scholarly journals Biochemical Characterization of Bovine Brain Myristoyl-CoA:Protein N-Myristoyltransferase Type 2

2009 ◽  
Vol 2009 ◽  
pp. 1-9 ◽  
Author(s):  
Ponniah Selvakumar ◽  
Ashakumary Lakshmikuttyamma ◽  
Rajendra K. Sharma
Keyword(s):  
Metal Ion ◽  
Biochemical Characterization ◽  
Kinetic Studies ◽  
Bovine Brain ◽  
Covalent Attachment ◽  
Reading Frame ◽  
Gene Coding ◽  
Acid Protein

Protein N-myristoylation is a lipidic modification which refers to the covalent attachment of myristate, a 14-carbon saturated fatty acid, to the N-terminal glycine residue of a number of mammalian, viral, and fungal proteins. In this paper, we have cloned the gene coding for myristoyl-CoA:protein N-myristoyltransferase (NMT) fromBos tarusbrain. The open reading frame codes for a 410-amino-acid protein and overexpressed inEscherichia coli. Kinetic studies suggested that bovine brain NMT2 and human NMT1 show significant differences in their peptide substrate specificities. The metal ionCa2+had stimulatory effects on NMT2 activity whileMn2+andZn2+inhibited the enzyme activity. In addition, NMT2 activity was inhibited by various organic solvents and other detergents while NMT1 had a stimulatory effect. Biochemical characterization suggested that both forms of NMT have unique characteristics. Further analysis towards functional role NMT2 will lead the development of therapeutic target for the progression of various diseases such as cancer, cardiovascular diseases, and neurodegenerative diseases.

scholarly journals Functional and biochemical characterization of a recombinant Arabidopsis thaliana 3-deoxy-D-manno-octulosonate 8-phosphate synthase

2004 ◽  
Vol 381 (1) ◽  
pp. 185-193 ◽  
Author(s):  
Jing WU ◽  
Mayur A. PATEL ◽  
Appavu K. SUNDARAM ◽  
Ronald W. WOODARD
Keyword(s):  
Arabidopsis Thaliana ◽  
Molecular Mass ◽  
Biochemical Characterization ◽  
Dipicolinic Acid ◽  
Kinetic Studies ◽  
Maximum Activity ◽  
Reading Frame ◽  
Sequential Mechanism ◽  
Phosphate Synthase

An open reading frame, encoding for KDOPS (3-deoxy-D-manno-octulosonate 8-phosphate synthase), from Arabidopsis thaliana was cloned into a T7-driven expression vector. The protein was overexpressed in Escherichia coli and purified to homogeneity. Recombinant A. thaliana KDOPS, in solution, displays an apparent molecular mass of 76 kDa and a subunit molecular mass of 31.519 kDa. Unlike previously studied bacterial KDOPSs, which are tetrameric, A. thaliana KDOPS appears to be a dimer in solution. The optimum temperature of the enzyme is 65 °C and the optimum pH is 7.5, with a broad peak between pH 6.5 and 9.5 showing 90% of maximum activity. The enzyme cannot be inactivated by EDTA or dipicolinic acid treatment, nor it can be activated by a series of bivalent metal ions, suggesting that it is a non-metallo-enzyme, as opposed to the initial prediction that it would be a metallo-enzyme. Kinetic studies showed that the enzyme follows a sequential mechanism with Km=3.6 μM for phosphoenolpyruvate and 3.8 μM for D-arabinose 5-phosphate and kcat=5.9 s−1 at 37 °C. On the basis of the characterization of A. thaliana KDOPS and phylogenetic analysis, plant KDOPSs may represent a new, distinct class of KDOPSs.

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.

Antigenic and Biochemical Characterization of Poliovirus Type 2 Isolated from Two Cases of Paralytic Disease

Intervirology ◽  
1987 ◽  
Vol 27 (4) ◽  
pp. 196-204 ◽  
Author(s):  
Lucia Fiore ◽  
Alessandra Pierangeli ◽  
Franco Lombard ◽  
Regina Santoro ◽  
Radu Crainic ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Poliovirus Type

scholarly journals Purification and biochemical characterization of non-myristoylated recombinant pp60c-src kinase

1992 ◽  
Vol 287 (3) ◽  
pp. 985-993 ◽  
Author(s):  
N B Lydon ◽  
B Gay ◽  
H Mett ◽  
B Murray ◽  
J Liebetanz ◽  
...  
Keyword(s):  
Metal Ion ◽  
Biochemical Characterization ◽  
Src Kinase ◽  
Sf9 Cells ◽  
Peptide Substrates ◽  
Biophysical Study ◽  
Stable Product ◽  
Full Activation

To obtain sufficient material for the biochemical and biophysical study of pp60c-src, we have utilized a recombinant pp60c-src baculovirus lacking the myristoylation site at codon 2. On infection of Sf9 cells, this virus produced large amounts of soluble non-myristoylated pp60c-src. The use of non-myristoylated pp60c-src (1) increases production of pp60c-src compared with the wild-type protein, (2) facilitates purification, (3) yields a stable product and (4) allows biochemical studies in the absence of detergents. Up to 20 mg of pp60c-src of greater than 95% purity has been purified from 6 litres of Sf9 cells grown in a bioreactor. One major and multiple minor forms of pp60c-src were separated by Mono Q f.p.l.c. Isoelectric focusing of purified pp60c-src species revealed heterogeneity, some of which could be attributed to differences in the tyrosine phosphorylation state of the enzyme. Kinetic analysis of non-myristoylated pp60c-src kinase in the presence of Mg2+ gave Km values for angiotensin II and ATP of 2 mM and 30 microM respectively and a Vmax. of 620 nmol/min per mg. The kinetic constants and metal ion preferences of a number of copolymers and peptide substrates have been compared. Polylysine and poly(GLAT), which was not phosphorylated by the pp60c-src kinase, dramatically activated autophosphorylation of Tyr-416, suggesting a conformation modulation of pp60c-src by charged polymers. This finding implies that Tyr-527 dephosphorylation is not sufficient for full activation of pp60c-src in vitro.

scholarly journals Genetic Context and Biochemical Characterization of the IMP-18 Metallo-β-Lactamase Identified in aPseudomonas aeruginosaIsolate from the United States

2010 ◽  
Vol 55 (1) ◽  
pp. 140-145 ◽  
Author(s):  
Luisa Borgianni ◽  
Silvia Prandi ◽  
Laurie Salden ◽  
Gisela Santella ◽  
Nancy D. Hanson ◽  
...  
Keyword(s):  
United States ◽  
Biochemical Characterization ◽  
Gene Cassette ◽  
The United States ◽  
Turnover Rates ◽  
Reading Frame ◽  
Class 1 ◽  
Original Array ◽  
Genetic Context

ABSTRACTThe production of metallo-β-lactamase (MBL) is an important mechanism of resistance to β-lactam antibiotics, including carbapenems. Despite the discovery and emergence of many acquired metallo-β-lactamases, IMP-type determinants (now counting at least 27 variants) remain the most prevalent in some geographical areas. In Asian countries, and notably Japan, IMP-1 and its closely related variants are most widespread. Some other variants have been detected in other countries and show either an endemic (e.g., IMP-13 in Italy) or sporadic (e.g., IMP-12 in Italy or IMP-18 in the United States) occurrence. The IMP-18-producingPseudomonas aeruginosastrain PS 297 from the southwestern United States carried at least two class 1 integrons. One was identical to In51, while the other, named In133and carrying theblaIMP-18gene cassette in the third position, showed an original array of five gene cassettes, includingaacA7,qacF,aadA1, and an unknown open reading frame (ORF). Interestingly. In133differed significantly from In96, theblaIMP-18-carrying integron identified in aP. aeruginosaisolate from Mexico. The meropenem and ertapenem MIC values were much lower forEscherichia colistrains producing IMP-18 (0.06 and 0.12 μg/ml, respectively) than for strains producing IMP-1 (2 μg/ml for each). Kinetic data obtained with the purified enzyme revealed lower turnover rates of IMP-18 than of other IMP-type enzymes with most substrates.

Cloning and characterization of an alternative splicing transcript of the gene coding for human cytidine deaminase

2007 ◽  
Vol 85 (1) ◽  
pp. 96-102 ◽  
Author(s):  
Bianca Cristina Garcia Lisboa ◽  
Tamara da Rocha Machado ◽  
Daniel Carvalho Pimenta ◽  
Sang Won Han
Keyword(s):  
Alternative Splicing ◽  
Dna Sequences ◽  
Genomic Sequence ◽  
Cytidine Deaminase ◽  
Alternative Form ◽  
Nih3t3 Cells ◽  
Reading Frame ◽  
Gene Coding ◽  
Identification And Characterization

Human cytidine deaminase (HCD) catalyzes the deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. The genomic sequence of HCD is formed by 31 kb with 4 exons and several alternative splicing signals, but an alternative form of HCD has yet to be reported. Here we describe the cloning and characterization of a small form of HCD, HSCD, and it is likely to be a product of alternative splicing of HCD. The alignment of DNA sequences shows that the HSCD matches HCD in 2 parts, except for a deletion of 170 bp. Based on the HCD genome organization, exons 1 and 4 should be joined and all sequences of introns and exons 2 and 3 should be deleted by splicing. This alternative splicing shifted the translation of the reading frame from the point of splicing. The estimated molecular mass is 9.8 kDa, and this value was confirmed by Western blot and mass spectroscopy after expressing the gene fused with glutathionine-S-transferase in the pGEX vector. The deletion and shift of the reading frame caused a loss of HCD activity, which was confirmed by enzyme assay and also with NIH3T3 cells modified to express HSCD and challenged against cytosine arabinoside. In this work we describe the identification and characterization of HSCD, which is the product of alternative splicing of the HCD gene.

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.

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