catesbeiana), purified by affinity chromatography with oligo(dT)-cellulose columns. The cDNA was synthesized and cloned in Escherichia coli using EcoRI linkers and pBR322 as vector. The cDNA library was screened by hybridization with 32P-labeled duck growth hormone (GH) cDNA. A positive clone was selected and sequenced. The full-length bullfrog GH cDNA contains 950 nucleotide pairs with an open reading frame coding for the precursor GH of 215 amino-acid residues. The partial amino-acid sequence from the protein confirms that derived from the cDNA, with Phe as the first residue in the mature bullfrog GH preceded by a 25-residue hydrophobic signal peptide. The bullfrog GH shares sequence homology with those of other vertebrate species in the following order: duck (61% protein sequence homology; 67% cDNA homology), rat (56%; 61%), human (47%; 57%) and salmon (42%; 50%). 相似文献
13.
Nucleotide sequences of the cDNA and an intronless pseudogene for human lactate dehydrogenase-A isozyme 总被引:9,自引:0,他引:9
Eight cDNA clones for lactate dehydrogenase-A isozyme (LDH-A) were isolated from a human fibroblast cDNA library, characterized, and no sequence heterogeneity was found. Four cDNA clones appear to contain nearly full-length cDNA inserts and the complete nucleotide sequence of 1710 base pairs consists of the protein-coding sequence (999 base pairs), the 5' (97 base pairs) and 3' (565 base pairs) untranslated regions and poly(dA) tail (49 base pairs). The predicted amino acid sequence of the human LDH-A polypeptide shows 92% homology (27 differences out of 331 amino acids compared) with that of the pig LDH-A subunit determined by direct protein sequencing [Kiltz et al. (1977) Hoppe-Seyler's Z. Physiol. Chem. 358, 123-127]. Human genomic clones containing an LDH-A pseudogene were isolated and the nucleotide sequence of 1635 base pairs from an intronless pseudogene was determined. The presence of two termination codons, two deletions of three nucleotides each and the replacement of three arginine residues at the active site (nos 98, 105 and 168) by other amino acids renders its coding region incapable of producing a functional LDH-A protein. A comparison between human LDH-A cDNA and the pseudogene sequences reveals 12.9% differences (114 transitions, 65 transversions and 36 deletions/insertions). Further, only four out of the 25 dCpdG dinucleotides present in the cDNA sequence remain unchanged, although the sequences possess 87.1% homology. 相似文献
14.
A cDNA clone encoding rat alpha 1-antitrypsin has been isolated from a lambda gt-11 rat liver cDNA library using an antigen-overlay immunoscreening method. The nucleotide sequence of this cDNA clone is 1306 base pairs in length and has a coding region of 1224 base pairs which can be translated into an alpha 1-antitrypsin precursor protein consisting of 408 amino acid residues. The cDNA sequence contains a termination codon, TAA, at position 1162 and a polyadenylation signal sequence, AATAAT, at position 1212. The calculated molecular weight of the translated mature protein is 43,700 with 387 amino acid residues; this differs from purified rat alpha 1-antitrypsin's apparent molecular weight of 54,000 because of glycosylation. Five potential glycosylation sites were identified on the basis of the cDNA sequence. The translated mature protein sequence from the cDNA clone matches completely with the N-terminal 33 amino acids of purified rat alpha 1-antitrypsin, which has an N-terminal Glu. The cDNA encoding rat alpha 1-antitrypsin shares 70% and 80% sequence identity with its human and mouse counterparts, respectively. The reactive center sequence of rat alpha 1-antitrypsin is highly conserved with respect to human alpha 1-antitrypsin, both having Met-Ser at the P1 and P1' residues. Genomic Southern blot analysis yielded a simple banding pattern, suggesting that the rat alpha 1-antitrypsin gene is single-copy. Northern blot analysis using the cDNA probe showed that rat alpha 1-antitrypsin is expressed at high levels in the liver and at low levels in the submandibular gland and the lung.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
15.
Identification of regions functioning in substrate interaction of rabbit liver cytochrome P-450 (laurate (omega-1)-hydroxylase) 总被引:1,自引:0,他引:1
The nucleotide sequence of cDNA for rabbit liver cytochrome P-450 (laurate (omega-1) hydroxylase) was replaced with that for rabbit liver cytochrome P-450 (testosterone 16 alpha-hydroxylase) in various regions coding for the amino acid sequence between residues 43 and 261. Six chimeric cDNAs thus constructed were cloned into expression vector pAAH5, and expressed in Saccharomyces cerevisiae AH22 cells under the control of yeast ADH1 promoter. Chimeric P-450s synthesized in the transformed yeast cells were purified partially and their catalytic and spectral properties were examined and compared with those of the chimeric P-450 which is considered to possess the same catalytic properties as the wild-type P-450. In the oxidized state the chimeric P-450s exhibited a low-and high-spin mixed-type absorption spectrum of cytochrome P-450 and the spectrum was converted to a typical high-spin type on addition of laurate or caprate, indicating the binding of the fatty acids to the substrate site of the chimeric P-450s. However the affinities of the fatty acids for the chimeras devoid of the sequence of P-450 (laurate (omega-1)-hydroxylase) in either of the regions spanning residues 90-125 and 210-261 were 10 to 20 times lower than those for the chimeras containing the sequence of the wild-type P-450 in both regions. The latter chimeras have about the same affinities as the chimera which is essentially the wild-type P-450.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
16.
Complete primary structure of the transacylase (E2b) subunit of the human branched chain alpha-keto acid dehydrogenase complex 总被引:4,自引:0,他引:4
Y Nobukuni H Mitsubuchi F Endo I Matsuda 《Biochemical and biophysical research communications》1989,161(3):1035-1041
We isolated from a placental cDNA library by immunoscreening a cDNA clone encoding the transacylase (E2b) precursor of the human branched chain alpha-keto acid dehydrogenase (BCKDH) complex. The cDNA insert consists of 2,649 base pairs with an open reading frame of 1,431 base pairs which can be translated into 477 amino acids and a 3'-untranslated region of 1,205 base pairs. The deduced amino acid sequence includes a leader peptide of 56 amino acid residues, a lipoyl-bearing domain, a E3-binding domain and an inner core domain. A mature human E2b subunit is likely to contain 421 amino acid residues with a calculated Mr 46,322. The nucleotide sequence of the open reading frame and the deduced amino acid sequence of the human E2b shows 91.6% and 92.0% homology with those of the bovine E2b subunit, respectively. 相似文献
17.
A cDNA coding for human sex hormone binding globulin. Homology to vitamin K-dependent protein S 总被引:4,自引:0,他引:4
Affinity purified antibodies to human sex hormone binding globulin (SHBG) were used in screening a human liver cDNA library, constructed in the expression vector lambda gt11. One clone, identified as producing recombinant SHBG, carried a cDNA insert of 1.1 kb. The nucleotide sequence of the insert had an open reading frame coding for 356 amino acid residues. The coding sequence was followed by a short 3'-region of 19 non-translated nucleotides and a poly(A) tail. Confirmation that the cDNA clone represented human SHBG was obtained by the finding of a complete agreement in amino acid sequence with several peptide fragments generated from purified SHBG by proteolytic cleavage. The primary structure of SHBG shows a considerable homology to that of protein S, a vitamin K-dependent protein with functions in the coagulation system. 相似文献
18.
The complete amino acid sequence of human retinal S-antigen (48 kDa protein), a retinal protein involved in the visual process has been determined by cDNA sequencing. The largest cDNA was 1590 base pairs (bp) and it contained an entire coding sequence. The similarity of nucleotide sequence between the human and bovine is approximately 80%. The predicted amino acid sequence indicates that human S-antigen has 405 residues and its molecular mass is 45050 Da. The amino acid sequence homology between human and bovine is 81%. There is no overall sequence similarity between S-antigen and other proteins listed in the National Biomedical Research Foundation (NBRF) protein data base. However, local regions of sequence homology with alpha-transducin (T alpha) are apparent including the putative rhodopsin binding and phosphoryl binding sites. In addition, human S-antigen has sequences identical to bovine uveitopathogenic sites, indicating that some types of human uveitis may in part be related to the animal model of experimental autoimmune uveitis (EAU). 相似文献
19.
Y Yabusaki H Murakami K Nakamura N Nomura M Shimizu K Oeda H Ohkawa 《Journal of biochemistry》1984,96(3):793-804
Double-stranded DNA complementary to the partially purified mRNA prepared from 3-methylcholanthrene (MC)-treated rat liver was constructed and cloned in Escherichia coli. Twenty clones were verified to carry a complementary DNA (cDNA) insert coding for MC-inducible cytochrome P-450 by positive hybridization translation assay and immunochemical assay with anti-cytochrome P-450 antibody. The identified cDNA clones were divided into at least two groups on the basis of comparison of restriction maps of the cDNA inserts. A clone pAU157 whose cDNA insert was approximately 2.7 kb in length contained nearly full-length mRNA information for cytochrome P-450MC or P-450c, which is the major form of MC-inducible cytochrome P-450. Other cDNA clones pTZ286-pTZ330 contained the 1.2 kb sequence complementary to cytochrome P-450d mRNA. RNA blot analysis revealed that pAU157 and pTZ286-pTZ330 cDNA clones were derived from 22S and 18S mRNAs, respectively, both of which were induced in rat liver by MC treatment. Sequence analysis revealed that there were closely homologous sequence regions in pAU157 and pTZ286-pTZ330 cDNA inserts and most of the homologous sequences were localized in two limited coding regions of the two cytochrome P-450 species. pAU157 encoded the total amino acid sequence of cytochrome P-450MC or P-450c and pTZ286-pTZ330 coded for the C-terminal 368 amino acid residues of cytochrome P-450d. Two highly homologous regions were found in the amino acid sequences of these cytochrome P-450 species. 相似文献
20.
Molecular cloning of a cDNA coding for human leukotriene A4 hydrolase. Complete primary structure of an enzyme involved in eicosanoid synthesis 总被引:7,自引:0,他引:7
M Minami S Ohno H Kawasaki O R?dmark B Samuelsson H J?rnvall T Shimizu Y Seyama K Suzuki 《The Journal of biological chemistry》1987,262(29):13873-13876
We have isolated a near full-length cDNA encoding human leukotriene A4 hydrolase, which synthesizes a potent chemotactic and spasmogenic compound, leukotriene B4. A human spleen cDNA library was screened with a 48-mer oligonucleotide probe, synthesized according to the partial amino acid sequence of the human leukocyte enzyme. The nucleotide sequence of the cDNA had an open reading frame of 1,833 base pairs, which contained regions coding for the N-terminal amino acid sequence, the amino acid sequence for the probe design, and several other peptide sequences of the enzyme. The complete primary structure of the enzyme composed of 610 amino acid residues (molecular weight, 69,153) was deduced from the cDNA. 相似文献