Isolation and characterization of the muscle-specific isoform of creatine kinase from the zebrafish, Danio rerio

2001 ◽  
Vol 79 (6) ◽  
pp. 779-782 ◽  
Author(s):  
Gregory Harder ◽  
Ross McGowan
Keyword(s):  
Creatine Kinase ◽  
Amino Acid ◽  
Danio Rerio ◽  
Cdna Sequence ◽  
Reading Frame ◽  
Isolation And Characterization ◽  
Acid Protein ◽  
Amino Acid Levels ◽  
Amino Acid Protein

We have isolated and characterized a cDNA sequence corresponding to the zebrafish muscle-specific isoform of creatine kinase. The sequence is 1552 bases in length and contains an open reading frame capable of producing a 381 amino acid protein. The sequence is very similar to muscle-specific creatine kinases isolated from other species at both the nucleotide and amino acid levels but contains some differences from a previously reported zebrafish clone.Key words: creatine kinase, muscle isoform, zebrafish, Danio rerio.

scholarly journals Characterization of the Gallate Dioxygenase Gene: Three Distinct Ring Cleavage Dioxygenases Are Involved in Syringate Degradation by Sphingomonas paucimobilis SYK-6

2005 ◽  
Vol 187 (15) ◽  
pp. 5067-5074 ◽  
Author(s):  
Daisuke Kasai ◽  
Eiji Masai ◽  
Keisuke Miyauchi ◽  
Yoshihiro Katayama ◽  
Masao Fukuda
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Terminal Region ◽  
Ring Cleavage ◽  
Sphingomonas Paucimobilis ◽  
Acid Protein ◽  
Demethylation Pathway ◽  
Dioxygenase Gene ◽  
Amino Acid Protein

ABSTRACT Sphingomonas paucimobilis SYK-6 converts vanillate and syringate to protocatechuate (PCA) and 3-O-methylgallate (3MGA) in reactions with the tetrahydrofolate-dependent O-demethylases LigM and DesA, respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, whereas 3MGA is metabolized via three distinct pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and 3MGA O-demethylase (LigM) are involved. In the 3MGA O-demethylation pathway, LigM converts 3MGA to gallate, and the resulting gallate appears to be degraded by a dioxygenase other than LigAB or DesZ. Here, we isolated the gallate dioxygenase gene, desB, which encodes a 418-amino-acid protein with a molecular mass of 46,843 Da. The amino acid sequences of the N-terminal region (residues 1 to 285) and the C-terminal region (residues 286 to 418) of DesB exhibited ca. 40% and 27% identity with the sequences of the PCA 4,5-dioxygenase β and α subunits, respectively. DesB produced in Escherichia coli was purified and was estimated to be a homodimer (86 kDa). DesB specifically attacked gallate to generate 4-oxalomesaconate as the reaction product. The Km for gallate and the V max were determined to be 66.9 ± 9.3 μM and 42.7 ± 2.4 U/mg, respectively. On the basis of the analysis of various SYK-6 mutants lacking the genes involved in syringate degradation, we concluded that (i) all of the three-ring cleavage dioxygenases are involved in syringate catabolism, (ii) the pathway involving LigM and DesB plays an especially important role in the growth of SYK-6 on syringate, and (iii) DesB and LigAB are involved in gallate degradation.

scholarly journals The bglA Gene of Aspergillus kawachii Encodes Both Extracellular and Cell Wall-Bound β-Glucosidases

1999 ◽  
Vol 65 (12) ◽  
pp. 5546-5553 ◽  
Author(s):  
Kazuhiro Iwashita ◽  
Tatsuya Nagahara ◽  
Hitoshi Kimura ◽  
Makoto Takano ◽  
Hitoshi Shimoi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Amino Acid Sequences ◽  
Enzyme Assays ◽  
Partial Amino Acid Sequence ◽  
Reading Frame ◽  
Acid Protein ◽  
Aspergillus Kawachii ◽  
Amino Acid Protein

ABSTRACT We cloned the genomic DNA and cDNA of bglA, which encodes β-glucosidase in Aspergillus kawachii, based on a partial amino acid sequence of purified cell wall-bound β-glucosidase CB-1. The nucleotide sequence of the cloned bglA gene revealed a 2,933-bp open reading frame with six introns that encodes an 860-amino-acid protein. Based on the deduced amino acid sequence, we concluded that the bglA gene encodes cell wall-bound β-glucosidase CB-1. The amino acid sequence exhibited high levels of homology with the amino acid sequences of fungal β-glucosidases classified in subfamily B. We expressed the bglA cDNA inSaccharomyces cerevisiae and detected the recombinant β-glucosidase in the periplasm fraction of the recombinant yeast.A. kawachii can produce two extracellular β-glucosidases (EX-1 and EX-2) in addition to the cell wall-bound β-glucosidase.A. kawachii in which the bglA gene was disrupted produced none of the three β-glucosidases, as determined by enzyme assays and a Western blot analysis. Thus, we concluded that thebglA gene encodes both extracellular and cell wall-bound β-glucosidases in A. kawachii.

Heterologous Expression and Characterization of Tyrosine Decarboxylase from Enterococcus faecalis R612Z1 and Enterococcus faecium R615Z1

2014 ◽  
Vol 77 (4) ◽  
pp. 592-598 ◽  
Author(s):  
FANG LIU ◽  
WENJUAN XU ◽  
LIHUI DU ◽  
DAOYING WANG ◽  
YONGZHI ZHU ◽  
...  
Keyword(s):  
Amino Acid ◽  
Heterologous Expression ◽  
Molecular Mass ◽  
Enterococcus Faecalis ◽  
Enterococcus Faecium ◽  
Maximal Activity ◽  
Tyrosine Decarboxylase ◽  
Acid Protein ◽  
Amino Acid Protein

Tyrosine decarboxylase (TDC) is responsible for tyramine production and can catalyze phenylalanine to produce β-phenylethylamine. Enterococcus strains are a group of bacteria predominantly producing tyramine and β-phenylethylamine in water-boiled salted duck. In this study, the heterologous expression and characterization of two TDCs from Enterococcus faecalis R612Z1 (612TDC) and Enterococcus faecium R615Z1 (615TDC) were studied. The recombinant putative proteins of 612TDC and 615TDC were heterologously expressed in Escherichia coli. 612TDC is a 620-amino-acid protein with a molecular mass of 70.0 kDa, whereas 615TDC is a 625-amino-acid protein with a molecular mass of 70.3 kDa. Both 612TDC and 615TDC showed an optimum temperature of 25°C for the tyrosine and phenylalanine substrates. However, 612TDC revealed maximal activity at pH 5.5, whereas 615TDC revealed maximal activity at pH 6.0. Kinetic studies showed that 612TDC and 615TDC exhibited higher specificity for tyrosine than for phenylalanine. The catalysis abilities of both 612TDC and 615TDC for phenylalanine were restrained significantly with the increase in NaCl concentration, but this was not the case for tyrosine. This study revealed that the enzyme properties of the purified recombinant 612TDC and 615TDC were similar, although their amino acid sequences had 84% identity.

Cloning and characterization of a 2,3-oxidosqualene cyclase from Eleutherococcus trifoliatus

Holzforschung ◽  
2013 ◽  
Vol 67 (4) ◽  
pp. 463-471 ◽  
Author(s):  
Li-Ting Ma ◽  
Sheng-Yang Wang ◽  
Yen-Hsueh Tseng ◽  
Yi-Ru Lee ◽  
Fang-Hua Chu
Keyword(s):  
Physiological Role ◽  
Active Site Residues ◽  
Reading Frame ◽  
Oxidosqualene Cyclases ◽  
Oxidosqualene Cyclase ◽  
Acid Protein ◽  
Leaf Tissues ◽  
New Perspective ◽  
Amino Acid Protein

Abstract The 2,3-oxidosqualene cyclases (OSCs) are a family of enzymes that have an important role in plant triterpene biosynthesis. In this study, an OSC gene designed EtLUS from Eleutherococcus trifoliatus has been cloned. EtLUS includes a 2292-bp open reading frame and encodes a 763-amino acid protein. EtLUS has an MLCYCR motif, which is conserved in lupeol synthases. Comparison of active-site residues and gene expression in yeast showed that EtLUS synthesizes lupeol. However, EtLUS has the highest sequence identity with β-amyrin synthases from Araliaceae rather than lupeol synthases, adding new perspective to the evolution of the OSCs of Araliaceae. Furthermore, EtLUS is upregulated in leaf tissues under methyl jasmonate treatment, which can be interpreted that lupeol and its derivatives play an ecological and physiological role in plant defense against pathogens and insect herbivores.

scholarly journals Identification and characterization of a cDNA encoding mouse CAP: a homolog of the yeast adenylyl cyclase associated protein

1993 ◽  
Vol 105 (3) ◽  
pp. 777-785 ◽  
Author(s):  
A.B. Vojtek ◽  
J.A. Cooper
Keyword(s):  
Adenylyl Cyclase ◽  
Leading Edge ◽  
Northern Analysis ◽  
Reading Frame ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Acid Protein ◽  
Carboxy Terminal ◽  
Amino Acid Protein

CAP, an adenylyl cyclase associated protein, is present in Saccharomyces cerevisiae and Schizosaccharomyces pombe. In both organisms, CAP is bifunctional: the N-terminal domain binds to adenylyl cyclase, thereby enabling adenylyl cyclase to respond appropriately to upstream regulatory signals, such as RAS in S. cerevisiae; the C-terminal domain is required for cellular morphogenesis. Here, we describe the isolation of a cDNA encoding a CAP homolog from a higher eukaryote. The mouse CAP cDNA contains an open reading frame capable of encoding a 474 amino acid protein. The protein encoded by the mouse CAP cDNA shows extensive homology to the yeast CAP proteins, particularly in the central poly-proline rich region and in the C-terminal domain. By northern analysis, the CAP message appears to be ubiquitous, but not uniform. By indirect immunofluorescence, ectopically expressed mouse CAP protein is found in the cytoplasm of fibroblasts and, in migrating cells, at the leading edge. Expression of the mouse CAP cDNA in S. cerevisiae complements defects associated with loss of the yeast CAP carboxy-terminal domain. Hence, the function of the CAP carboxy-terminal domain has been conserved from yeast to mouse.

scholarly journals Molecular Cloning and Characterization of a cDNA Encoding Sedoheptulose-1,7-bisphosphatase from Mulberry (Morus alba var. multicaulis)

2008 ◽  
Vol 57 (1-6) ◽  
pp. 152-157 ◽  
Author(s):  
X. Ji ◽  
Y. Gai ◽  
J. Ma ◽  
C. Zheng ◽  
Z. Mu
Keyword(s):  
Morus Alba ◽  
Evolutionary Analysis ◽  
Comparison Analysis ◽  
Reading Frame ◽  
Acid Protein ◽  
Fructose 1,6 Bisphosphatase ◽  
Rapid Amplification ◽  
Molecular Evolutionary Analysis ◽  
Amino Acid Protein

Abstract A full-length cDNA encoding sedoheptulose-1,7-bisphosphatase (SBPase; EC 3.1.3.37) was cloned from mulberry (Morus alba var. multicaulis) by rapid amplification of cDNA ends (RACE). The cDNA consisted of 1,527 nucleotides with an open reading frame (ORF) of 1,179 nucleotides encoding a 393 amino acid protein of approximately 42.6 kDa. Sequence comparison analysis showed that mulberry SBPase (MSBPase) had high homology to other plant counterparts. Phylogenetic and molecular evolutionary analysis revealed that MSBPase fell into plant SBPase group. Moreover, SBPase and fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) shared 28-32% identical residues, suggesting that the two enzymes originated from the same evolution branch. Molecular modeling indicated that each subunit of MSBPase was composed of α-helices and β-sheets joined by turns and loops, and folded into a structure of hexahedron shape which was very similar to FBPase.

scholarly journals Molecular Characterization of OXA-20, a Novel Class D β-Lactamase, and Its Integron from Pseudomonas aeruginosa

1998 ◽  
Vol 42 (8) ◽  
pp. 2074-2083 ◽  
Author(s):  
Thierry Naas ◽  
Wladimir Sougakoff ◽  
Anne Casetta ◽  
Patrice Nordmann
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Clavulanic Acid ◽  
Antibiotic Resistance Gene ◽  
Amino Acid Identity ◽  
Acid Identity ◽  
Class D ◽  
Acid Protein ◽  
Amino Acid Protein

ABSTRACT The Pseudomonas aeruginosa Mus clinical isolate produces OXA-18, a pI 5.5 class D extended-spectrum β-lactamase totally inhibited by clavulanic acid (L. N. Philippon, T. Naas, A.-T. Bouthors, V. Barakett, and P. Nordmann, Antimicrob. Agents Chemother. 41:2188–2195, 1997). A second β-lactamase was cloned, and the recombinant Escherichia coli clone pPL10 expressed a pI 7.4 β-lactamase which conferred high levels of amoxicillin and ticarcillin resistance and which was partially inhibited by clavulanic acid. The 2.5-kb insert from pPL10 was sequenced, and a 266-amino-acid protein (OXA-20) was deduced; this protein has low amino acid identity with most of the class D β-lactamases except OXA-2, OXA-15, and OXA-3 (75% amino acid identity with each). OXA-20 is a restricted-spectrum oxacillinase and is unusually inhibited by clavulanic acid. OXA-20 is a peculiar β-lactamase because its translation initiates with a TTG (leucine) codon, which is rarely used as a translational origin in bacteria. Exploration of the genetic environment of oxa20revealed the presence of the following integron features: (i) a second antibiotic resistance gene, aacA4; (ii) anintI1 gene; and (iii) two 59-base elements, each associated with either oxa20 or aacA4. This integron is peculiar because it lacks the 3′ conserved region, and therefore is not a sul1-associated integron like most of them, and because its 3′ end is located within tnpR, a gene involved in the transposition of Tn5393, a gram-negative transposon.P. aeruginosa Mus produces two novel and unrelated oxacillinases, OXA-18 and OXA-20, both of which are inhibited by clavulanic acid.

scholarly journals Characterization of an Endo-β-1,6-Galactanase from Streptomyces avermitilis NBRC14893

2008 ◽  
Vol 74 (8) ◽  
pp. 2379-2383 ◽  
Author(s):  
Hitomi Ichinose ◽  
Toshihisa Kotake ◽  
Yoichi Tsumuraya ◽  
Satoshi Kaneko
Keyword(s):  
Signal Sequence ◽  
Recombinant Enzyme ◽  
Streptomyces Avermitilis ◽  
Glycoside Hydrolase Family ◽  
Reading Frame ◽  
Secretion Signal ◽  
Acid Protein ◽  
Hydrolysis Of ◽  
Amino Acid Protein

ABSTRACT The putative endo-β-1,6-galactanase gene from Streptomyces avermitilis was cloned and expressed in Escherichia coli, and the enzymatic properties of the recombinant enzyme were characterized. The gene consisted of a 1,476-bp open reading frame and encoded a 491-amino-acid protein, comprising an N-terminal secretion signal sequence and glycoside hydrolase family 5 catalytic module. The recombinant enzyme, Sa1,6Gal5A, catalyzed the hydrolysis of β-1,6-linked galactosyl linkages of oligosaccharides and polysaccharides. The enzyme produced galactose and a range of β-1,6-linked galacto-oligosaccharides, predominantly β-1,6-galactobiose, from β-1,6-galactan chains. There was a synergistic effect between the enzyme and Sa1,3Gal43A in degrading tomato arabinogalactan proteins. These results suggest that Sa1,6Gal5A is the first identified endo-β-1,6-galactanase from a prokaryote.

scholarly journals Molecular Cloning and Biochemical Characterization of a Recombinant Sterol 3-O-Glucosyltransferase fromGymnema sylvestreR.Br. Catalyzing Biosynthesis of Steryl Glucosides

2014 ◽  
Vol 2014 ◽  
pp. 1-14 ◽  
Author(s):  
Pragya Tiwari ◽  
Rajender Singh Sangwan ◽  
Asha ◽  
B. N. Mishra ◽  
Farzana Sabir ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Present Report ◽  
Hydroxyl Group ◽  
Biological Origin ◽  
Reading Frame ◽  
Steryl Glucosides ◽  
Acid Protein ◽  
Phytochemical Profiles ◽  
Amino Acid Protein

Gymnema sylvestreR.Br., a pharmacologically important herb vernacularly called Gur-Mar (sugar eliminator), is widely known for its antidiabetic action. This property of the herb has been attributed to the presence of bioactive triterpene glycosides. Although some information regarding pharmacology and phytochemical profiles of the plant are available, no attempts have been made so far to decipher the biosynthetic pathway and key enzymes involved in biosynthesis of steryl glucosides. The present report deals with the identification and catalytic characterization of a glucosyltransferase, catalyzing biosynthesis of steryl glycosides. The full length cDNA (2572 bp) contained an open reading frame of 2106 nucleotides that encoded a 701 amino acid protein, falling into GT-B subfamily of glycosyltransferases. The GsSGT was expressed inEscherichia coliand biochemical characterization of the recombinant enzyme suggested its key role in the biosynthesis of steryl glucosides with catalytic preference for C-3 hydroxyl group of sterols. To our knowledge, this pertains to be the first report on cloning and biochemical characterization of a sterol metabolism gene fromG. sylvestreR.Br. catalyzing glucosylation of a variety of sterols of biological origin from diverse organisms such as bacteria, fungi, and plants.

scholarly journals The Legionella pneumophila iraAB Locus Is Required for Iron Assimilation, Intracellular Infection, and Virulence

2000 ◽  
Vol 68 (3) ◽  
pp. 1069-1079 ◽  
Author(s):  
V. K. Viswanathan ◽  
Paul H. Edelstein ◽  
C. Dumais Pope ◽  
Nicholas P. Cianciotto
Keyword(s):  
Amino Acid ◽  
Legionella Pneumophila ◽  
Open Reading Frame ◽  
Host Cells ◽  
Reading Frame ◽  
Intracellular Infection ◽  
Acid Protein ◽  
Iron Deficient ◽  
Iron Assimilation ◽  
Amino Acid Protein

ABSTRACT Legionella pneumophila, a facultative intracellular parasite of human alveolar macrophages and protozoa, causes Legionnaires' disease. Using mini-Tn10 mutagenesis, we previously isolated a L. pneumophila mutant that was hypersensitive to iron chelators. This mutant, NU216, and its allelic equivalent, NU216R, were also defective for intracellular infection, particularly in iron-deficient host cells. To determine whether NU216R was attenuated for virulence, we assessed its ability to cause disease in guinea pigs following intratracheal inoculation. NU216R-infected animals yielded 1,000-fold fewer bacteria from their lungs and spleen compared to wild-type-130b-infected animals that had received a 50-fold-lower dose. Moreover, NU216R-infected animals subsequently cleared the bacteria from these sites. While infection with 130b resulted in high fever, weight loss, and ruffled fur, inoculation with NU216R did not elicit any signs of disease. DNA sequence analysis revealed that the transposon insertion in NU216R lies in the first open reading frame of a two-gene operon. This open reading frame (iraA) encodes a 272-amino-acid protein that shows sequence similarity to methyltransferases. The second open reading frame (iraB) encodes a 501-amino-acid protein that is highly similar to di- and tripeptide transporters from both prokaryotes and eukaryotes. Southern hybridization analyses determined that theiraAB locus was largely limited to strains of L. pneumophila, the most pathogenic of the Legionellaspecies. A newly derived mutant containing a targeted disruption ofiraB showed reduced ability to grow under iron-depleted extracellular conditions, but it did not have an infectivity defect in the macrophage-like U937 cells. These data suggest thatiraA is critical for virulence of L. pneumophila while iraB is involved in a novel method of iron acquisition which may utilize iron-loaded peptides.

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