scholarly journals Purification of D-alanine carboxypeptidase from Escherichia coli B on a penicillin-Sepharose column

1975 ◽  
Vol 147 (1) ◽  
pp. 131-137 ◽  
Author(s):  
M Gorecki ◽  
A Bar-Eli ◽  
Y Burstein ◽  
A Patchornik ◽  
E B Chain
Keyword(s):  
Escherichia Coli ◽  
Protein Band ◽  
Enzymic Activity ◽  
Coli Strain ◽  
Sepharose Column ◽  
Absolute Requirement ◽  
Single Protein ◽  
One Step ◽  
Triton X ◽  
Escherichia Coli B

1. A soluble D-alanine carboxypeptidase from Escherichia coli strain B was purified on a p-aminobenzylpenicillin-Sepharose column. This one-step chromatography followed by an (NH4)2SO4 precipitation yielded an enzyme purified 1200-fold and some of its properties are reported. 2. The pure D-alanine carboxypeptidase was devoid of D-alanine carboxypeptidase II activity and migrated as a single protein band on analytical disc gel electrophoresis. 3. Triton X-100 in the purification procedure is an absolute requirement for obtaining a stable enzyme. 4. The enzymic activity of D-alanine carboxypeptidase was greatly affected in solution of high salt concentrations and varied somewhat with the nature of the cation tested.

scholarly journals Glycosylation of ribonuclease A catalysed by rabbit liver extracts

1975 ◽  
Vol 146 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Z Khalkhall ◽  
R D Marshall
Keyword(s):  
Metal Ion ◽  
Ribonuclease A ◽  
Enzymic Activity ◽  
Rabbit Liver ◽  
Absolute Requirement ◽  
Pancreatic Ribonuclease A ◽  
Triton X ◽  
Increased Activity ◽  
Asparagine Residue

Crude extracts of rabbit liver catalyse in vitro the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to bovine pancreatic ribonuclease A. The enzymic activity is contained in rough endoplasmic reticulum. It has an absolute requirement for a bivalent metal ion: Co-2+ greater than Mn-2+ greater than Ni-2+. Mg-2+ is ineffective. There is enzymic activity in the absence of detergent, but increased activity is observed in the presence of Triton X-100. The site of glycosylation of ribonuclease A is asparagine-34, and glycosylation occurs only at this point. These findings agree with the hypothesis that the sequence Asn-X-Thr(Ser) (where X may be one of a number of types of amino acid) is a necessary, but not sufficient, condition for N-acetylglucosaminylation of a protein-bound asparagine residue.

scholarly journals Physiochemical properties of a heat-stable enterotoxin produced by Escherichia coli of human origin

1980 ◽  
Vol 28 (3) ◽  
pp. 1051-1053
Author(s):  
G L Madsen ◽  
F C Knoop
Keyword(s):  
Escherichia Coli ◽  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Molecular Sieve ◽  
Protein Band ◽  
Biologically Active ◽  
Enterotoxigenic Escherichia Coli ◽  
Physiochemical Properties ◽  
Heat Stable ◽  
Single Protein

Concentrated cell-free filtrates, prepared from a human strain of enterotoxigenic Escherichia coli, were subjected to isoelectric focusing, molecular sieve chromatography, and polyacrylamide disc gel electrophoresis. Isoelectric focusing in a pH 3 to 5 gradient resulted in two biologically active peaks, I and II, that electrofocused at pI 1.5 and 3.8, respectively. Molecular sieve chromatography of the major enterotoxic peak (II) at pH 3.8 indicated a molecular weight of 2,500. Polyacrylamide disc gel electrophoresis of ST revealed a single protein band containing enterotoxic activity.

scholarly journals Absolute requirement for polyamines for growth of Escherichia coli mutants (mnmE/G) defective in modification of the wobble anticodon of transfer-RNA

2019 ◽  
Vol 366 (10) ◽  
Author(s):  
Christopher Keller ◽  
Manas Chattopadhyay ◽  
Herbert Tabor
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Protein Biosynthesis ◽  
Transfer Rna ◽  
Coli Strain ◽  
E Coli ◽  
Wobble Position ◽  
Inhibition Of Growth ◽  
Absolute Requirement

Abstract The genes mnmE and mnmG are responsible for the modification of uridine 34, ‘the wobble position’ of many aminoacyl-tRNAs. Deletion of these genes affects the strength of the codon-anticodon interactions of the aminoacyl-tRNAs with the mRNAs and the ribosomes. However, deletion of these genes does not usually have a significant effect on the growth rate of the standard Escherichia coli strains. In contrast, we have found that if the host E. coli strain is deficient in the synthesis of polyamines, deletion of the mnmE or mnmG gene results in complete inhibition of growth unless the medium contains polyamines. The finding of an absolute requirement for polyamines in our current work will be significant in studies on polyamine function, in studies on the function of the mnmE/G genes, and in studies on the role of aminoacyl-tRNAs in protein biosynthesis.

scholarly journals The bromoperoxidase from the lichen Xanthoria parietina is a novel vanadium enzyme

1987 ◽  
Vol 248 (1) ◽  
pp. 277-279 ◽  
Author(s):  
H Plat ◽  
B E Krenn ◽  
R Wever
Keyword(s):  
High Temperatures ◽  
Thermal Denaturation ◽  
Protein Band ◽  
Enzymic Activity ◽  
Single Protein Band ◽  
Xanthoria Parietina ◽  
Single Protein

A novel bromoperoxidase was isolated from the lichen Xanthoria parietina. The enzyme contained vanadium, which is essential for enzymic activity. Under denaturating conditions the preparation showed a single protein band with an Mr of 65,000. Thermal-denaturation studies showed that this bromoperoxidase could tolerate high temperatures. The affinity of the enzyme for its substrate bromide is high; the Km for bromide was 29 microM. Excess halides (50 mM) inhibited enzymic activity considerably.

scholarly journals Characterization of an EnterotoxigenicEscherichia coli Strain from Africa Expressing a Putative Colonization Factor

1999 ◽  
Vol 67 (8) ◽  
pp. 4019-4026 ◽  
Author(s):  
Sami B. Khalil ◽  
Frederick J. Cassels ◽  
Hind I. Shaheen ◽  
Lewis K. Pannell ◽  
Nemat El-Ghorab ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Immunoblot Analysis ◽  
Protein Band ◽  
Coli Strain ◽  
Spectrometric Analysis ◽  
Dot Blot ◽  
Single Protein Band ◽  
Single Protein

ABSTRACT An enterotoxigenic Escherichia coli (ETEC) strain of serotype O114:H− that expressed both heat-labile and heat-stable enterotoxins and tested negative for colonization factors (CF) was isolated from a child with diarrhea in Egypt. This strain, WS0115A, induced hemagglutination of bovine erythrocytes and adhered to the enterocyte-like cell line Caco-2, suggesting that it may elaborate novel fimbriae. Surface-expressed antigen purified by differential ammonium sulfate precipitation and column chromatography yielded a single protein band with M r 14,800 when resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (16% polyacrylamide). A monoclonal antibody against this putative fimbrial antigen was generated and reacted with strain WS0115A and also with CS1-, CS17-, and CS19-positive strains in a dot blot assay. Reactivity was temperature dependent, with cells displaying reactivity when grown at 37°C but not when grown at 22°C. Immunoblot analysis of a fimbrial preparation from strain WS0115A showed that the monoclonal antibody reacted with a single protein band. Electron microscopy and immunoelectron microscopy revealed fimbria-like structures on the surface of strain WS0115A. These structures were rigid and measured 6.8 to 7.4 nm in diameter. Electrospray mass-spectrometric analysis showed that the mass of the purified fimbria was 14,965 Da. The N-terminal sequence of the fimbria established that it was a member of the CFA/I family, with sequence identity to the amino terminus of CS19, a new CF recently identified in India. Cumulatively, our results suggest that this fimbria is CS19. Screening of a collection of ETEC strains isolated from children with diarrhea in Egypt found that 4.2% of strains originally reported as CF negative were positive for this CF, suggesting that it is biologically relevant in the pathogenesis of ETEC.

scholarly journals Hydrolysis of fucosyl-GM1 ganglioside by purified pellet-associated human brain and human liver α-l-fucosidases without activator proteins or detergents

1990 ◽  
Vol 266 (2) ◽  
pp. 491-496 ◽  
Author(s):  
R L Hopfer ◽  
S W Johnson ◽  
M Masserini ◽  
A Giuliani ◽  
J A Alhadeff
Keyword(s):  
Human Brain ◽  
Specific Activity ◽  
Protein Band ◽  
Enzymic Activity ◽  
Gm1 Ganglioside ◽  
Ph Optimum ◽  
Activator Proteins ◽  
Minor Protein ◽  
A Minor ◽  
Triton X

Pellet-associated human brain alpha-L-fucosidase was solubilized with 0.5% (w/v) Triton X-100 and purified by affinity chromatography on agarose-6-aminohexanoyl-fucosamine resin. The procedure resulted in a 290,000-fold purification, a 58% yield and a final specific activity of 11,500 nmol/min per mg of protein. Isoelectric focusing indicated that all six major isoforms (with pI values between 4.1 and 5.3) present in crude brain pellet preparations were purified by the affinity technique. SDS/PAGE indicated the presence of one subunit (54 kDa) and a minor protein band at 67 kDa, which presumably is a contaminant since it was not immunoreactive on Western blotting. The pH optimum of the brain enzyme and its apparent Km for the synthetic substrate 4-methylumbelliferyl alpha-L-fucopyranoside were 5.5 and 0.07 mM respectively. Pellet-associated human brain and liver alpha-L-fucosidases were both capable of hydrolysing fucosyl-GM1 ganglioside without activator proteins or detergents. Linear hydrolysis rates were found only for short incubation times (1-5 min). Optimal enzymic activity at 37 degrees C was found at pH 3.4 for both alpha-L-fucosidases, with no activity at pH values above 4.0.

NITROFURAN DERIVATIVES AS RADIOMIMETIC AGENTS: CROSS-RESISTANCE STUDIES WITH ESCHERICHIA COLI

1965 ◽  
Vol 11 (2) ◽  
pp. 185-191 ◽  
Author(s):  
D. R. McCalla
Keyword(s):  
Escherichia Coli ◽  
Ultraviolet Light ◽  
Coli Strain ◽  
Cross Resistance ◽  
Resistance Pattern ◽  
E Coli ◽  
Mutant Strains ◽  
Nitrofuran Derivatives ◽  
Escherichia Coli B

Mutants of Escherichia coli B selected on the basis of resistance to nitrofurazone, NFT, or ultraviolet light proved to be resistant to all three of these agents. Further tests showed that these mutants are also resistant to three other nitrofuran derivatives, nitrofurantoin, nihydrazone, and furazolidone, but not to nifuroxime. The mutants are also resistant to the chemically dissimilar radiomimetic agent, proflavin. Strain B and all the mutants derived from it are equally sensitive to streptomycin, a non-radiomimetic antibiotic. E. coli strain B/r (resistant to radiation and radiomimetic chemicals) shows the same resistance pattern as the new mutant strains. The results are discussed in terms of what is known concerning the basis for the resistance of strain B/r to ultraviolet light. It is concluded that radiomimetic nitrofurans probably exert their effect on E. coli through damage to DNA.

scholarly journals Solubilization and one-step purification of mannosylphosphodolichol synthase from Trichoderma reesei.

1996 ◽  
Vol 43 (2) ◽  
pp. 397-401 ◽  
Author(s):  
J S Kruszewska ◽  
U Perlińska-Lenart ◽  
G Palamarczyk
Keyword(s):  
Protein Kinase ◽  
Molecular Mass ◽  
Protease Inhibitors ◽  
Protein Band ◽  
Dependent Protein Kinase ◽  
Absolute Requirement ◽  
Sds Page ◽  
Dependent Protein ◽  
One Step ◽  
Lipophilic Substrate

Mannosylphosphodolichol synthase (MPD-synthase) (EC 2.4.1.830) catalyzing formation of MPD from GDPMan and dolichylphosphate (PD) has been purified from T. reesei cellular membranes almost to homogeneity. Selective solubilization of the enzyme was followed by one step purification on Phenyl-Sepharose column. SDS/ PAGE of the purified enzyme fraction revealed the presence of a protein band of 31 kDa corresponding to the apparent molecular mass of the MPD-synthase purified from S. cerevisiae [Babczinski, P. et al. (1980) Eur. J. Biochem. 105, 509-515; Haselbeck A. (1989) Eur. J. Biochem. 181, 663-668]. During solubilization, the enzyme was stabilized by the presence of a lipophilic substrate dolichylphosphate and phospholipids as well as by protease inhibitors. The Phenyl-Sepharose purified enzyme had an absolute requirement for dolichylphosphate and was activated by cAMP dependent protein kinase.

scholarly journals Molecular characterization of the Acremonium chrysogenum cefG gene product: the native deacetylcephalosporin C acetyltransferase is not processed into subunits

1999 ◽  
Vol 337 (3) ◽  
pp. 379-385 ◽  
Author(s):  
Javier VELASCO ◽  
Santiago GUTIERREZ ◽  
Sonia CAMPOY ◽  
Juan F. MARTIN
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Protein Band ◽  
Immunoaffinity Chromatography ◽  
Enzymic Activity ◽  
Acremonium Chrysogenum ◽  
Wild Type ◽  
E Coli ◽  
Protein Levels

Constructions starting at each of the three in-frame ATG codons of the Acremonium chrysogenum cefG gene (Met1, Met46 and Met60) were expressed in Escherichia coli, obtaining proteins of 49, 44 and 43 kDa, respectively. All three proteins showed deacetylcephalosporin C (DAC) acetyltransferase activity. The native A. chrysogenum DAC acetyltransferase was purified to electrophoretic homogeneity by immunoaffinity chromatography. It showed a molecular mass of 50 kDa by filtration in calibrated Sephadex G-75 SF or Superose 12 (FPLC) columns. The N-terminal end of the pure DAC acetyltransferase was Met-Leu-Pro-Ser-Ala-Gln-Val-Ala-Arg-Leu, which matched perfectly the deduced amino acid sequence starting at Met1. The putative α- and β-subunits of DAC acetyltransferase were also obtained in E. coli but showed no enzymic activity either separately or in combination. Immunoblotting (Western) analysis revealed that the 50 kDa DAC acetyltransferase showed high protein levels in A. chrysogenum cultures at 72 and 96 h and decreased sharply thereafter, but in all cases no detectable processing of the enzyme into subunits was found. Three different A. chrysogenum strains (including the wild-type Brotzu strain and two high-cephalosporin-producing mutants) showed the same unprocessed 50 kDa DAC acetyltransferase. The non-producer mutant ATCC 20371 showed no DAC acetyltransferase protein band but formed a normal transcript of 1.4 kb.

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