scholarly journals Preparative isolation of the two forms of pig pancreatic pro-(carboxypeptidase A) and their monomeric carboxypeptidases A

1985 ◽  
Vol 229 (3) ◽  
pp. 605-609 ◽  
Author(s):  
M Vilanova ◽  
J Vendrell ◽  
M T López ◽  
C M Cuchillo ◽  
F X Avilés
Keyword(s):  
Amino Acid ◽  
Specific Activity ◽  
High Specific Activity ◽  
High Yield ◽  
Binary Complex ◽  
Acid Hydrolase ◽  
Carboxypeptidase A ◽  
Preparative Isolation

A method is reported for the preparative isolation of the two forms of pro-(carboxypeptidase A) from pig pancreas: the monomer and the binary complex with pro-(proteinase E). This method, which is mainly based on chromatography on DEAE-Sepharose at pH 5.7, allows these proenzymes to be prepared more quickly and in safer conditions than with other reported methods. Undegraded and homogeneous carboxypeptidase A1 and A2 species (peptidyl-L-amino acid hydrolase, EC 3.4.17.1), in monomeric forms with high specific activity, are also obtained in high yield by controlled trypsin activation of either of the pro-(carboxypeptidases A) followed by chromatography on DEAE-Sepharose at pH 5.8 under dissociating conditions (7 M-urea).

Synthesis and N.M.R. Spectra of Substituted Aminoiodoacridines

1979 ◽  
Vol 32 (12) ◽  
pp. 2637 ◽  
Author(s):  
RF Martin ◽  
DP Kelly
Keyword(s):  
Electron Density ◽  
Electrophilic Substitution ◽  
Specific Activity ◽  
Diazonium Salt ◽  
High Specific Activity ◽  
High Yield ◽  
Model Compounds ◽  
Iodide Ion ◽  
Ion Substitution ◽  
Chloramine T

3-Amino-6-iodoacridine (10), 3,6-diiodoacridine (11) and 9-amino-2-ethoxy-6-iodoacridine (14) were prepared by iodide ion substitution of the corresponding diazonium salt whereas 3,6-diamino-4,5-diiodoacridine (12) and 6,9-diamino-2-ethoxy-5-iodoacridine (13) were prepared by direct iodination with iodide ion in the presence of chloramine-T. The latter reaction proceeded in relatively high yield and has been used for the synthesis of high specific activity 125I-labelled compounds (12), (13). The 1H and 13C N.M.R. spectra of (10)-(14) and model compounds indicate higher electron density at C4(C5) than at C2(C7) in 3(6)-amino-substituted acridines in agreement with the observed pattern of electrophilic substitution.

scholarly journals A New Technic for the Production of an in Vivo Labeled Fibrinogen

Blood ◽  
1967 ◽  
Vol 29 (4) ◽  
pp. 517-525 ◽  
Author(s):  
HENRY GANS ◽  
JAMES MC LEOD ◽  
JAMES T. LOWMAN
Keyword(s):  
Amino Acid ◽  
Specific Activity ◽  
High Specific Activity ◽  
Entire Period ◽  
Turnover Rates ◽  
Prolonged Infusion ◽  
Slow Infusion

Abstract The fact that in vitro labeled proteins, as a rule, exhibit faster turnover rates than in vivo labeled materials led us to explore means of obtaining in vivo labeled fibrinogen of high specific activity. It was found that defibrination of the rat provides a stimulus for the liver to regenerate fibrinogen at an accelerated rate. Administration of seleno75 methionine shortly after thrombin-induced defibrination of the animal resulted in the incorporation of large quantities of the label. The rate of incorporation was further increased if the amino acid was administered as a slow infusion during the entire period of fibrinogen regeneration. In addition, prior nephrectomy of the animal would appear to result in a slight increase in specific activity of the fibrinogen preparation obtained. The results of these studies indicate that defibrination, nephrectomy, and the prolonged infusion of the labeled amino acid selenomethionine provided us with a technic for obtaining a biosynthetically labeled, γ-emitting, fibrinogen preparation of high specific activity.

Preparation of 14C-Labeled Penicillic Acid with High Specific Activity and Yield

1977 ◽  
Vol 40 (2) ◽  
pp. 90-93 ◽  
Author(s):  
DOUGLAS L. PARK ◽  
PHILIP B. MISLIVEC ◽  
JAMES L. HEATH
Keyword(s):  
Acid Production ◽  
Specific Activity ◽  
High Specific Activity ◽  
High Yield ◽  
Liquid Media ◽  
Penicillic Acid ◽  
Stationary Culture ◽  
Radioactive Compound ◽  
Growth Substrates ◽  
Label Incorporation

14C-Labeled penicillic acid was produced by stationary culture incubation of Penicillium cyclopium (NRRL 1888) on a modified Raulin-Thom broth medium containing 14C-labeled acetate. Approximately 1.2 g of radioactive compound, with a specific activity of 23.0 μCi/mmole, was produced in 9 days in 1500 ml of the broth. Incorporation of the isotope into penicillic acid was 11. 9%. Production of the radiolabeled compound with high specific activity was achieved by correlating the monitoring of expired 14C-CO2 with production of penicillic acid during the fermentation. The effects of various growth substrates, pH, and incubation times on production of non-labeled penicillic acid also were investigated. Results show that sterile rice is an excellent substrate, that among liquid media examined, higher yields were obtained in stationary rather than in shake cultures, and that higher yields of penicillic acid were obtained at pH 3.5 or lower. Simultaneous monitoring of penicillic acid production and 14C-label incorporation is essential to detect and isolate a high yield of labeled compound with high specific activity.

Directed evolution to produce an alkalophilic variant from a Neocallimastix patriciarum xylanase

2001 ◽  
Vol 47 (12) ◽  
pp. 1088-1094 ◽  
Author(s):  
Yew-Loom Chen ◽  
Tsung-Yin Tang ◽  
Kuo-Joan Cheng
Keyword(s):  
Amino Acid ◽  
Directed Evolution ◽  
Catalytic Domain ◽  
Specific Activity ◽  
Synergistic Effects ◽  
High Specific Activity ◽  
Site Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Neocallimastix Patriciarum

The catalytic domain of a xylanase from the anaerobic fungus Neocallimastix patriciarum was made more alkalophilic through directed evolution using error-prone PCR. Transformants expressing the alkalophilic variant xylanases produced larger clear zones when overlaid with high pH, xylan-containing agar. Eight amino acid substitutions were identified in six selected mutant xylanases. Whereas the wild-type xylanase exhibited no activity at pH 8.5, the relative and specific activities of the six mutants were higher at pH 8.5 than at pH 6.0. Seven of the eight amino acid substitutions were assembled in one enzyme (xyn-CDBFV) by site-directed mutagenesis. Some or all of the seven mutations exerted positive and possibly synergistic effects on the alkalophilicity of the enzyme. The resulting composite mutant xylanase retained a greater proportion of its activity than did the wild type at pH above 7.0, maintaining 25% of its activity at pH 9.0, and its retention of activity at acid pH was no lower than that of the wild type. The composite xylanase (xyn-CDBFV) had a relatively high specific activity of 10 128 µmol glucose·min–1·(mg protein)–1 at pH 6.0. It was more thermostable at 60°C and alkaline tolerant at pH 10.0 than the wild-type xylanase. These properties suggest that the composite mutant xylanase is a promising and suitable candidate for paper pulp bio-bleaching.Key words: xylanase, Neocallimastix patriciarum, alkalophilicity, random mutagenesis, directed evolution.

scholarly journals High yield and high specific activity synthesis of [18F]fallypride in a batch microfluidic reactor for micro-PET imaging

2014 ◽  
Vol 50 (10) ◽  
pp. 1192-1194 ◽  
Author(s):  
Muhammad Rashed Javed ◽  
Supin Chen ◽  
Jack Lei ◽  
Jeffrey Collins ◽  
Maxim Sergeev ◽  
...  
Keyword(s):  
Pet Imaging ◽  
Specific Activity ◽  
High Specific Activity ◽  
High Yield ◽  
Microfluidic Reactor

scholarly journals High yield synthesis of high specific activity R-(−)-[11C]epinephrine for routine PET studies in humans

1993 ◽  
Vol 20 (8) ◽  
pp. 939-944 ◽  
Author(s):  
Pulak K. Chakraborty ◽  
David L. Gildersleeve ◽  
Douglas M. Jewett ◽  
Steve A. Toorongian ◽  
Michael R. Kilbourn ◽  
...  
Keyword(s):  
Specific Activity ◽  
High Specific Activity ◽  
High Yield

scholarly journals Both Subunits of ATP-Citrate Lyase from Chlorobium tepidum Contribute to Catalytic Activity

2006 ◽  
Vol 188 (18) ◽  
pp. 6544-6552 ◽  
Author(s):  
Wonduck Kim ◽  
F. Robert Tabita
Keyword(s):  
Catalytic Mechanism ◽  
Specific Activity ◽  
High Specific Activity ◽  
Small Subunit ◽  
High Yield ◽  
Chlorobium Tepidum ◽  
Atp Citrate Lyase ◽  
Citrate Lyase ◽  
Green Sulfur

ABSTRACT ATP-citrate lyase (ACL) is an essential enzyme of the reductive tricarboxylic acid (RTCA) pathway of CO2 assimilation. The RTCA pathway occurs in several groups of autotrophic prokaryotes, including the green sulfur bacteria. ACL catalyzes the coenzyme A (CoA)-dependent and MgATP-dependent cleavage of citrate into oxaloacetate and acetyl-CoA, representing a key step in the RTCA pathway. To characterize this enzyme from the green sulfur bacterium Chlorobium tepidum and determine the role of its two distinct polypeptide chains, recombinant holo-ACL as well as its two individual subunit polypeptides were synthesized in Escherichia coli. The recombinant holoenzyme, prepared from coexpressed large and small ACL genes, and the individual large and small subunit polypeptides, prepared from singly expressed genes, were all purified to homogeneity to high yield. Purified recombinant holo-ACL was isolated at high specific activity, and its k cat was comparable to that of previously prepared native C. tepidum ACL. Moreover, the purified recombinant large and small subunit polypeptides were able to reconstitute the holo-ACL in vitro, with activity levels approaching that of recombinant holo-ACL prepared from coexpressed genes. Stoichiometric amounts of each subunit protein were required to maximize the activity and form the most stable structure of reconstituted holo-ACL. These results suggested that this reconstitution system could be used to discern the catalytic role of specific amino acid residues on each subunit. Reconstitution and mutagenesis studies together indicated that residues of each subunit contributed to different aspects of the catalytic mechanism, suggesting that both subunit proteins contribute to the active site of C. tepidum ACL.

scholarly journals DNA sequence homology between the terminal inverted repeats of Shope fibroma virus and an endogenous cellular plasmid species

1986 ◽  
Vol 6 (1) ◽  
pp. 265-276
Author(s):  
C Upton ◽  
G McFadden
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Sequence Homology ◽  
Inverted Repeat ◽  
Specific Activity ◽  
High Specific Activity ◽  
Cesium Chloride ◽  
Open Reading Frame ◽  
Terminal Inverted Repeat ◽  
Reading Frame

DNA hybridization experiments indicate that the genome of a tumorigenic poxvirus. Shope fibroma virus (SFV), possesses sequence homology with DNA isolated from uninfected rabbit cells. Southern blotting experiments, either with high-complexity rabbit DNA as probe and SFV restriction fragments as targets or with high-specific activity, 32P-labeled, cloned SFV sequences as probes and rabbit DNA as target, indicate that the homologous sequences map at two locations within the viral genome, one in each copy of the terminal inverted repeat sequences. Unexpectedly, Southern blots revealed that the homologous host sequences reside in a rabbit extrachromosomal DNA element. This autonomous low-molecular-weight DNA species could be specifically amplified by cycloheximide treatment and was shown by isopycnic centrifugation in cesium chloride-ethidium bromide to consist predominantly of covalently closed circular DNA molecules. DNA sequencing of pSIC-9, a cloned 1.9-kilobase fragment of the rabbit plasmid species, indicated extensive homology at the nucleotide level over a 1.5-kilobase stretch of the viral terminal inverted repeat. Analysis of open reading frames in both the plasmid and SFV DNA revealed that (i) the N-terminal 157-amino acid sequence of a potential 514-amino acid SFV polypeptide is identical to the N-terminal 157 amino acids of one pSIC-9 open reading frame, and (ii) a second long pSIC-9 open reading frame of 361 amino acids, although significantly diverged from the comparable nucleotide sequence in the virus, possessed considerable homology to a family of cellular protease inhibitors, including alpha 1-antichymotrypsin, alpha 1-antitrypsin, and antithrombin III. The potential role of such cellular plasmid-like DNA species as a mediator in the exchange of genetic information between the host cell and a cytoplasmically replicating poxvirus is discussed.

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 purification in high yield and characterization of rat hepatic glucokinase

1976 ◽  
Vol 153 (2) ◽  
pp. 363-373 ◽  
Author(s):  
M J Holroyde ◽  
M B Allen ◽  
A C Storer ◽  
A S Warsy ◽  
J M E Chesher ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Affinity Chromatography ◽  
Amino Acid Composition ◽  
Specific Activity ◽  
Sedimentation Equilibrium ◽  
High Yield ◽  
Equilibrium Studies ◽  
Enzyme Specific Activity

A new improved procedure for the purification of rat hepatic glucokinase (ATP-d-glucose 6-phosphotransferase, EC 2.7.1.2) is given. A key step is affinity chromatography on Sepharose-N-(6-aminohexanoyl)-2-amino-2-deoxy-d-glucopyranose. A homogeneous enzyme, specific activity 150 units/mg of protein, is obtained in about 40% yield. The molecular weight of the pure enzyme was determined by several procedures. In particular, sedimentation-equilibrium studies under a variety of conditions indicate a molecular weight of 48000 and no evidence for dimerization; reports in the literature of other values are discussed in the light of this evidence on the pure enzyme. The amino acid composition suggests that hepatic glucokinase is closely related to rat brain hexokinase and also the wheat “light” hexokinases.

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