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1.
A cDNA clone for muscle adenylate kinase was isolated from a cDNA library of chick skeletal muscle poly(A)+ RNA, and the DNA sequence was determined. The cDNA insert had 854 nucleotides, which consisted of the 5'-untranslated sequence of 57 nucleotides, the sequence of 582 nucleotides coding for 194 amino acids, and the 3'-untranslated sequence of 163 nucleotides and the poly(A) tail of 52 nucleotides. The amino acid sequence predicted from the nucleotide sequence was highly homologous with the reported sequences of human, calf, porcine, and rabbit muscle adenylate kinases. RNA blot analysis of poly(A)+ RNA from various chicken tissues revealed a single species of mRNA of approximately 850 nucleotides and its tissue-specific distribution. The induction of muscle adenylate kinase mRNA synthesis during the chick embryogenesis was also demonstrated by the blot analysis. Southern blot analysis indicated a single gene for muscle adenylate kinase in the chicken genome. 相似文献
2.
Chicken muscle adenylate kinase was produced in a large amount in Escherichia coli cells harboring an expression plasmid, pKK-cAKl-1. The plasmid was constructed by placing the cDNA sequence for chicken muscle adenylate kinase after the tac promoter. After induction by isopropyl-beta-D-thiogalactopyranoside, the enzyme protein amounted to about 10% of the bacterial proteins. The enzyme was readily purified in two steps by using phosphocellulose and Sephadex G-100 columns. The apparent molecular weight of the enzyme produced in E. coli was estimated to be 22,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in agreement with the value deduced from the cDNA sequence. Ten amino acids in the NH2-terminal region were determined, and were identical with the sequence deduced from the cDNA sequence except that the terminal methionine was absent. Michaelis constants for ATP, ADP, and AMP of the enzyme thus synthesized were essentially identical to those determined with the enzyme in crude extracts of chicken skeletal muscle. 相似文献
3.
Isolation, characterization, and expression in Escherichia coli of a cDNA encoding rat heme oxygenase-2 总被引:15,自引:0,他引:15
In a recent study (Cruse, I., and Maines, M.D. (1988) J. Biol. Chem. 263, 3348-3353), we reported the isolation of a small cDNA fragment encoding a portion of heme oxygenase-2 through immunological screening of a rat testis cDNA library in lambda gt11. We have now used this 274-base pair (bp) cDNA fragment as a hybridization probe for rescreening of the same library, and have thereby recovered a number of additional positive isolates. Of these, three candidates of approximately 900, 1100, and 1300 bp, respectively, were subsequently subcloned and sequenced. Although differing in length, the sequences of these clones were found to be otherwise identical. Moreover, the length of isolate 18B, 1284 bp, corresponded well with that of the single mRNA species (approximately 1300-1350 nucleotides) detected through Northern blot hybridization analysis of rat testis total and poly(A)+RNA. This full- or near full-length cDNA encodes a 315-amino acid protein with a molecular weight of 35,757, in good agreement with the 36,000 estimated molecular weight of heme oxygenase-2. When expressed in Escherichia coli, cDNA encodes a protein that cross-reacts with heme oxygenase-2 antiserum (as assayed by Western immunoblotting) and yields high levels of heme oxygenase activity in bacterial soluble cell extracts. Finally, computer analysis of the heme oxygenase-2 cDNA sequence indicates that the predicted amino acid sequence and hydropathy profile of the heme oxygenase-2 protein exhibit similarity with heme oxygenase-1. 相似文献
4.
Isolation and characterization of the gene and cDNA encoding human mitochondrial creatine kinase 总被引:6,自引:0,他引:6
R C Haas C Korenfeld Z F Zhang B Perryman D Roman A W Strauss 《The Journal of biological chemistry》1989,264(5):2890-2897
Creatine kinase (CK; EC 2.7.3.2) isoenzymes play prominent roles in energy metabolism. Nuclear genes encode three known CK subunits: cytoplasmic muscle (MCK), cytoplasmic brain (BCK), and mitochondrial (MtCK). We have isolated the gene and cDNA encoding human placental MtCK. By using a dog heart MCK cDNA-derived probe, the 7.0-kb EcoRI fragment from one cross-hybridizing genomic clone was isolated and its complete nucleotide sequence determined. A region of this clone encoded predicted amino acid sequence identical to residues 15-26 of the human heart MtCK NH2-terminal protein sequence. The human placental MtCK cDNA was isolated by hybridization to a genomic fragment encoding this region. The human placental MtCK gene contains 9 exons encoding 416 amino acids, including a 38-amino acid transit peptide, presumably essential for mitochondrial import. Residues 1-14 of human placental MtCK cDNA-derived NH2-terminal sequence differ from the human heart MtCK protein sequence, suggesting that tissue-specific MtCK mRNAs are derived from multiple MtCK genes. RNA blot analysis demonstrated abundant MtCK mRNA in adult human ventricle and skeletal muscle, low amounts in placenta and small intestine, and a dramatic increase during in vitro differentiation induced by serum-deprivation in the non-fusing mouse smooth muscle cell line, BC3H1. These findings demonstrate coordinate regulation of MtCK and cytosolic CK gene expression and support the phosphocreatine shuttle hypothesis. 相似文献
5.
The amino acid sequence of all but a few N-terminal residues of the beta subunit of rat liver ATP synthase has been determined from cDNA clones. Rat liver F1-beta is shown to contain 17 amino acid differences from that reported for F1-beta of bovine heart, 2 differences of which involve differences in charge. This may account in part for the observation that bovine heart F1 binds nucleotides with much greater affinity than the rat liver enzyme. Rat liver F1-beta also contains homologous regions with another nucleotide binding protein, adenylate kinase, for which high-resolution structural studies are available. Adjacent to one of these homologous regions is an eight amino acid stretch which bears striking homology to the phosphorylation region of the (Na+,K+)-ATPase. The combination of these two homology regions may constitute at least part of a nucleotide binding domain in F1-beta. Significantly, both rat liver and bovine heart beta contain these regions of homology, whereas the 17 amino acid differences between the two enzymes lie outside this region. The possibility of a second nucleotide binding domain which differs between the two enzymes is discussed. A cDNA clone containing all the regions of homology as well as 11 of the 17 amino acid differences between the bovine heart and rat liver beta subunits has been ligated into the bacterial expression vector pKK223-3. After transformation of a protease-deficient strain of Escherichia coli, this cDNA clone is expressed as a 36-kilodalton protein.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
6.
H J Kim S Nishikawa T Tanaka S Uesugi H Takenaka M Hamada S A Kuby 《Protein engineering》1989,2(5):379-386
An artificial gene coding for the human muscle-type cytosolic adenylate kinase (hAK1) was chemically synthesized and directly expressed in Escherichia coli under the control of trp promoter. The DNA duplex of 596 bp was designed and constructed from 40 oligonucleotide fragments of typically 30 nucleotides in length. Twelve unique restriction sites were fairly evenly spaced in the synthetic gene to facilitate site-specific mutagenesis at any part of this recombinant protein. The genes for mutant hAK1 (Tyr 95----Phe 95, Y95F hAK1; Arg 97----Ala 97, R97A hAK1) were constructed by cassette mutagenesis and utilized restriction sites incorporated in the hAK1 gene. The recombinant hAK1 was purified to homogeneity by a two-step chromatographic procedure with a good yield, and showed the same adenylate kinase activity as that of authentic hAK1. Preliminary kinetic studies show that the enzymatic activity (Vmax app,cor/Et) of Y95F hAK1 was slightly greater than that of recombinant hAK1, whereas R97A hAK1 still possessed approximately 4% of recombinant hAK1 activity. These results suggest that the Arg-97 residue is important but not essential for catalytic activity, and that Tyr-95 can be replaced by phenylalanine without substantial effects on the enzymatic activity. Moreover, preliminary estimates of the apparent kinetic parameters suggest that these residues are not required for MgATP binding, and therefore they do not appear to be part of the MgATP binding site. 相似文献
7.
Two distinct types of fumarase were purified to homogeneity from aerobically grown Escherichia coli W cells. The amino acid sequences of their NH2-terminals suggest that the two enzymes are the products of the fumA gene (FUMA) and fumC gene (FUMC), respectively. FUMA was separated from FUMC by chromatography on a Q-Sepharose column, and was further purified to homogeneity on Alkyl-Superose, Mono Q, and Superose 12 columns. FUMA is a dimer composed of identical subunits (Mr = 60,000). Although the activity of FUMA rapidly decreased during storage, reactivation was attained by anaerobic incubation with Fe2+ and thiols. Studies on the inactivation and reactivation of FUMA suggested that oxidation and the concomitant release of iron inactivated the enzyme in a reversible manner. While the inactivated FUMA was EPR-detectable, through a signal with g perpendicular = 2.02 and g = 2.00, the active enzyme was EPR-silent. These results suggested FUMA is a member of the 4Fe-4S hydratases represented by aconitase. After the separation of FUMC from FUMA, purification of the former enzyme was accomplished by chromatography on Phenyl-Superose and Matrex Gel Red A columns. FUMC was stable, Fe-independent and quite similar to mammalian fumarases in enzymatic properties. 相似文献
8.
9.
Isolation and expression in Escherichia coli of a cDNA clone encoding human beta-glucuronidase 总被引:9,自引:0,他引:9
K S Guise R G Korneluk J Waye A M Lamhonwah F Quan R Palmer R E Ganschow W S Sly R A Gravel 《Gene》1985,34(1):105-110
Mucopolysaccharidosis type VII is a lysosomal storage disease resulting from a deficiency of beta-glucuronidase (BG) activity. To facilitate the investigation of mutation in the disease and provide molecular diagnostic tools for affected families, we have isolated human BG cDNA clones. The SV40-transformed human fibroblast cDNA library of Okayama and Berg [Mol. Cell. Biol. 3 (1982) 280-289] was screened with a fragment of a murine BG cDNA clone (pGUS-1). The 17 human cDNA clones (pHUG) isolated were identical by restriction mapping, varying only in length. The pHUG clones show 80% DNA sequence homology with pGUS-1 in a 198-bp PvuII-SstI restriction fragment. Both pGUS-1 and the pHUG clones contained an open reading frame (ORF) throughout the sequenced region with a predicted amino acid sequence homology of 73%. Expression in Escherichia coli of a 1150-bp fragment of pHUG-1 subcloned in pUC9 resulted in an isopropyl-thio-beta-galactoside (IPTG)-inducible 35-kDal fusion protein which was specifically immunoprecipitated by goat anti-human BG immunoglobulin G (IgG). This evidence provides direct confirmation that the pHUG cDNA clones correspond to human BG. 相似文献
10.
I Saint Girons A M Gilles D Margarita S Michelson M Monnot S Fermandjian A Danchin O Barzu 《The Journal of biological chemistry》1987,262(2):622-629
The adk gene encoding adenylate kinase in Escherichia coli was cloned in pBR322. Adenylate kinase represented about 4% of total proteins in extracts of cells containing the pBR322:adk plasmid. This allowed preparation of more than 90% pure enzyme in a single-step purification procedure. Amino acid analysis, high performance liquid chromatography separation of trypsin digests, sequence analysis of most peptides, and determination of the N-terminal sequence of the whole protein confirmed the primary structure of E. coli adenylate kinase predicted from the nucleotide sequence of the adk gene (Brune, M., Schumann, R., and Wittinghofer, F. (1985) Nucleic Acids Res. 13, 7139-7151). 2-Nitro-5-thiocyanatobenzoic acid reacted with the single cysteine residue of E. coli adenylate kinase. The cyanylated protein was cleaved upon exposure to alkaline pH, yielding two peptides corresponding to residues 1-76 and 77-214, respectively. A mixture of purified peptides tended to reassociate, recovering both catalytic activity and binding properties for adenine nucleotides. E. coli adenylate kinase has a broader specificity for nucleoside monophosphates than does the mammalian enzyme. In addition to 2'-dAMP, other nucleoside monophosphates such as 3'-dAMP, adenine-9-beta-D-arabinofuranoside 5'-monophosphate, and 7-deazaadenosine (tubercidine) 5'-monophosphate were able to replace AMP as substrate. 相似文献
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12.
Adenylate kinase from E. coli (strains CR341 and CR341 T28, a temperature-sensitive mutant) was purified by a two-step chromatographic procedure. The enzyme from crude extracts of both mutant and parent strain was bound to blue-Sepharose at pH 7.5, thereafter specifically eluted with 0.05 mM P1,P5-di(adenosine-5')pentaphosphate. A second chromatography on Sephadex G-100 yielded pure enzyme. E. coli adenylate kinase was strongly inhibited by P1,P5-di(adenosine-5')pentaphosphate (Ki 0.6 microM for adenylate kinase of strain CR341 and 2.1 microM in the case of mutant enzyme). After denaturation in 6 M guanidinium hydrochloride both mutant and parent adenylate kinase returned rapidly to the native, active state by dilution of guanidinium hydrochloride. 相似文献
13.
M Monnot A M Gilles I S Girons S Michelson O Barzu S Fermandjian 《The Journal of biological chemistry》1987,262(6):2502-2506
We examined by circular dichroism (CD) spectroscopy in far- and near-ultraviolet three different molecular forms of Escherichia coli adenylate kinase: the wild type protein, the enzyme carboxymethylated at a single cysteine residue (Cys-77), and the thermosensitive adenylate kinase. The thermosensitive enzyme differs from the wild type protein in that a serine is substituted for a proline residue at position 87 (Gilles, A.-M., Saint Girons, I., Monnot, M., Fermandjian, S., Michelson, S., and Barzu, O. (1986) Proc. Natl. Acad. Sci. U. S. A., 83, 5798-5802). We also examined the CD spectra of isolated peptides resulting from chemical cleavage of adenylate kinase at Cys-77 (C1, residues 1-76; C2, residues 77-214). The secondary structure composition of wild type bacterial adenylate kinase (50% alpha-helix and 15% beta-sheet) was close to that derived from x-ray analysis of pig muscle enzyme (Schulz, G.E., Elzinga, M., Marx, F., and Schirmer, R. H. (1974) Nature 250, 120-123). Carboxymethylation of wild type protein did not greatly affect the CD spectrum. The secondary structure of the thermosensitive adenylate kinase was observed to be significantly different from that of the wild type enzyme (reduction in alpha-helix content to 39%). Changes in ellipticities at 222 nm as a function of temperature indicated that the melting temperature for thermosensitive adenylate kinase was 38 degrees C and that for the wild type enzyme was 54 degrees C. Isolated C1 and C2 peptides had a large proportion of unordered structures. When mixed, C1 and C2 fragments reassociated into structures resembling native, uncleaved adenylate kinase. The recovery of ordered structures, indicated by CD spectroscopy, paralleled the recovery of catalytic activity. 相似文献
14.
Isolation and characterization of an Escherichia coli mutant deficient in dTMP kinase activity 总被引:4,自引:3,他引:1
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Escherichia coli LD0181 is sensitive to 15 micrograms of 2',3'-dideoxythymidine per ml. A derivative that was resistant to 40 micrograms of the same chemical per ml at 30 degrees C and that had lost the ability to grow on enriched medium at 42 degrees C was isolated after nitroso-guanidine mutagenesis. This mutant, TD105, produced a dTMP kinase with 25-fold lower specific activity and a 5-fold higher Km for dTMP than the parental strain. The dTMP pool in TD105 was 4.4-fold higher than in the parent. In addition to temperature sensitivity and resistance to 2',3'-dideoxythymidine, the mutant exhibited a hypersensitivity to 5-bromo-2'-deoxyuridine. All three of these phenotypes are cotransducible. The tmk gene was mapped by cotransduction to approximately 30 min on the E. coli map. 相似文献
15.
Y Shirasu H Yoshida T Mikayama S Matsuki J Tanaka H Ikenaga 《Journal of biochemistry》1986,99(6):1707-1712
We have cloned a DNA that is complementary to the messenger RNA that encodes porcine pancreatic elastase 1 from pancreas using rat pancreatic elastase 1 cDNA as a probe. This complementary DNA contains the entire protein coding region of 798 nucleotides which encodes an elastase of 266 amino acids, and 22 and 136 nucleotides of the 5' and 3'-untranslated sequences. When this deduced amino acid sequence was compared with known amino acid sequences, a carboxy-terminal 240 amino acids long peptide was found to be identical with a mature form of porcine pancreatic elastase 1, except for two amino acids. The porcine enzyme contains the same number of amino acid residues as the rat enzyme, and their amino acid sequences are 85% homologous. Taking the above findings together, we conclude that the cloned cDNA encodes a mature enzyme of 240 amino acids including a leader and activation peptide of 26 amino acids. We expressed the cloned porcine pancreatic elastase 1 cDNA in E. coli as a lac-fused protein. The resulting fused protein showed enzymatic activity and immunoreactivity toward anti-elastase serum. 相似文献
16.
The first enzyme responsible for assimilating levoglucosan in Aspergillus niger CBX-209 was corroborated to be levoglucosan kinase that catalyzes the transfer of a phosphate group from ATP to levoglucosan to yield a glucose 6-phosphate in the presence of magnesium ion and ATP by FAB-mass spectrometric method combined with previous observations from HPLC and enzymological experiments. Levoglucosan kinase was purified to apparent homogeneity by using a combination of seven purification steps. SDS-PAGE revealed a single protein band of 56 KDa. It is a monomeric enzyme and maximal enzyme activity was measured at pH 9.3 and 30 degrees C. This kinase is stable below 20 degrees C at a quite broad pHs ranging from 6 to 10 and levoglucosan could protect the enzyme from thermal inactivation. Exclusive substrate specificity for levoglucosan suggested that not only the structure of the intramolecular glucosidic linkage but also the configuration of the pyranose frame would be specific for recognition by levoglucosan kinase. The K(m) values of this enzyme were 71.2mM for levoglucosan and 0.25 mM for ATP, determined by double reciprocal plottings and ADP inhibited on the enzyme activity competitively with a Ki value of 0.20mM. A cDNA library from A. niger was constructed in Escherichia coli DH5alpha. The library was screened for levoglucosan kinase gene on NCE selective medium and three positive recombinants were selected after a five day culture. Detection of activities of levoglucosan kinase in the cell extracts indicated that levoglucosan kinase gene (lgk) was expressed by the recombinant strain of E. coli DH5alpha. 相似文献
17.
Isolation and characterization of homogeneous acetate kinase from Salmonella typhimurium and Escherichia coli 总被引:7,自引:0,他引:7
Acetate kinase from Salmonella typhimurium and Escherichia coli was purified to electrophoretic homogeneity. The amino acid compositions of both proteins were similar, and the apparent molecular weights were the same, about 40,000 for the putative monomers. The native proteins gave higher molecular weights, suggesting that the enzymes may be oligomers, perhaps with two polypeptide subunits. Steady-state kinetic studies were performed with the enzymes isolated from both organisms and the kinetic constants were determined. The Km values were 0.07 and 7 mM for ATP and acetate, respectively. In contrast to earlier studies using less pure preparations, the homogeneous enzymes from both strains were active only with acetate but not with propionate or butyrate. The enzyme activity was cold-labile, and the length of reactivation time in the presence of Mg X ATP and acetate was dependent on protein concentration, suggesting that the monomer may not be catalytically active. The enzyme was phosphorylated with [gamma-32P]ATP and the phosphoprotein was isolated. Phosphoacetate kinase was capable of transferring the phosphate group to either ADP or acetate. The accompanying paper (Fox, D. K., Meadow, N. D., and Roseman, S. (1986) J. Biol. Chem. 261, 13498-13503) shows that the phosphoryl group of phosphoacetate kinase can also be reversibly transferred to Enzyme I of the phosphoenolpyruvate:glycose phosphotransferase system. 相似文献
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19.
Inessa A. Khmel Victor M. Bondarenko Irina M. Manokhina Eugenia I. Basyuk Anastasia Z. Metlitskaya Valentina A. Lipasova Yulia M. Romanova 《FEMS microbiology letters》1993,111(2-3):269-274
Abstract A screening of 11956 enterobacteria isolates resulted in selection of seven active microcin-producing strains. The microcins were shown to be peptides or their derivatives with a rather broad spectrum of activity, mainly against Gram-negative bacteria. According to cross-immunity criteria, the microcins studied belonged to two of the previously suggested types, B (five strains) and C (two strains). Those of type B could be further classified into two subtypes on the account of differences in the spectrum of antibacterial activity. In five cases out of seven the microcin-producing ability has been attributed to plasmids that the strains harboured. The effect of microcins on sensitive cells was shown to depend on ompR and ompF gene products. 相似文献
20.
A cDNA clone encoding L-galactono-gamma-lactone (GAL) dehydrogenase (EC 1.3.2.3) was isolated from tobacco leaves. The cDNA clone contained an open reading frame encoding the protein of 501 amino acids with a calculated molecular mass of 56,926 Da, preceded by a putative mitochondrial targeting signal consisting of 86 amino acid residues. In fact, GAL dehydrogenase was localized in the mitochondria of tobacco cells. The deduced amino acid sequence of the cDNA showed 77 and 82% homology to cauliflower and sweet potato GAL dehydrogenases, respectively. Southern blot analysis showed that tobacco contains one copy of the gene for the enzyme. Northern blot analysis showed that GAL dehydrogenase mRNA (2.0 kb) is expressed in the leaves, stems, and roots in almost equal quantities. We introduced the cDNA clone encoding tobacco GAL dehydrogenase into a pET expression vector to overexpress this protein in Escherichia coli. The partially purified recombinant enzyme was used for comparative studies on the native enzymes from tobacco and other sources; its enzymatic properties were similar to those of other GAL dehydrogenases. 相似文献