scholarly journals The Escherichia coli yjfP Gene Encodes a Carboxylesterase Involved in Sugar Utilization during Diauxie

2015 ◽  
Vol 25 (6) ◽  
pp. 412-422 ◽  
Author(s):  
Nat Johns ◽  
Algevis Wrench ◽  
Flavia Loto ◽  
Ricardo Valladares ◽  
Graciela Lorca ◽  
...  
Keyword(s):  
Western Blot ◽  
Wild Type Strain ◽  
Metabolizable Energy ◽  
Lag Phase ◽  
Purification And Characterization ◽  
Qrt Pcr ◽  
Gene And Protein Expression ◽  
Diauxic Lag ◽  
Detoxification Process

<b><i>Background:</i></b> Acetylation and efflux of carbohydrates during cellular metabolism is a well-described phenomenon associated with a detoxification process to prevent metabolic congestion. It is still unclear why cells discard important metabolizable energy sources in the form of acetylated compounds. <b><i>Methods:</i></b> We describe the purification and characterization of an approximately 28-kDa intracellular carboxylesterase (YjfP) and the analysis of gene and protein expression by qRT-PCR and Western blot. <b><i>Results:</i></b> qRT-PCR and Western blot, respectively, showed that y<i>jfP</i> is upregulated during the diauxic lag in cells growing with a mixture of glucose and lactose. The β-galactosidase activity in the &#x0394;<i>yjfP</i> strain was both delayed and half the magnitude of that of the wild-type strain. YjfP-hyperproducing strains displayed a long lag phase when cultured with glucose and then challenged to grow with lactose or galactose as the sole carbon source. <b><i>Conclusion:</i></b> Our results suggest that YjfP controls the intracellular concentration of acetyl sugars by redirecting them to the main metabolic circuits. Instead of detoxification, we propose that sugar acetylation is utilized by the cell for protection and to prevent the metabolism of a necessary minimal intracellular sugar pool. Those sugars can eventually be exported as a side effect of these mechanisms.

Purification and characterization of a β-glucosidase from Streptomyces lividans 66

1990 ◽  
Vol 36 (1) ◽  
pp. 53-56 ◽  
Author(s):  
Anca Mihoc ◽  
Dieter Kluepfel
Keyword(s):  
High Performance Liquid Chromatography ◽  
Molecular Sieve ◽  
Type Strain ◽  
High Performance ◽  
Wild Type Strain ◽  
Streptomyces Lividans ◽  
Wild Type ◽  
Purification And Characterization ◽  
Anion Exchange Column

An intracellular β-1, 4-D-glucosidase (EC 3.2.1.21) was isolated from the mutant strain HP-3 of Streptomyces lividans 66 which produced about 12 times more enzyme than the wild-type strain. The purification was carried out by anion exchange column chromatography followed by high-performance liquid chromatography on DEAE and on molecular sieve columns. The enzyme is glycosylated and has an apparent Mr of 51 000 and a pI of 4.3. Its activity was optimal at pH 6.5 and at a temperature of 40 °C. The Km and the Vmax on cellobiose were 3.1 mM and 65.6 μmol min−1 mg−1 of enzyme. Key words: β-glucosidase, Streptomyces lividans, purification, characterization.

scholarly journals ERK Phosphorylation Regulates the Aml1/Runx1 Splice Variants and the TRP Channels Expression during the Differentiation of Glioma Stem Cell Lines

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2052
Author(s):  
Giorgio Santoni ◽  
Massimo Nabissi ◽  
Consuelo Amantini ◽  
Matteo Santoni ◽  
Lucia Ricci-Vitiani ◽  
...  
Keyword(s):  
Stem Cells ◽  
Protein Expression ◽  
Western Blot ◽  
Trp Channels ◽  
Splice Variants ◽  
Annexin V ◽  
Qrt Pcr ◽  
Trpv1 Channels ◽  
Erk Phosphorylation ◽  
Gene And Protein Expression

The identification of cancer stem cells in brain tumors paved the way for new therapeutic approaches. Recently, a role for the transcriptional factor Runx1/Aml1 and the downstream ion channel genes in brain cancer development and progression has been suggested. This study aimed to explore the expression and the role of Runx1/Aml1, its Aml1b and Aml1c splice variants and the downstream TRPA1 and TRPV1 ion channels in undifferentiated and day-14 differentiated neural stem cells (NSCs and D-NSCs) and glioblastoma stem cells (GSCs and D-GSCs) lines with different proneural (PN) or mesenchymal (MES) phenotype. Gene and protein expression were evaluated by qRT-PCR, cytofluorimetric, western blot and confocal microscopy analyses. Moreover, by western blot, we observed that ERK phosphorylation enhances the Aml1b and Aml1c protein expression during glioma differentiation. Furthermore, the agonists of TRPA1 and TRPV1 channels stimulated apoptosis/necrosis in GSCs and D-GSCs as evaluated by Annexin V and PI staining and cytofluorimetric analysis. Finally, by qRT-PCR, the modulation of Wnt/β catenin, FGF, and TGFβ/SMAD signaling pathways in PN- and MES-GSCs was reported. Overall, our results provide new evidence regarding Runx1/Aml1 isoform overexpression and modulation in TRP channel expression during gliomagenesis, thus offering new directions for glioblastoma therapy.

scholarly journals Purification and characterization of a C5a-inactivating enzyme from human peritoneal fluid

Blood ◽  
1995 ◽  
Vol 85 (12) ◽  
pp. 3503-3509 ◽  
Author(s):  
SK Ayesh ◽  
Y Azar ◽  
II Barghouti ◽  
JM Ruedi ◽  
BM Babior ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Bovine Serum Albumin ◽  
Familial Mediterranean Fever ◽  
Western Blot ◽  
Specific Activity ◽  
Purification And Characterization ◽  
Inhibitory Antibody ◽  
Mediterranean Fever ◽  
Crude Material

Earlier work has suggested that familial Mediterranean fever, an inherited disorder characterized by sporadic episodes of inflammation involving the pleural and peritoneal cavities and the joints, is caused by the lack of a C5a inactivator normally found in serosal fluid. We have purified this inactivator from ascites fluid and obtained a protein of molecular weight 53 to 56 kD with a specific activity 10,000- fold greater than the crude material. On Western blot, an inhibitory antibody recognized a single antigenic species at the same molecular weight. The enzyme had no activity against denatured bovine serum albumin. With recombinant C5a as substrate, the Km and Vm were 3.4 mumol/L and 52 nmol C5a/min/mg protein, respectively.

scholarly journals Purification and characterization of two isoforms of Acanthamoeba profilin.

1986 ◽  
Vol 102 (1) ◽  
pp. 221-226 ◽  
Author(s):  
D A Kaiser ◽  
M Sato ◽  
R F Ebert ◽  
T D Pollard
Keyword(s):  
Molecular Weight ◽  
Actin Filaments ◽  
High Performance ◽  
Reversed Phase ◽  
Lag Phase ◽  
Purification And Characterization ◽  
Spontaneous Polymerization ◽  
Isoelectric Points ◽  
Pointed End

Acanthamoeba profilin purified according to E. Reichstein and E.D. Korn (1979, J. Biol. Chem. 254:6174-6179) consists of two isoforms (profilin-I and-II) with approximately the same molecular weight and reactivity to a monoclonal antibody but different isoelectric points and different mobilities on carboxymethyl-agarose chromatography and reversed-phase high-performance liquid chromatography. The isoelectric points of profilin-I is approximately 5.5 and that of profilin-II is greater than or equal to 9.0. Tryptic peptides from the two proteins are substantially different, which suggests that there are major differences in their sequences. At similar concentrations, both profilins prolong the lag phase at the outset of spontaneous polymerization and inhibit the extent of polymerization. Both forms also inhibit elongation weakly at the barbed end and strongly at the pointed end of actin filaments.

scholarly journals Biosynthesis of proline in Pseudomonas aeruginosa. Partial purification and characterization of γ-glutamyl kinase

1979 ◽  
Vol 181 (1) ◽  
pp. 215-222 ◽  
Author(s):  
R V Krishna ◽  
T Leisinger
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Kinase Activity ◽  
Wild Type Strain ◽  
Deae Cellulose ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Gamma Glutamyl ◽  
Hydrolysis Of ◽  
Molecular Sieving

A gamma-glutamyl kinase (ATP-L-glutamate 5-phosphotransferase) was purified about 85-fold from crude extracts of Pseudomonas aeruginosa strain PAO 1 by (NH4)2SO4 precipitation, molecular-sieving by Sephadex G-150 and DEAE-cellulose chromatography. The molecular weight of this enzyme was 84,000. The preparation catalysed formation of gamma-glutamyl hydroxamate from L-glutamate, ATP and Mg2+ or Mn2+ with concomitant hydrolysis of ATP to ADP + Pi. L-Proline inhibited the gamma-glutamyl kinase activity by 50% at 5 mM and almost completely at 30 mM. The inhibition of L-proline was non-competitive, wherease L-methionine-DL-sulphoximine inhibited the enzyme competitively. Proline was found to inhibit the gamma-glutamyl kinase activity of the wild-type strain and of representatives of two of the three transductional classes of proline-auxotrophic mutants. Strain PAO 879, a mutant representing the third transductional class of proline auxotrophs, lacked proline-inhibitible gamma-glutamyl kinase. Thiol-blocking reagents inhibited the gamma-glutamyl kinase and this effect was prevented by dithiothreitol.

Purification and characterization of phosphoglucomutase from peas

1994 ◽  
Vol 92 (3) ◽  
pp. 479-486 ◽  
Author(s):  
Cynthia M. Galloway ◽  
W. Mack Dugger
Keyword(s):  
Purification And Characterization
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