scholarly journals Isolation, amino acid analyses and refolding of subunits of pig heart succinyl-CoA synthetase

1988 ◽  
Vol 250 (2) ◽  
pp. 429-434 ◽  
Author(s):  
J S Nishimura ◽  
J Ybarra ◽  
T Mitchell ◽  
P M Horowitz
Keyword(s):  
Amino Acid ◽  
Specific Activity ◽  
Guanidine Hydrochloride ◽  
Beta Subunit ◽  
Native Enzyme ◽  
Tryptophan Residue ◽  
Beta Subunits ◽  
Post Translational Modification ◽  
E Coli ◽  
Succinyl Coa Synthetase

For the first time, pig heart succinyl-CoA synthetase has been refolded from its isolated subunits after denaturation. Amino acid analyses of pig heart succinyl-CoA synthetase and its subunits were performed. Subunits were isolated by gel filtration in neutral 6 M-urea. The amino acid composition of the native enzyme bears a strong resemblance to that of the Escherichia coli enzyme. Application of the various methods for comparing amino acid compositions [Cornish-Bowden (1983) Methods Enzymol. 91, 60-75] shows that the degree of relatedness between the alpha-subunits of the pig heart and E. coli enzymes and between the beta-subunits of the two synthetases is intermediate between ‘strong’ and ‘weak’. As for the E. coli synthetase, it is unlikely that the alpha-subunit arises from the larger beta-subunit by post-translational modification. The pig heart enzyme contains a single tryptophan residue, which is located in the beta-subunit. Excitation of the enzyme at 295 nm resulted in a typical tryptophan emission spectrum. Refolding of enzyme denatured in 6 M-guanidine hydrochloride or of alpha- and beta-subunits isolated in this solvent required the presence of either ethylene glycol or glycerol, optimally at 20-25% (v/v). GTP-Mg2+ did not stimulate reactivation of the enzyme, in contrast with the result obtained with ATP-Mg2+ in the reconstitution of the enzyme from E. coli. Yields of 60% and 40% were obtained in the refolding of denatured enzyme and isolated subunits respectively. The fluorescence spectrum of the refolded protein was essentially the same as that of native enzyme. Unrecovered activity could not be accounted for in the form of protein aggregates. The specific activity of refolded enzyme that had been separated from inactive protein on a Bio-Sil TSK 250 column was the same as that of native enzyme. Km values for GTP of 27 microM and 14 microM were determined for native and refolded enzyme respectively.

scholarly journals Investigations into the post-translational modification and mechanism of isopenicillin N:acyl-CoA acyltransferase using electrospray mass spectrometry

1993 ◽  
Vol 294 (2) ◽  
pp. 357-363 ◽  
Author(s):  
R T Aplin ◽  
J E Baldwin ◽  
P L Roach ◽  
C V Robinson ◽  
C J Schofield
Keyword(s):  
Mass Spectrometry ◽  
Electrospray Mass Spectrometry ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
British Library ◽  
Beta Subunits ◽  
Post Translational Modification ◽  
Acetic Acids

Electrospray mass spectrometry (e.s.m.s.) was used to confirm the position of the post-translational cleavage of the isopenicillin N:acyl-CoA acyltransferase preprotein to give the alpha- and beta-subunits. The e.s.m.s. studies suggested partial modification of the alpha-subunit in vivo by exogenously added substituted acetic acids. E.s.m.s. has also allowed the observation in vitro of the transfer of the acyl group from several acyl-CoAs to the beta-subunit. N.m.r. data for the CoA species have been deposited as Supplementary Publication SUP 500173 (2 pages) at the British Library Document Supply Centre (DSC), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, from whom copies can be obtained on the terms indicated in Biochem. J. (1993) 289, 9.

scholarly journals Cloning, Nucleotide Sequence, and Overexpression of the l-Rhamnose Isomerase Gene from Pseudomonas stutzeri in Escherichia coli

2004 ◽  
Vol 70 (6) ◽  
pp. 3298-3304 ◽  
Author(s):  
Khim Leang ◽  
Goro Takada ◽  
Akihiro Ishimura ◽  
Masashi Okita ◽  
Ken Izumori
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Pseudomonas Stutzeri ◽  
Fold Increase ◽  
E Coli ◽  
Apparent Homogeneity ◽  
Volumetric Yield

ABSTRACT The gene encoding l-rhamnose isomerase (l-RhI) from Pseudomonas stutzeri was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the l-RhI gene revealed an open reading frame of 1,290 bp coding for a protein of 430 amino acid residues with a predicted molecular mass of 46,946 Da. A comparison of the deduced amino acid sequence with sequences in relevant databases indicated that no significant homology has previously been identified. An amino acid sequence alignment, however, suggested that the residues involved in the active site of l-RhI from E. coli are conserved in that from P. stutzeri. The l-RhI gene was then overexpressed in E. coli cells under the control of the T5 promoter. The recombinant clone, E. coli JM109, produced significant levels of l-RhI activity, with a specific activity of 140 U/mg and a volumetric yield of 20,000 U of soluble enzyme per liter of medium. This reflected a 20-fold increase in the volumetric yield compared to the value for the intrinsic yield. The recombinant l-RhI protein was purified to apparent homogeneity on the basis of three-step chromatography. The purified recombinant enzyme showed a single band with an estimated molecular weight of 42,000 in a sodium dodecyl sulfate-polyacrylamide gel. The overall enzymatic properties of the purified recombinant l-RhI protein were the same as those of the authentic one, as the optimal activity was measured at 60�C within a broad pH range from 5.0 to 11.0, with an optimum at pH 9.0.

scholarly journals Purification, characterization, DNA sequence and cloning of a pimeloyl-CoA synthetase from Pseudomonas mendocina 35

1999 ◽  
Vol 340 (3) ◽  
pp. 793-801 ◽  
Author(s):  
Andrew BINIEDA ◽  
Martin FUHRMANN ◽  
Bruno LEHNER ◽  
Claudine REY-BERTHOD ◽  
Séverine FRUTIGER-HUGHES ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dna Sequence ◽  
Specific Activity ◽  
Amino Acid Sequences ◽  
Active Enzyme ◽  
Gene Probe ◽  
Pimelic Acid ◽  
Pseudomonas Mendocina ◽  
Ph Optimum ◽  
E Coli

A pimeloyl-CoA synthetase from Pseudomonas mendocina 35 was purified and characterized, the DNA sequence determined, and the gene cloned into Escherichia coli to yield an active enzyme. The purified enzyme had a pH optimum of ≈ 8.0, Km values of 0.49 mM for pimelic acid, 0.18 mM for CoA and 0.72 mM for ATP, a subunit Mr of ≈ 80000 as determined by SDS/PAGE, and was found to be a tetramer by gel-filtration chromatography. The specific activity of the purified enzyme was 77.3 units/mg of protein. The enzyme was not absolutely specific for pimelic acid. The relative activity for adipic acid (C6) was 72% and for azaleic acid (C9) was 18% of that for pimelic acid (C7). The N-terminal amino acid was blocked to amino acid sequencing, but controlled proteolysis resulted in three peptide fragments for which amino acid sequences were obtained. An oligonucleotide gene probe corresponding to one of the amino acid sequences was synthesized and used to isolate the gene (pauA, imelic cid-tilizing ) coding for pimeloyl-CoA synthetase. The pauA gene, which codes for a protein with a theoretical Mr of 74643, was then sequenced. The deduced amino acid sequence of the enzyme showed similarity to hypothetical proteins from Archaeoglobus fulgidus, Methanococcus jannaschii, Pyrococcus horikoshii, E. coli and Streptomyces coelicolor, and some limited similarity to microbial succinyl-CoA synthetases. The similarity with the protein from A. fulgidus was especially strong, thus indicating a function for this unidentified protein. The pauA gene was cloned into E. coli, where it was expressed and resulted in an active enzyme.

scholarly journals Neuropeptide W/neuropeptide B receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

2019 ◽  
Vol 2019 (4) ◽  
Author(s):  
Anthony P. Davenport ◽  
Janet J. Maguire ◽  
Gurminder Singh
Keyword(s):  
Amino Acid ◽  
Tryptophan Residue ◽  
Post Translational Modification ◽  
Tissue Extracts ◽  
Hypothalamic Tissue

The neuropeptide BW receptor 1 (NPBW1, provisional nomenclature [5]) is activated by two 23-amino-acid peptides, neuropeptide W (neuropeptide W-23) and neuropeptide B (neuropeptide B-23) [20, 6]. C-terminally extended forms of the peptides (neuropeptide W-30 and neuropeptide B-29) also activate NPBW1 [2]. Unique to both forms of neuropeptide B is the N-terminal bromination of the first tryptophan residue, and it is from this post-translational modification that the nomenclature NPB is derived. These peptides were first identified from bovine hypothalamus and therefore are classed as neuropeptides. Endogenous variants of the peptides without the N-terminal bromination, des-Br-neuropeptide B-23 and des-Br-neuropeptide B-29, were not found to be major components of bovine hypothalamic tissue extracts. The NPBW2 receptor is activated by the short and C-terminal extended forms of neuropeptide W and neuropeptide B [2].

scholarly journals Characterization of an Atypical Superoxide Dismutase from Sinorhizobium meliloti

1999 ◽  
Vol 181 (15) ◽  
pp. 4509-4516 ◽  
Author(s):  
Renata Santos ◽  
Stephane Bocquet ◽  
Alain Puppo ◽  
Danièle Touati
Keyword(s):  
Superoxide Dismutase ◽  
Amino Acid ◽  
Sinorhizobium Meliloti ◽  
Genomic Library ◽  
Specific Activity ◽  
Growth Conditions ◽  
Aerobic Bacterium ◽  
Degenerate Primers ◽  
E Coli ◽  
Acid Protein

ABSTRACT Sinorhizobium meliloti Rm5000 is an aerobic bacterium that can live free in the soil or in symbiosis with the roots of leguminous plants. A single detectable superoxide dismutase (SOD) was found in free-living growth conditions. The corresponding gene was isolated from a genomic library by using a sod fragment amplified by PCR from degenerate primers as a probe. ThesodA gene was located in the chromosome. It is transcribed monocistronically and encodes a 200-amino-acid protein with a theoretical M r of 22,430 and pI of 5.8.S. meliloti SOD complemented a deficient E. coli mutant, restoring aerobic growth of a sodA sodB recA strain, when the gene was expressed from the synthetictac promoter but not from its own promoter. Amino acid sequence alignment showed great similarity with Fe-containing SODs (FeSODs), but the enzyme was not inactivated by H2O2. The native enzyme was purified and found to be a dimeric protein, with a specific activity of 4,000 U/mg. Despite its Fe-type sequence, atomic absorption spectroscopy showed manganese to be the cofactor (0.75 mol of manganese and 0.24 mol of iron per mol of monomer). The apoenzyme was prepared from crude extracts of S. meliloti. Activity was restored by dialysis against either MnCl2 or Fe(NH4)2(SO4)2, demonstrating the cambialistic nature of the S. melilotiSOD. The recovered activity with manganese was sevenfold higher than with iron. Both reconstituted enzymes were resistant to H2O2. Sequence comparison with 70 FeSODs and MnSODs indicates that S. meliloti SOD contains several atypical residues at specific sites that might account for the activation by manganese and resistance to H2O2of this unusual Fe-type SOD.

scholarly journals Construction of Deoxyriboaldolase-Overexpressing Escherichia coli and Its Application to 2-Deoxyribose 5-Phosphate Synthesis from Glucose and Acetaldehyde for 2′-Deoxyribonucleoside Production

2003 ◽  
Vol 69 (7) ◽  
pp. 3791-3797 ◽  
Author(s):  
Nobuyuki Horinouchi ◽  
Jun Ogawa ◽  
Takafumi Sakai ◽  
Takako Kawano ◽  
Seiichiro Matsumoto ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Specific Activity ◽  
Cell Extract ◽  
Predicted Amino Acid Sequence ◽  
Enzymatic Reactions ◽  
Chromosomal Dna ◽  
Gene Encoding ◽  
Reading Frame ◽  
E Coli

ABSTRACT The gene encoding a deoxyriboaldolase (DERA) was cloned from the chromosomal DNA of Klebsiella pneumoniae B-4-4. This gene contains an open reading frame consisting of 780 nucleotides encoding 259 amino acid residues. The predicted amino acid sequence exhibited 94.6% homology with the sequence of DERA from Escherichia coli. The DERA of K. pneumoniae was expressed in recombinant E. coli cells, and the specific activity of the enzyme in the cell extract was as high as 2.5 U/mg, which was threefold higher than the specific activity in the K. pneumoniae cell extract. One of the E. coli transformants, 10B5/pTS8, which had a defect in alkaline phosphatase activity, was a good catalyst for 2-deoxyribose 5-phosphate (DR5P) synthesis from glyceraldehyde 3-phosphate and acetaldehyde. The E. coli cells produced DR5P from glucose and acetaldehyde in the presence of ATP. Under the optimal conditions, 100 mM DR5P was produced from 900 mM glucose, 200 mM acetaldehyde, and 100 mM ATP by the E. coli cells. The DR5P produced was further transformed to 2′-deoxyribonucleoside through coupling the enzymatic reactions of phosphopentomutase and nucleoside phosphorylase. These results indicated that production of 2′-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase is possible with the addition of a suitable energy source, such as ATP.

scholarly journals Cloning, Expression, and Characterization of a Novel Thermophilic Monofunctional Catalase fromGeobacillussp. CHB1

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Xianbo Jia ◽  
Jichen Chen ◽  
Chenqiang Lin ◽  
Xinjian Lin
Keyword(s):  
Amino Acid ◽  
Specific Activity ◽  
Fermentation Broth ◽  
Residual Activity ◽  
Industrial Applications ◽  
Activity Level ◽  
High Yield ◽  
E Coli ◽  
Wide Range ◽  
Clone And Express

Catalases are widely used in many scientific areas. A catalase gene (Kat) fromGeobacillussp. CHB1 encoding a monofunctional catalase was cloned and recombinant expressed inEscherichia coli(E. coli), which was the first time to clone and express this type of catalase ofgenus Geobacillusstrains as far as we know. ThisKatgene was 1,467 bp in length and encoded a catalase with 488 amino acid residuals, which is only 81% similar to the previously studiedBacillussp. catalase in terms of amino acid sequence. Recombinant catalase was highly soluble inE. coliand made up 30% of the totalE. coliprotein. Fermentation broth of the recombinantE. colishowed a high catalase activity level up to 35,831 U/mL which was only lower than recombinantBacillussp. WSHDZ-01 among the reported catalase production strains. The purified recombinant catalase had a specific activity of 40,526 U/mg andKmof 51.1 mM. The optimal reaction temperature of this recombinant enzyme was 60°C to 70°C, and it exhibited high activity over a wide range of reaction temperatures, ranging from 10°C to 90°C. The enzyme retained 94.7% of its residual activity after incubation at 60°C for 1 hour. High yield and excellent thermophilic properties are valuable features for this catalase in industrial applications.

scholarly journals The Schizosaccharomyces pombe casein kinase II alpha and beta subunits: evolutionary conservation and positive role of the beta subunit.

1994 ◽  
Vol 14 (1) ◽  
pp. 576-586 ◽  
Author(s):  
I Roussou ◽  
G Draetta
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Schizosaccharomyces Pombe ◽  
Sequence Similarity ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Beta Subunit ◽  
Beta Subunits ◽  
High Sequence Similarity

Casein kinase II is a key regulatory enzyme involved in many cellular processes, including the control of growth and cell division. We report the molecular cloning and sequencing of cDNAs encoding the alpha and the beta subunits of casein kinase II of Schizosaccharomyces pombe. The deduced amino acid sequence of Cka1, the alpha catalytic subunit, shows high sequence similarity to alpha subunits identified in other species. The amino acid sequence of Ckb1, the S. pombe beta subunit, is 57% identical to that of the human beta subunit. Cka1 overexpression results in no detectable phenotype. In contrast, Ckb1 overexpression inhibits cell growth and cytokinesis, with formation of multiseptated cells. Disruption of the ckb1+ gene causes a cold-sensitive phenotype and abnormalities in cell shape. In these cells, the casein kinase II activity is reduced to undetectable levels, demonstrating that Ckb1 is required for enzyme activity in vivo. In agreement with this, the activity measured in a strain expressing high levels of Cka1 is enhanced only when the Ckb1 protein is coexpressed. Altogether, our data suggest that Ckb1 is a positive regulator of the enzyme activity, and that it plays a role in mediating the interaction of casein kinase II with downstream targets and/or with additional regulators.

scholarly journals Distinct mutations in two patients with leukocyte adhesion deficiency and their functional correlates.

1990 ◽  
Vol 172 (1) ◽  
pp. 335-345 ◽  
Author(s):  
A J Wardlaw ◽  
M L Hibbs ◽  
S A Stacker ◽  
T A Springer
Keyword(s):  
Amino Acid ◽  
Leukocyte Adhesion ◽  
Expression System ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Beta Subunits ◽  
Cdna Clones ◽  
Loss Of Function ◽  
Leukocyte Adhesion Deficiency ◽  
Mammalian Expression

Two patients with leukocyte adhesion deficiency (LAD), one with a moderate phenotype (patient 14) and one with a severe phenotype (patient 2) who had been shown to have a normal sized beta subunit protein precursor, were analyzed in an attempt to determine the molecular basis for their disease. RNase mapping located possible mutations to two distinct but adjacent regions of the beta subunit cDNA. Sequencing of patient-derived cDNA clones in this region revealed a C for T difference at amino acid 149 in patient 14 which resulted in the substitution of a leucine for a proline, and an A for G substitution at amino acid 169 in patient 2 which mutated a glycine to an arginine. The mutated amino acids are in a region of the cDNA that is highly conserved between the beta subunits of the integrin family and are identical in all known integrin beta subunits. Co-transfection of the beta subunit cDNA containing the patient 2 mutation with the wild-type alpha subunit of LFA-1 in a mammalian expression system resulted in no expression of LFA-1. In the case of the mutation in patient 14 there was markedly diminished expression of LFA-1 with loss of function and loss of the epitope for a number of anti-beta mAbs. Normal half-life of the mutant beta subunits, and previous demonstration of a lack of alpha/beta complex formation during biosynthesis in patient cells, suggest a defect in association with the alpha subunit. Association with beta is required for expression of the alpha subunit of LFA-1. Loss of functional expression with both of these beta subunit mutations suggests that they lie in a site critical for association with the alpha subunit.

Identification of a second region of the beta-subunit involved in regulation of calcium channel inactivation

1996 ◽  
Vol 271 (5) ◽  
pp. C1539-C1545 ◽  
Author(s):  
N. Qin ◽  
R. Olcese ◽  
J. Zhou ◽  
O. A. Cabello ◽  
L. Birnbaumer ◽  
...  
Keyword(s):  
Amino Acid ◽  
Calcium Channel ◽  
Splice Variants ◽  
Terminal Segment ◽  
Beta Subunit ◽  
Beta Subunits ◽  
High Sequence Identity ◽  
Channel Inactivation ◽  
Calcium Channel Inactivation ◽  
Common Effect

Previous studies have shown that NH2 termini of the type 1 and 2 beta-subunits modulate the rate at which the neuronal alpha 1E calcium channel inactivates in response to voltage and that they do so independently of their common effect to stimulate activation by voltage (R. Olcese, N. Qin, T. Schneider, A. Neely, X. Wei, E. Stefani, and L. Birnbaumer, Neuron 13: 1433-1438, 1994). By constructing NH2-terminal deletions of several splice variants of beta-subunits, we have now found differences in the way they affect the rate of alpha 1E inactivation that lead us to identify a second domain that also regulates the rate of voltage-induced inactivation of the Ca2+ channel. This second domain, named segment 3, lies between two regions of high-sequence identity between all known beta-subunits and exists in two lengths (long and short), each encoded in a separate exon. Beta-Subunits with the longer 45- to 53-amino acid version cause the channel to inactivate more slowly than subunits with the shorter 7-amino acid version. As is the case for the NH2 terminus, the segment 3 does not affect the regulation of channel activation by the beta-subunit. In addition, the effect of the NH2-terminal segment prevails over that of the internal segment. This raises the possibility that phosphorylation, other types of posttranslational modification, or interaction with other auxiliary calcium channel subunits may be necessary to unmask the regulatory effect of the internal segment.

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