scholarly journals The ς70 Transcription Factor TyrR Has Zinc-Stimulated Phosphatase Activity That Is Inhibited by ATP and Tyrosine

2000 ◽  
Vol 182 (4) ◽  
pp. 1053-1061 ◽  
Author(s):  
Shimin Zhao ◽  
Qin Zhu ◽  
Ronald L. Somerville
Keyword(s):  
Amino Acid ◽  
Phosphatase Activity ◽  
Sequence Similarity ◽  
Zinc Ion ◽  
Amino Acid Residues ◽  
Protein Residues ◽  
E Coli ◽  
C Terminus ◽  
Atp Analogues ◽  
High Degree

ABSTRACT The TyrR protein of Escherichia coli (513 amino acid residues) is the chief transcriptional regulator of a group of genes that are essential for aromatic amino acid biosynthesis and transport. The TyrR protein can function either as a repressor or as an activator. The central region of the TyrR protein (residues 207 to 425) is similar to corresponding polypeptide segments of the NtrC protein superfamily. Like the NtrC protein, TyrR has intrinsic ATPase activity. Here, we report that TyrR possesses phosphatase activity. This activity is subject to inhibition by l-tyrosine and its analogues and by ATP and ATP analogues. Zinc ion (2 mM) stimulated the phosphatase activity of the TyrR protein by a factor of 57. The phosphatase-active site of TyrR was localized to a 31-kDa domain (residues 191 to 467) of the protein. However, mutational alteration of distant amino acid residues at both the N terminus and the C terminus of TyrR altered the phosphatase activity. Haemophilus influenzae TyrR (318 amino acid residues), a protein with a high degree of sequence similarity to the C terminus of the E. coli TyrR protein, exhibited a phosphatase activity similar to that of E. coliTyrR.

scholarly journals Molecular cloning and sequence analysis of the cDNA for ancrod, a thrombin-like enzyme from the venom of Calloselasma rhodostoma

1993 ◽  
Vol 294 (2) ◽  
pp. 387-390 ◽  
Author(s):  
L C Au ◽  
S B Lin ◽  
J S Chou ◽  
G W Teh ◽  
K J Chang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Serine Proteases ◽  
Sequence Similarity ◽  
Active Form ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Binding Reaction ◽  
Venom Glands ◽  
High Degree

The 1.54 kb cDNA for ancrod, a thrombin-like enzyme, was cloned from a lambda ZAP cDNA library derived from the venom glands of Calloselasma (Agkistrodon) rhodostoma. The cDNA sequence reveals that ancrod is synthesized as a pre-zymogen of 258 amino acids, including a putative secretory peptide of 18 amino acids and a proposed zymogen peptide of 6 amino-acid residues. The amino-acid sequence of the predicted active form of the enzyme exhibits a high degree of sequence similarity to those of mammalian serine proteases (trypsin and pancreatic kallikrein) and other thrombin-like enzymes (batroxobin and flavoxobin). Key amino-acid residues (His43, Asp88, Ser182 and Asp176) that are thought to be involved in the substrate cleavage and in the substrate-binding reaction are conserved. Ancrod contains 13 cysteine residues. Based on alignment with the amino-acid sequences of trypsin and batroxobin, six disulphide bridges can be predicted to be present in the ancrod protein. The existence of a free cysteine, which changes the common sequence surrounding the Ser182 active site from Gly-Asp-Ser-Gly-Gly-Pro to Cys-Asp-Ser-Gly-Gly-Pro, is unusual for a serine protease.

scholarly journals Sequence around the Active Center Cystine of Lipoamide Dehydrogenase from Pig Heart, Comparison with the E. coli Enzyme

1972 ◽  
Vol 27 (9) ◽  
pp. 1078-1080 ◽  
Author(s):  
C. H. Williams ◽  
L. D. Arscott
Keyword(s):  
Amino Acid ◽  
Active Center ◽  
Active Site ◽  
Polypeptide Chain ◽  
Amino Acid Residues ◽  
Concerted Action ◽  
E Coli ◽  
Lipoamide Dehydrogenase ◽  
Flavin Binding ◽  
High Degree

Catalysis by lipoamide dehydrogenase involves the concerted action of the flavin and a cystine residue. Peptides containing this cystine residue have been previously isolated from E. coli and now from pig heart. The sequences of amino acid residues reveal a high degree of homology indicating a strict conservation of the region around the active site cystine during the long evolutionary period between these two species. The peptide sequences suggest a likely conformation of the polypeptide chain in the region of the flavin as well as the forces involved in substrate and flavin binding.

scholarly journals Primary- and secondary-structural analysis of a unique prokaryotic metallothionein from a Synechococcus sp. cyanobacterium

1988 ◽  
Vol 251 (3) ◽  
pp. 691-699 ◽  
Author(s):  
R W Olafson ◽  
W D McCubbin ◽  
C M Kay
Keyword(s):  
Amino Acid ◽  
Metal Binding ◽  
Helical Structure ◽  
Basic Amino Acid ◽  
Cysteine Residues ◽  
Amino Acid Residues ◽  
C Terminus ◽  
Empirical Relations ◽  
Heavy Metal Binding ◽  
Synechococcus Sp

Biochemical and physiological studies of Synechococcus cyanobacteria have indicated the presence of a low-Mr heavy-metal-binding protein with marked similarity to eukaryotic metallothioneins (MTs). We report here the characterization of a Synechococcus prokaryotic MT isolated by gel-permeation and reverse-phase chromatography. The large number of variants of this molecule found during chromatographic separation could not be attributed to the presence of major isoproteins as assessed by amino acid analysis and amino acid sequencing of isoforms. Two of the latter were shown to have identical primary structures that differed substantially from the well-described eukaryotic MTs. In addition to six long-chain aliphatic residues, two aromatic residues were found adjacent to one another near the centre of the molecule, making this the most hydrophobic MT to be described. Other unusual features included a pair of histidine residues located in repeating Gly-His-Thr-Gly sequences near the C-terminus and a complete lack of association of hydroxylated residues with cysteine residues, as is commonly found in eukaryotes. Similarly, aside from a single lysine residue, no basic amino acid residues were found adjacent to cysteine residues in the sequence. Most importantly, sequence alignment analyses with mammalian, invertebrate and fungal MT sequences showed no statistically significant homology aside from the presence of Cys-Xaa-Cys structures common to all MTs. On the other hand, like other MTs, the prokaryotic molecule appears to be free of alpha-helical structure but has a considerable amount of beta-structure, as predicted by both c.d. measurements and the Chou & Fasman empirical relations. Considered together, these data suggested that some similarity between the metal-thiolate clusters of the prokaryote and eukaryote MTs may exist.

Expression and partial purification of human pro-opiomelanocortin in Escherichia coli

1989 ◽  
Vol 3 (2) ◽  
pp. 105-112 ◽  
Author(s):  
T. S. Grewal ◽  
P. J. Lowry ◽  
D. Savva
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Fusion Protein ◽  
Western Blot ◽  
Blot Analysis ◽  
Expression Vectors ◽  
Partial Purification ◽  
Amino Acid Residues ◽  
E Coli ◽  
Dna Fragment

ABSTRACT A large portion of the human pro-opiomelanocortin (POMC) peptide corresponding to amino acid residues 59–241 has been cloned and expressed in Escherichia coli. A 1·0 kb DNA fragment encoding this peptide was cloned into the expression vectors pUC8 and pUR291. Plasmid pJMBG51 (a pUC8 recombinant) was found to direct the expression of a 24 kDa peptide. The recombinant pUR291 (pJMBG52) was shown to produce a β-galactosidase fusion protein of 140 kDa. Western blot analysis showed that both the 24 kDa and 140 kDa peptides are recognized by antibodies raised against POMC-derived peptides. The β-galactosidase fusion protein has been partially purified from crude E. coli cell lysates using affinity chromatography on p-aminobenzyl-1-thio-β-d-galactopyranoside agarose.

scholarly journals Deletion of the Actin-Associated Tropomyosin Tpm3 Leads to Reduced Cell Complexity in Cultured Hippocampal Neurons—New Insights into the Role of the C-Terminal Region of Tpm3.1

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 715
Author(s):  
Tamara Tomanić ◽  
Claire Martin ◽  
Holly Stefen ◽  
Esmeralda Parić ◽  
Peter Gunning ◽  
...  
Keyword(s):  
Amino Acid ◽  
Hippocampal Neurons ◽  
Molecular Mechanisms ◽  
Growth Cones ◽  
Amino Acid Residues ◽  
Terminal Amino Acid ◽  
C Terminus ◽  
Primary Mouse ◽  
Terminal Amino

Tropomyosins (Tpms) have been described as master regulators of actin, with Tpm3 products shown to be involved in early developmental processes, and the Tpm3 isoform Tpm3.1 controlling changes in the size of neuronal growth cones and neurite growth. Here, we used primary mouse hippocampal neurons of C57/Bl6 wild type and Bl6Tpm3flox transgenic mice to carry out morphometric analyses in response to the absence of Tpm3 products, as well as to investigate the effect of C-terminal truncation on the ability of Tpm3.1 to modulate neuronal morphogenesis. We found that the knock-out of Tpm3 leads to decreased neurite length and complexity, and that the deletion of two amino acid residues at the C-terminus of Tpm3.1 leads to more detrimental changes in neurite morphology than the deletion of six amino acid residues. We also found that Tpm3.1 that lacks the 6 C-terminal amino acid residues does not associate with stress fibres, does not segregate to the tips of neurites, and does not impact the amount of the filamentous actin pool at the axonal growth cones, as opposed to Tpm3.1, which lacks the two C-terminal amino acid residues. Our study provides further insight into the role of both Tpm3 products and the C-terminus of Tpm3.1, and it forms the basis for future studies that aim to identify the molecular mechanisms underlying Tpm3.1 targeting to different subcellular compartments.

scholarly journals Cloning, post-translational modifications, heterologous expression and ligand-binding of boar salivary lipocalin

2000 ◽  
Vol 350 (2) ◽  
pp. 369-379 ◽  
Author(s):  
Dietrich LOEBEL ◽  
Andrea SCALONI ◽  
Sara PAOLINI ◽  
Carlo FINI ◽  
Lino FERRARA ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sequence Similarity ◽  
Three Dimensional ◽  
Protein Isoforms ◽  
Cysteine Residues ◽  
Protein Chemistry ◽  
Single Individual ◽  
Amino Acid Residues ◽  
Three Dimensional Model ◽  
Submaxillary Glands

Boar submaxillary glands produce the sex-specific salivary lipocalin (SAL), which binds steroidal sex pheromones as endogenous ligands. The cDNA encoding SAL was cloned and sequenced. From a single individual, two protein isoforms, differing in three amino acid residues, were purified and structurally characterized by a combined Edman degradation/MS approach. These experiments ascertained that the mature polypeptide is composed of 168 amino acid residues, that one of the three putative glycosylation sites is post-translationally modified and the structure of the bound glycosidic moieties. Two of the cysteine residues are paired together in a disulphide bridge, whereas the remaining two occur as free thiols. SAL bears sequence similarity to other lipocalins; on this basis, a three-dimensional model of the protein has been built. A SAL isoform was expressed in Escherichiacoli in good yields. Protein chemistry and CD experiments verified that the recombinant product shows the same redox state at the cysteine residues and that the same conformation is observed as in the natural protein, thus suggesting similar folding. Binding experiments on natural and recombinant SAL were performed with the fluorescent probe 1-aminoanthracene, which was efficiently displaced by the steroidal sex pheromone, as well as by several odorants.

scholarly journals Isolation and amino-acid sequence of two inhibitors of serine proteinases, members of the squash inhibitor family, from Echinocystis lobata seeds.

1996 ◽  
Vol 43 (3) ◽  
pp. 507-513 ◽  
Author(s):  
D Stachowiak ◽  
A Polanowski ◽  
G Bieniarz ◽  
T Wilusz
Keyword(s):  
Amino Acid ◽  
Proteinase Inhibitors ◽  
Sequence Similarity ◽  
Serine Proteinase ◽  
Exchange Chromatography ◽  
Association Constants ◽  
Cathepsin G ◽  
Amino Acid Residues ◽  
Mature Seeds ◽  
Echinocystis Lobata

Two serine proteinase inhibitors (ELTI I and ELTI II) have been isolated from mature seeds of Echinocystis lobata by ammonium sulfate fractionation, methanol precipitation, ion exchange chromatography, affinity chromatography on immobilized anhydrotrypsin and HPLC. ELTI I and ELTI II consist of 33 and 29 amino-acid residues, respectively. The primary structures of these inhibitors are as follows: ELTI I KEEQRVCPRILMRCKRDSDCLAQCTCQQSGFCG ELTI II RVCPRILMRCKRDSDCLAQCTCQQSGFCG The inhibitors show sequence similarity with the squash inhibitor family. ELTI I differs from ELTI II only by the presence of the NH2-terminal tetrapeptide Lys-Glu-Glu-Gln. The association constants (Ka) of ELTI I and ELTI II with bovine-trypsin were determined to be 6.6 x 10(10) M-1, and 3.1 x 10(11) M-1, whereas the association constants of these inhibitors with cathepsin G were 1.2 x 10(7) M-1, and 1.1 x 10(7) M-1, respectively.

The amino acid sequence of yeast phosphoglycerate mutase

1982 ◽  
Vol 215 (1198) ◽  
pp. 19-44 ◽  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Proteolytic Enzymes ◽  
Phosphoglycerate Mutase ◽  
Crystallographic Structure ◽  
X Ray ◽  
C Terminus ◽  
Detailed Interpretation ◽  
High Degree

The complete amino acid sequence of yeast phosphoglycerate mutase comprising 241 residues has been determined. The sequence was deduced from the two cyanogen bromide fragments, and from the peptides derived from these fragments after digestion by a number of proteolytic enzymes. Determination of this sequence now allows a detailed interpretation of the existing high-resolution X-ray crystallographic structure. A comparison of the sequence reported here with the sequences of peptides from phosphoglycerate mutases from other species, and with the sequence of erythrocyte diphosphoglycerate mutase, indicates that these enzymes have a high degree of structural homology. Autolysis of phosphoglycerate mutase by yeast extracts leads to the complete loss of mutase activity, and the formation of electrophoretically distinguishable forms (R. Sasaki, E. Sugimoto & H. Chiba, Archs Biochem. Biophys. 115, 53-61 (1966)). It is apparent from the amino acid sequence that these changes are due to the loss of an 8─12 residue peptide from the C-terminus.

Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus

1989 ◽  
Vol 9 (1) ◽  
pp. 83-91
Author(s):  
S Miyazawa ◽  
T Osumi ◽  
T Hashimoto ◽  
K Ohno ◽  
S Miura ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
Coenzyme A ◽  
Terminal Region ◽  
Proteinase K ◽  
Amino Acid Residues ◽  
C Terminus ◽  
Targeting Signal ◽  
Acyl Coenzyme A

To identify the topogenic signal of peroxisomal acyl-coenzyme A oxidase (AOX) of rat liver, we carried out in vitro import experiments with mutant polypeptides of the enzyme. Full-length AOX and polypeptides that were truncated at the N-terminal region were efficiently imported into peroxisomes, as determined by resistance to externally added proteinase K. Polypeptides carrying internal deletions in the C-terminal region exhibited much lower import activities. Polypeptides that were truncated or mutated at the extreme C terminus were totally import negative. When the five amino acid residues at the extreme C terminus were attached to some of the import-negative polypeptides, the import activities were rescued. Moreover, the C-terminal 199 and 70 amino acid residues of AOX directed fusion proteins with two bacterial enzymes to peroxisomes. These results are interpreted to mean that the peroxisome targeting signal of AOX residues at the C terminus and the five or fewer residues at the extreme terminus have an obligatory function in targeting. The C-terminal internal region also has an important role for efficient import, possibly through a conformational effect.

Expression of a UDPglucose pyrophosphorylase cDNA during fruit ripening of banana (Musa acuminata)

2000 ◽  
Vol 27 (12) ◽  
pp. 1151 ◽  
Author(s):  
Eng-Chong Pua ◽  
Stevenson Szu-Wei Lim ◽  
Pei Liu ◽  
Jian-Zhong Liu
Keyword(s):  
Sequence Similarity ◽  
Soluble Sugars ◽  
Transcript Abundance ◽  
Reproductive Organs ◽  
Banana Fruit ◽  
Amino Acid Residues ◽  
Promoting Effect ◽  
Udpglucose Pyrophosphorylase ◽  
Exogenous Ethylene ◽  
High Degree

We report the isolation of a banana cDNA, designated MWUGPA, encoding uridine diphosphoryl (UDP)-glucose pyrophosphorylase (UGPase, EC.2.7.7.9) that catalyses the reversible conversion between glucose 1-phosphate and UDPglucose in plants and animals. Furthermore, UGPase expression in fruit during ripening and in response to exogenous ethylene and sugars was also investigated. MWUGPA encodes a polypeptide of 467 amino acid residues and shares a high degree of sequence similarity (85–90%) with other plant UGPase homologs. In northern blot analysis, a 1.7-kb UGPase transcript was detected in both the vegetative and reproductive organs, but the former was considerably less abundant than the latter. In fruit, the level of accumulated transcripts was higher in pulp than peel at all ripening stages. Transcript abundance in both fruit tissues was relatively constant during ripen-ing, but pulp transcripts surged in the ‘more green than yellow’ category fruit when ethylene also increased. Further analysis revealed that UGPase expression in fruit was ethylene-inducible, but the response was tissue-specific, as evidenced by the promoting effect of exogenous ethylene on accumulation of UGPase transcripts in pulp but not peel. Exogenous application of sucrose and fructose also increased UGPase transcript abundance in leaf and fruit tissues, especially pulp, whereas exogenous glucose had little or no effect. The results of this study indicate that ethy-lene and soluble sugars may play a regulatory role in UGPase expression during ripening in banana fruit.

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