Phosphofructokinase from Escherichia coli: Further evidence for identical subunits

1978 ◽  
Vol 56 (8) ◽  
pp. 836-838 ◽  
Author(s):  
Bruce N. Thornburgh ◽  
Licia Liu Wu ◽  
Charles C. Griffin
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Affinity Chromatography ◽  
Tryptic Peptide ◽  
Peptide Mapping

A procedure for the purification of Escherichia coli phosphofructokinase by affinity chromatography is described. The results of amino acid analyses of the purified protein and tryptic peptide mapping suggest that the tetrameric phosphofructokinase is composed of chemically identical subunits. In addition, the reaction product, ADP, was observed to bind to 4.1 ± 0.1 equal and independent sites on the enzyme.

scholarly journals Cytotoxic Necrotizing Factor Type 2 Produced by Pathogenic Escherichia coli Deamidates a Gln Residue in the Conserved G-3 Domain of the Rho Family and Preferentially Inhibits the GTPase Activity of RhoA and Rac1

1999 ◽  
Vol 67 (12) ◽  
pp. 6550-6557 ◽  
Author(s):  
Motoyuki Sugai ◽  
Kiyotaka Hatazaki ◽  
Akira Mogami ◽  
Hiroyuki Ohta ◽  
Sylvie Y. Pérès ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Cultured Cells ◽  
Peptide Mapping ◽  
Small Gtpases ◽  
Amino Acid Sequences ◽  
Gtpase Activity ◽  
Toxic Activity ◽  
Cytotoxic Necrotizing Factor ◽  
Rho Family

ABSTRACT Cytotoxic necrotizing factor types 1 and 2 (CNF1 and -2) produced by pathogenic Escherichia coli strains have 90% conserved residues over 1,014-amino-acid sequences. Both CNFs are able to provoke a remarkable increase in F-actin structures in cultured cells and covalently modify the RhoA small GTPases. In this study, we demonstrated that CNF2 reduced RhoA GTPase activity in the presence and absence of P122RhoGAP. Subsequently, peptide mapping and amino acid sequencing of CNF2-modified FLAG-RhoA produced in E. coli revealed that CNF2 deamidates Q63 of RhoA-like CNF1. In vitro incubation of the C-terminal domain of CNF2 with FLAG-RhoA resulted also in deamidation of the FLAG-RhoA, suggesting that this region contains the enzymatic domain of CNF2. An oligopeptide antibody (anti-E63) which specifically recognized the altered G-3 domain of the Rho family reacted with glutathione S-transferase (GST)-RhoA and GST-Rac1 but not with GST-Cdc42 when coexpressed with CNF2. In addition, CNF2 selectively induced accumulation of GTP form of FLAG-RhoA and FLAG-Rac1 but not of FLAG-Cdc42 in Cos-7 cells. Taken together, these results indicate that CNF2 preferentially deamidates RhoA Q63 and Rac1 Q61 and constitutively activates these small GTPases in cultured cells. In contrast, anti-E63 reacted with GST-RhoA and GST-Cdc42 but not with GST-Rac1 when coexpressed with CNF1. These results indicate that CNF2 and CNF1 share the same catalytic activity but have distinct substrate specificities, which may reflect their differences in toxic activity in vivo.

scholarly journals The subunit structure of rabbit skeletal-muscle phosphofructokinase and the amino acid sequence of the tryptic peptide containing the highly reactive thiol group

1977 ◽  
Vol 163 (2) ◽  
pp. 309-316 ◽  
Author(s):  
I A Simpson ◽  
M R Hollaway ◽  
J Beard
Keyword(s):  
Skeletal Muscle ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Tryptic Peptide ◽  
Peptide Mapping ◽  
Thiol Group ◽  
Cysteine Residue ◽  
Amino Acid Sequences ◽  
Class I ◽  
Subunit Structure

1. The single highly reactive (class I) thiol group per 80000-mol.wt. subunit of skeletal-muscle phosphofructokinase was specifically carboxymethylated with iodo[2-14C]acetate, and after denaturation the remaining thiol groups were carboxymethylated with bromo[2-3H]acetate. After tryptic digestion and peptide ‘mapping’ it was found that the 14C radioactivity was in a spot that did not contain significant amounts of 3H radioactivity, so it is concluded that there is not a second, ‘buried’ cysteine residue within a sequence identical with that of the class-I cysteine peptide. 2. The total number of tryptic peptides as well as the number of those containing cysteine, histidine or tryptophan were inconsistent with the smallest polypeptide chain of phosphofructokinase (mol.wt. about 80000) being composed of two identical amino acid sequences. 3. The amino acid sequence of the tryptic peptide containing the class-I thiol group was shown to be Cys-Lys-Asp-Phe-Arg. This sequence is compared with part of the sequence containing the highly reactive thiol group of phosphorylase.

scholarly journals Bovine ß-Lactoglobulin E, F and G of Bali (Banteng) Cattle, Bos (Bihos) Javanicus

1981 ◽  
Vol 34 (2) ◽  
pp. 133 ◽  
Author(s):  
K Bell ◽  
HA McKenzie ◽  
DC Shaw
Keyword(s):  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Amino Acid Analysis ◽  
Tryptic Peptide ◽  
Bos Taurus ◽  
Peptide Mapping ◽  
Northern Territory ◽  
Aureus Strain ◽  
Residue Substitution ◽  
Additional Charge

An electrophoretic examination is made of samples of milk from eight Bali (banteng) cattle, Bos (Bibos) javanicus, in the Northern Territory of Australia. There are two new electrophoretically distinct bovine ft-Iactoglobulins, designated E and F, present in these samples. It is shown by amino acid analysis and tryptic peptide mapping of isolated samples that: (I) both E and F differ from the B variant of domestic cattle (Bos taurus) by + 1 G1y, -1 Glu at residue 157 or 158; and (2) the F variant has the additional charge differences from E and B due to + I Tyr, -1 Asp at residue 129 or 130. It is also shown by studies of peptides produced by the action of Staphylococcus aureus strain V8 protease and by more recent amino acid analyses that (1) the GlyjGlu substitution is at residue 158; (2) there is an additional Bali variant, designated ft-lactoglobulin G, differing from E by + 1 Met, -1 lie at residue 78, but having the same mobility as E; and (3) there is an hitherto undetected neutral residue substitution of + 1 Ser, -1 Pro at position 50 in the F variant. The relation of these variants to other known ft-lactoglobulin variants of the Bos genus is discussed.

scholarly journals Molecular Cloning and Characterization of Fengycin Synthetase Gene fenB from Bacillus subtilis

1998 ◽  
Vol 180 (5) ◽  
pp. 1338-1341 ◽  
Author(s):  
Guang-Huey Lin ◽  
Chyi-Liang Chen ◽  
Johannes Scheng-Ming Tschen ◽  
San-San Tsay ◽  
Yu-Sun Chang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Affinity Chromatography ◽  
Molecular Mass ◽  
Optimum Temperature ◽  
Turnover Number ◽  
Optimum Ph

ABSTRACT A fengycin synthetase gene, fenB, has been cloned and sequenced. The protein (FenB) encoded by this gene has a predicted molecular mass of 143.6 kDa. This protein was overexpressed inEscherichia coli and was purified to near homogeneity by affinity chromatography. Experimental results indicated that the recombinant FenB has a substrate specificity toward isoleucine with an optimum temperature of 25°C, an optimum pH of 4.5, aKm value of 922 μM, and a turnover number of 236 s−1. FenB also consists of a thioesterase domain, suggesting that this protein may be involved in the activation of the last amino acid of fengycin.

scholarly journals Amino acid substitutions and alteration in cation specificity in the melibiose carrier of Escherichia coli.

1988 ◽  
Vol 263 (28) ◽  
pp. 14276-14280 ◽  
Author(s):  
T Kawakami ◽  
Y Akizawa ◽  
T Ishikawa ◽  
T Shimamoto ◽  
M Tsuda ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Substitutions
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