THE LOCALIZATION AND PROPERTIES OF AN AMINOPEPTIDASE IN ESCHERICHIA COLI B

1963 ◽  
Vol 41 (1) ◽  
pp. 9-18 ◽  
Author(s):  
A. T. Matheson
Keyword(s):  
Membrane Fraction ◽  
Specific Activity ◽  
Soluble Fraction ◽  
Progressive Increase ◽  
Aminopeptidase Activity ◽  
Peptidase Activity ◽  
Phase Growth ◽  
Latent Form ◽  
E Coli ◽  
Escherichia Coli B

Two types of intracellular aminopeptidase activity are present in E. coli B. One type, present in the 'soluble' fraction, is completely inactivated by chymotrypsin or trypsin; the other, in the particulate fractions ('ribosome' and 'membrane'), is resistant to these enzymes. The 'ribosomal' peptidase activity is present partially in a latent form which becomes activated on disruption of the ribosome structure. During the transition from log phase to post-log phase growth there is a progressive increase in the specific activity of the peptidase in the 'soluble' and 'membrane' fraction and a corresponding decrease in the 'ribosome' fraction.

THE LOCALIZATION AND PROPERTIES OF AN AMINOPEPTIDASE IN ESCHERICHIA COLI B

1963 ◽  
Vol 41 (1) ◽  
pp. 9-18 ◽  
Author(s):  
A. T. Matheson
Keyword(s):  
Membrane Fraction ◽  
Specific Activity ◽  
Soluble Fraction ◽  
Progressive Increase ◽  
Aminopeptidase Activity ◽  
Peptidase Activity ◽  
Phase Growth ◽  
Latent Form ◽  
E Coli ◽  
Escherichia Coli B

Two types of intracellular aminopeptidase activity are present in E. coli B. One type, present in the 'soluble' fraction, is completely inactivated by chymotrypsin or trypsin; the other, in the particulate fractions ('ribosome' and 'membrane'), is resistant to these enzymes. The 'ribosomal' peptidase activity is present partially in a latent form which becomes activated on disruption of the ribosome structure. During the transition from log phase to post-log phase growth there is a progressive increase in the specific activity of the peptidase in the 'soluble' and 'membrane' fraction and a corresponding decrease in the 'ribosome' fraction.

Activity and location of two enzyme fractions during the culture cycle of Schizosaccharomyces pombe

1970 ◽  
Vol 16 (3) ◽  
pp. 187-191 ◽  
Author(s):  
Gail Dolan Rock ◽  
B. F. Johnson
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Cell Walls ◽  
Membrane Fraction ◽  
Soluble Fraction ◽  
Total Activity ◽  
Aminopeptidase Activity ◽  
Peptidase Activity ◽  
E Coli ◽  
Culture Cycle ◽  
Ribonuclease I

When cell walls of Schizosaccharomyces pombe were removed, the protoplast contained most of the ribonuclease but only about 50% of the aminopeptidase activity. In cell homogenates approximately 75% of the total peptidase activity was in the soluble fraction; the membrane fraction retained an average of 25% while the ribosomes had less than 1% of the total activity. The RNase activity was highest in stationary phase, aminopeptidase at mid log phase. Properties of the soluble aminopeptidase were similar in many respects to those of the enzyme from Escherichia coli. In contrast, 90% of the ribonuclease activity was attached to the membrane fraction and up to 10% was found on the ribosomes. The ribosome-bound ribonuclease required incubation in 4 M urea for activation; however, the purified ribonuclease had properties similar to the ribonuclease I of E. coli.

Localization of the Basic Aminopeptidases in Escherichia coli

1971 ◽  
Vol 49 (12) ◽  
pp. 1340-1346 ◽  
Author(s):  
A. T. Matheson ◽  
L. P. Visentin ◽  
A. Boutet ◽  
C. F. Rollin
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Ribosomal Subunit ◽  
Growth Conditions ◽  
Aminopeptidase Activity ◽  
E Coli ◽  
Ribosomal Particle ◽  
K 12 ◽  
Ribonuclease I ◽  
Similar Growth

The basic aminopeptidase activity was found in both the 30 S and 50 S ribosomal subunit of Escherichia coli B; the specific activity of the enzyme in the 30 S particle was about six times that found in the 50 S particle. Although about 50% of the enzyme was released from E. coli B ribosomes on dissociation into the subunits, this was not observed with E. coli Q-13, a ribonuclease I-less strain. The partition of the basic aminopeptidase activity between the soluble and ribosomal fractions (isolated in 60 mM KCl) of E. coli was determined by the strain of E. coli used, as well as the stage and temperature of growth. Under similar growth conditions, a significantly greater amount of enzyme was found in the ribosomal fraction of E. coli B than in the corresponding fraction of E. coli K-12. When E. coli B cells were disrupted in 300 mM KCl, over 90% of the enzyme was found in the soluble fraction. The remaining 10% on the ribosomal particle had enriched activity towards methionyl peptides suggesting that more than one enzyme is present in the basic aminopeptidase fraction.

scholarly journals PepN is the major aminopeptidase in Escherichia coli: insights on substrate specificity and role during sodium-salicylate-induced stress

Microbiology ◽  
2003 ◽  
Vol 149 (12) ◽  
pp. 3437-3447 ◽  
Author(s):  
Dilip Chandu ◽  
Dipankar Nandi
Keyword(s):  
Escherichia Coli ◽  
Sodium Salicylate ◽  
Physiological Role ◽  
Negative Regulator ◽  
Aminopeptidase Activity ◽  
Peptidase Activity ◽  
E Coli ◽  
Induced Stress ◽  
Hydrolysis Of

PepN and its homologues are involved in the ATP-independent steps (downstream processing) during cytosolic protein degradation. To obtain insights into the contribution of PepN to the peptidase activity in Escherichia coli, the hydrolysis of a selection of endopeptidase and exopeptidase substrates was studied in extracts of wild-type strains and two pepN mutants, 9218 and DH5αΔpepN. Hydrolysis of three of the seven endopeptidase substrates tested was reduced in both pepN mutants. Similar studies revealed that hydrolysis of 10 of 14 exopeptidase substrates studied was greatly reduced in both pepN mutants. This decreased ability to cleave these substrates is pepN-specific as there is no reduction in the ability to hydrolyse exopeptidase substrates in E. coli mutants lacking other peptidases, pepA, pepB or pepE. PepN overexpression complemented the hydrolysis of the affected exopeptidase substrates. These results suggest that PepN is responsible for the majority of aminopeptidase activity in E. coli. Further in vitro studies with purified PepN revealed a preference to cleave basic and small amino acids as aminopeptidase substrates. Kinetic characterization revealed the aminopeptidase cleavage preference of E. coli PepN to be Arg>Ala>Lys>Gly. Finally, it was shown that PepN is a negative regulator of the sodium-salicylate-induced stress in E. coli, demonstrating a physiological role for this aminoendopeptidase under some stress conditions.

scholarly journals Identification of a Novel Membrane-Associated Gene Product That Suppresses Toxicity of a TrfA Peptide from Plasmid RK2 and Its Relationship to the DnaA Host Initiation Protein

2003 ◽  
Vol 185 (6) ◽  
pp. 1817-1824 ◽  
Author(s):  
Peter D. Kim ◽  
Trevor Banack ◽  
Daniel M. Lerman ◽  
Jeremiah C. Tracy ◽  
Johanna Eltz Camara ◽  
...  
Keyword(s):  
Membrane Fraction ◽  
Soluble Fraction ◽  
Host Protein ◽  
Broad Host Range ◽  
Terminal Portion ◽  
Inner Membrane ◽  
E Coli ◽  
Amino Terminal ◽  
Broad Host Range Plasmid ◽  
Plasmid Rk2

ABSTRACT The toxicity of a peptide derived from the amino-terminal portion of 33-kDa TrfA, one of the initiation proteins encoded by the broad-host-range plasmid RK2, was suppressed by a host protein related to DnaA, the initiation protein of Escherichia coli. The newly identified 28.4-kDa protein, termed a DnaA paralog (Dp) because it is similar to a region of DnaA but likely has a different function in initiation of plasmid RK2 replication, interacts physically with the 33-kDa TrfA initiation protein, including the initiation-active monomeric form. The Dp has a cellular distribution similar to that of the 33-kDa TrfA initiation protein, being found primarily in the inner membrane fraction, with lesser amounts detected in the outer membrane fraction and almost none in the soluble fraction of E. coli. Maintenance and inner membrane-associated replication of plasmid RK2 were enhanced in a Dp knockout strain and inhibited in strains containing extra copies of the Dp gene or in membrane extracts to which a tagged form of Dp was added. Recently, the Dp was independently shown to help prevent overinitiation in E. coli and was termed Hda (S. Kato and T. Katayama, EMBO J. 20:4253-4262, 2001).

Sites of Adsorption of the Bacteriophages T3 and T5 on the Wall of Escherichia Coli B.

1967 ◽  
Vol 25 ◽  
pp. 96-97
Author(s):  
Manfred E. Bayer
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Electron Microscope ◽  
Uranyl Acetate ◽  
E Coli ◽  
Receptor Sites ◽  
Rigid Cell Wall ◽  
Host Cell Surface ◽  
Bacterial Viruses ◽  
Escherichia Coli B

Bacterial viruses adsorb specifically to receptors on the host cell surface. Although the chemical composition of some of the cell wall receptors for bacteriophages of the T-series has been described and the number of receptor sites has been estimated to be 150 to 300 per E. coli cell, the localization of the sites on the bacterial wall has been unknown.When logarithmically growing cells of E. coli are transferred into a medium containing 20% sucrose, the cells plasmolize: the protoplast shrinks and becomes separated from the somewhat rigid cell wall. When these cells are fixed in 8% Formaldehyde, post-fixed in OsO4/uranyl acetate, embedded in Vestopal W, then cut in an ultramicrotome and observed with the electron microscope, the separation of protoplast and wall becomes clearly visible, (Fig. 1, 2). At a number of locations however, the protoplasmic membrane adheres to the wall even under the considerable pull of the shrinking protoplast. Thus numerous connecting bridges are maintained between protoplast and cell wall. Estimations of the total number of such wall/membrane associations yield a number of about 300 per cell.

scholarly journals Triosephosphate isomerase deficiency: consequences of an inherited mutation at mRNA, protein and metabolic levels

2005 ◽  
Vol 392 (3) ◽  
pp. 675-683 ◽  
Author(s):  
Judit Oláh ◽  
Ferenc Orosz ◽  
László G. Puskás ◽  
László Hackler ◽  
Margit Horányi ◽  
...  
Keyword(s):  
Steady State ◽  
Triosephosphate Isomerase ◽  
Specific Activity ◽  
Energy State ◽  
Dihydroxyacetone Phosphate ◽  
Peptidase Activity ◽  
Mrna Levels ◽  
Regulatory Enzymes ◽  
Computational Data ◽  
Fructose 1,6 Bisphosphate

Triosephosphate isomerase (TPI) deficiency is a unique glycolytic enzymopathy coupled with neurodegeneration. Two Hungarian compound heterozygote brothers inherited the same TPI mutations (F240L and E145Stop), but only the younger one suffers from neurodegeneration. In the present study, we determined the kinetic parameters of key glycolytic enzymes including the mutant TPI for rational modelling of erythrocyte glycolysis. We found that a low TPI activity in the mutant cells (lower than predicted from the protein level and specific activity of the purified recombinant enzyme) is coupled with an increase in the activities of glycolytic kinases. The modelling rendered it possible to establish the steady-state flux of the glycolysis and metabolite concentrations, which was not possible experimentally due to the inactivation of the mutant TPI and other enzymes during the pre-steady state. Our results showed that the flux was 2.5-fold higher and the concentration of DHAP (dihydroxyacetone phosphate) and fructose 1,6-bisphosphate increased 40- and 5-fold respectively in the erythrocytes of the patient compared with the control. Although the rapid equilibration of triosephosphates is not achieved, the energy state of the cells is not ‘sick’ due to the activation of key regulatory enzymes. In lymphocytes of the two brothers, the TPI activity was also lower (20%) than that of controls; however, the remaining activity was high enough to maintain the rapid equilibration of triosephosphates; consequently, no accumulation of DHAP occurs, as judged by our experimental and computational data. Interestingly, we found significant differences in the mRNA levels of the brothers for TPI and some other, apparently unrelated, proteins. One of them is the prolyl oligopeptidase, the activity decrease of which has been reported in well-characterized neurodegenerative diseases. We found that the peptidase activity of the affected brother was reduced by 30% compared with that of his neurologically intact brother.

scholarly journals Identification and Function of the pdxY Gene, Which Encodes a Novel Pyridoxal Kinase Involved in the Salvage Pathway of Pyridoxal 5′-Phosphate Biosynthesis in Escherichia coli K-12

1998 ◽  
Vol 180 (7) ◽  
pp. 1814-1821 ◽  
Author(s):  
Yong Yang ◽  
Ho-Ching Tiffany Tsui ◽  
Tsz-Kwong Man ◽  
Malcolm E. Winkler
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Specific Activity ◽  
Salvage Pathway ◽  
Pyridoxal Kinase ◽  
Triple Mutant ◽  
Reading Frame ◽  
E Coli ◽  
Specific Activities ◽  
K 12

ABSTRACT pdxK encodes a pyridoxine (PN)/pyridoxal (PL)/pyridoxamine (PM) kinase thought to function in the salvage pathway of pyridoxal 5′-phosphate (PLP) coenzyme biosynthesis. The observation that pdxK null mutants still contain PL kinase activity led to the hypothesis that Escherichia coli K-12 contains at least one other B6-vitamer kinase. Here we support this hypothesis by identifying the pdxY gene (formally, open reading frame f287b) at 36.92 min, which encodes a novel PL kinase. PdxY was first identified by its homology to PdxK in searches of the complete E. coli genome. Minimal clones of pdxY + overexpressed PL kinase specific activity about 10-fold. We inserted an omega cassette intopdxY and crossed the resultingpdxY::ΩKanr mutation into the bacterial chromosome of a pdxB mutant, in which de novo PLP biosynthesis is blocked. We then determined the growth characteristics and PL and PN kinase specific activities in extracts ofpdxK and pdxY single and double mutants. Significantly, the requirement of the pdxB pdxK pdxY triple mutant for PLP was not satisfied by PL and PN, and the triple mutant had negligible PL and PN kinase specific activities. Our combined results suggest that the PL kinase PdxY and the PN/PL/PM kinase PdxK are the only physiologically important B6vitamer kinases in E. coli and that their function is confined to the PLP salvage pathway. Last, we show thatpdxY is located downstream from pdxH (encoding PNP/PMP oxidase) and essential tyrS (encoding aminoacyl-tRNATyr synthetase) in a multifunctional operon.pdxY is completely cotranscribed with tyrS, but about 92% of tyrS transcripts terminate at a putative Rho-factor-dependent attenuator located in thetyrS-pdxY intercistronic region.

scholarly journals EXPLORATION OF BROTH CHICKEN GUT AS GROWTH MEDIA OF Escherichia coli BL21 pET-Endo FOR ENDO-Β-1,4-D-XYLANASE PRODUCTION

2017 ◽  
Vol 13 (2) ◽  
pp. 191
Author(s):  
Anak Agung Istri Ratnadewi ◽  
Moch. Yoris Alidion ◽  
Agung Budi Santoso ◽  
Ika Oktavianawatia
Keyword(s):  
Large Scale ◽  
Incubation Temperature ◽  
Specific Activity ◽  
Hydrolytic Enzyme ◽  
Optimal Growth ◽  
Good Alternative ◽  
Growth Media ◽  
Gene Encoding ◽  
Xylanase Production ◽  
E Coli

<p>Endo-β-1,4-D-xylanase is a hydrolytic enzyme that breakdown the 1.4 chain of xylan polysaccharide. We have succes to transform the plasmid pET-Endo gene encoding endo-1,4-β-D-xylanase from Bacillus sp. originally from termites abdominal to E. coli BL21. The clone was ready for large scale of enzyme production. To reduce production cost, we look for subtitute media for the expensive Luria Berthani broth. Chicken guts broth is good alternative while rich of protein and very cheap. The content of N dissolved chicken guts broth reaches 87 % of LB broth. Growth of E. Coli BL21 in Chicken guts broth and LB broth (as control) was observed by Optical Density (OD) using spectrofotometer. Concentration of glucose added in broth and incubation temperature was varied. The result showed that optimal growth was in addition of 1.5 % glucose and incubated at  37 <sup>o</sup>C for 16 h. This optimal condition was used to grow E. coli BL21 pET-Endo for xylanase production. Enzyme purification was done by Ni-NTA affinity chromatography. Highest protein yield was 0.076 mg/mL obtained in 100 mM imidazole elucidation. The activity and specific activity of xylanase were estimated as 0.042 U/mL and 0.556 U/µg, respectively. The purification factor was 3.16 time and the molecular weight of enzyme was ± 30, 000 Dalton</p>

scholarly journals EXPRESSION OF b-XYLOSIDASE ENCODING GENE IN PHIS/ Bacillus megaterium MS SYSTEM

2015 ◽  
Vol 2 (1) ◽  
pp. 25 ◽  
Author(s):  
Sri Sumarsih
Keyword(s):  
Culture Medium ◽  
Culture Supernatant ◽  
Specific Activity ◽  
Agar Medium ◽  
Nitrilotriacetic Acid ◽  
Protoplast Transformation ◽  
E Coli ◽  
Recombinant Cells ◽  
Relative Molecular Weight ◽  
Encoding Gene

b-Xylosidase encoding gene from G. thermoleovorans IT-08 had been expressed in the pHIS1525/ B. megaterium MS941 system. The b-xylosidase gene (xyl) was inserted into plasmid pHIS1525 and propagated in E. coli DH10b. The recombinant plasmid was transformed into B. megaterium MS941 by protoplast transformation. Transformants were selected by growing the recombinant cells on solid LB medium containing tetracycline (10 µg/ ml). The expression of the b-xylosidase gene was assayed by overlaid the recombinant B. megaterium MS941 cell with agar medium containing 0.2% ethylumbelliferyl-b-D-xyloside (MUX). This research showed that the b-xylosidase gene was succesfully sub-cloned in pHIS1525 system and expressed by the recombinant B. megaterium MS941. Theaddition of 0.5% xylose into the culture medium could increase the activity of recombinantactivity of recombinant of recombinantb-xylosidase by 2.74 fold. The recombinant B. megaterium MS941 secreted 75.56% of the expressed b-xylosidase into culture medium. The crude extract b-xylosidase showed the optimum activity at 50° C and pH 6. The recombinant b-xylosidase was purified from culture supernatant by affinity chromatographic method using agarose containing Ni-NTA (Nickel-Nitrilotriacetic acid). The pure b-xylosidase showed a specific activity of 10.06 Unit/mg protein and relative molecular weight ± 58 kDa.

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