Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.11.18 (MAP)
7,412 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mutant human initiator tRNA genes carrying changes in each of the three features unique to eukaryotic initiator tRNAs have been constructed, and introduced into CV-1 monkey kidney cells using SV40 virus vectors. The mutant tRNA genes are expressed, and the mutant tRNAs can all be aminoacylated with both rabbit liver and Escherichia coli methionyl-tRNA synthetases. Based on aminoacylation levels, the tRNAs are expressed to 5-15-fold over the level of endogenous initiator tRNA. The activity of the mutant [35S]methionyl-tRNAs in initiation was studied in rabbit reticulocyte and wheat germ cell-free protein synthesis systems programmed with various mRNAs. Initiation is studied by using a mRNA that codes for a protein whose N-terminal methionine is stable and not removed by methionine aminopeptidase. Changing the A1:U72 base pair to a G1:C72 base pair greatly reduced activity of the tRNA in initiation. Changing the three consecutive G:C base pairs (G29G30G31:C39C40C41) in the anticodon stem to those found in elongator methionine tRNA also reduced initiation activity. Interestingly, changing the A54 and A60 residues in loop IV to T54 and U60 had less of an effect on activity. The tRNA with changes in all three conserved features had virtually no activity in initiation.
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PMID:The role of nucleotides conserved in eukaryotic initiator methionine tRNAs in initiation of protein synthesis. 822 87

The Streptococcus salivarius ptsH gene encoding histidine-containing phosphocarrier protein (HPr) of the phosphotransferase system (PTS) has been cloned, sequenced, and found to be part of a ptsH, ptsI operon. Upstream from ptsH, putative -35 and -10 boxes and a Shine-Dalgarno sequence highly similar to the Escherichia coli consensus regulatory elements were identified. A second promoter, located in the ptsH coding sequence was also observed and is sufficient for the expression of the S. salivarius ptsI gene, encoding enzyme I of the PTS in E. coli [Gagnon et al., Gene 121 (1992) 71-78]. The amino acid sequence of S. salivarius HPr, inferred from the ptsH sequence, shared identity varying between 37 and 76% with known HPr from other bacteria. Moreover, the S. salivarius HPr shared 78% identity with an HPr-like protein of Aspergillus fumigatus, a eukaroytic mold that does not possess a functional PTS. Expression analysis of S. salivarius HPr in E. coli demonstrated that (i) S. salivarius ptsH is expressed in E. coli under the control of its own promoter, (ii) S. salivarius HPr synthesized by E. coli is completely processed by methionine aminopeptidase, and (iii) S. salivarius HPr is phosphorylated in vivo by E. coli enzyme I. It was also observed that, in E. coli, the copy number of pUC18 bearing S. salivarius ptsH was reduced more than 25-fold, as compared to pUC18 without an insertion.
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PMID:Phosphotransferase system of Streptococcus salivarius: characterization of the ptsH gene and its product. 829 15

We have constructed a plasmid (pHE2) in which the synthetic human alpha- and beta-globin genes and the methionine aminopeptidase (Met-AP) gene from Escherichia coli are coexpressed under the control of separate tac promoters. The Hbs were expressed in E. coli JM109 and purified by fast protein liquid chromatography, producing two major components, a and b. Electrospray mass spectrometry shows that at least 98% and about 90% of the expressed alpha and beta chains of component a, respectively, have the expected masses. The remaining 10% of the beta chain in component a corresponds in mass to the beta chain plus methionine. In component b, both alpha and beta chains have the correct masses without detectable N-terminal methionine (< 2%). These results have been confirmed by Edman degradation studies of the amino-terminal sequences of the alpha and beta chains of these two recombinant Hb (rHb) samples. rHbs from components a and b exhibit visible optical spectra identical to that of human normal adult Hb (Hb A). Component a and Hb A have very similar oxygen-binding properties, but component b shows somewhat altered oxygen binding, especially at low pH values. 1H-NMR spectra of component a and Hb A are essentially identical, whereas those of component b exhibit altered ring current-shifted and hyperfine-shifted proton resonances, indicating altered heme conformation in the beta chain. These altered resonance patterns can be changed to those of Hb A by converting component b to the ferric state and then to the deoxy state and finally back to either the carbonmonoxy or oxy form. Thus, our E. coli expression system produces native, unmodified Hb A in high yield and can be used to produce desired mutant Hbs.
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PMID:Production of unmodified human adult hemoglobin in Escherichia coli. 836 71

Several mitochondrial genes from a large number of different fungi, mammals and plants have been sequenced but little is known about the corresponding translation products. We have affinity purified cytochrome c reductase from potato mitochondria and isolated the mitochondrially encoded cytochrome b protein. Amino-terminal sequencing reveals that the polypeptide does not start with a methionine. Comparison of the amino acid sequence with the recently published sequence of the gene encoding the cytochrome b apoprotein suggests that the N-formylmethionine is removed. This result provides the first evidence for the presence of a deformylase and a methionine aminopeptidase in mitochondria.
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PMID:Purification and sequencing of cytochrome b from potato reveals methionine cleavage of a mitochondrially encoded protein. 842 Jul 97

The X-ray structure of Escherichia coli methionine aminopeptidase (MAP) has been determined to 2.4-A resolution and refined to a crystallographic R-factor of 18.2%. The fold is novel and displays internal pseudo-2-fold symmetry which structurally relates the first and second halves of the polypeptide chain. The topology consists of a central antiparallel beta-sheet covered on one side by two pairs of alpha-helices and by a C-terminal loop. The other face of the beta-sheet, together with some irregular loops, forms the active site, which contains two cobalt ions 2.9 A apart. These metal ions are liganded by the side chains of Asp 97, Asp 108, Glu 204, Glu 235, and His 171 with approximate octahedral coordination. In terms of both the novel backbone fold and the constitution of the active site, MAP appears to represent a new class of proteolytic enzyme.
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PMID:Structure of the cobalt-dependent methionine aminopeptidase from Escherichia coli: a new type of proteolytic enzyme. 847 2

We previously characterized a methionine aminopeptidase (EC 3.4.11.18; Met-AP1; also called peptidase M) in Saccharomyces cerevisiae, which differs from its prokaryotic homologues in that it (i) contains an N-terminal zinc-finger domain and (ii) does not produce lethality when disrupted, although it does slow growth dramatically; it is encoded by a gene called MAP1. Here we describe a second methionine aminopeptidase (Met-AP2) in S. cerevisiae, encoded by MAP2, which was cloned as a suppressor of the slow-growth phenotype of the map1 null strain. The DNA sequence of MAP2 encodes a protein of 421 amino acids that shows 22% identity with the sequence of yeast Met-AP1. Surprisingly, comparison with sequences in the GenBank data base showed that the product of MAP2 has even greater homology (55% identity) with rat p67, which was characterized as an initiation factor 2-associated protein but not yet shown to have Met-AP activity. Transformants of map1 null cells expressing MAP2 in a high-copy-number plasmid contained 3- to 12-fold increases in Met-AP activity on different peptide substrates. The epitope-tagged suppressor gene product was purified by immunoaffinity chromatography and shown to contain Met-AP activity. To evaluate the physiological significance of Met-AP2, the MAP2 gene was deleted from wild-type and map1 null yeast strains. The map2 null strain, like the map1 null strain, is viable but with a slower growth rate. The map1, map2 double-null strains are nonviable. Thus, removal of N-terminal methionine is an essential function in yeast, as in prokaryotes, but yeast require two methionine aminopeptidases to provide the essential function which can only be partially provided by Met-AP1 or Met-AP2 alone.
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PMID:Amino-terminal protein processing in Saccharomyces cerevisiae is an essential function that requires two distinct methionine aminopeptidases. 861

We used chromosomal walking methods to isolate a 10.8-kb region from the major ribosomal protein (r-protein) gene cluster of Bacillus subtilis (Bs). The gene order in this region, given by gene product, was r-proteins L16-L29-S17-L14-L24-L5-S14-S8-L6-L18-S5-L30-L15-SecY-adenylate kinase (Adk)-methionine aminopeptidase (Map)-initiation factor 1 (IF1)-L36-S13-S11-alpha subunit of RNA polymerase-L17. The region cloned, therefore, contains the homologues for the last three genes of the Escherichia coli (Ec) S10 operon, together with entire spc and alpha operons. This Bs organization differs from the corresponding region in Ec by the inclusion of the genes encoding Adk, Map and IF1 between the genes encoding SecY and L36. Plasmid integration experiments indicated that all 22 genes comprise a single large transcriptional unit controlled from a major promoter which lies upstream from the gene encoding r-protein L16. Promoter probe experiments located lesser activities internal to this large transcriptional unit, the secY and map promoters. The secY promoter region (psecY) contained two activities, each principally functioning in the stationary growth phase when high protein export is required. Thus, the Bs S10-spc-alpha region differs from its Ec counterpart in both genetic and transcriptional organization. Given this difference in transcriptional organization, the mechanisms coordinating expression of the translational apparatus are also likely to differ between Ec and Bs.
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PMID:Genetic and transcriptional organization of the Bacillus subtilis spc-alpha region. 863 44

Aminopeptidases are exopeptidases that selectively release N-terminal amino acid residues from polypeptides and proteins. Bacteria display several aminopeptidasic activities which may be localised in the cytoplasm, on membranes, associated with the cell envelope or secreted into the extracellular media. Studies on the bacterial aminopeptide system have been carried out over the past three decades and are significant in fundamental and biotechnological domains. At present, about one hundred bacterial aminopeptidases have been purified and biochemically studied. About forty genes encoding aminopeptidases have also been cloned and characterised. Recently, the three-dimensional structure of two aminopeptidases, the methionine aminopeptidase from Escherichia coli and the leucine aminopeptidase from Aeromonas proteolytica, have been elucidated by crystallographic studies. Most of the quoted studies demonstrate that bacterial aminopeptidases generally show Michaelis-Menten kinetics and can be placed into either of two categories based on their substrate specificity: broad or narrow. These enzymes can also be classified by another criterium based on their catalytic mechanism: metallo-, cysteine- and serine-aminopeptidases, the former type being predominant in bacteria. Aminopeptidases play a role in several important physiological processes. It is noteworthy that some of them take part in the catabolism of exogenously supplied peptides and are necessary for the final steps of protein turnover. In addition, they are involved in some specific functions, such as the cleavage of N-terminal methionine from newly synthesised peptide chains (methionine aminopeptidases), the stabilisation of multicopy ColE1 based plasmids (aminopeptidase A) and the pyroglutamyl aminopeptidase (Pcp) present in many bacteria and responsible for the cleavage of the N-terminal pyroglutamate.
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PMID:Bacterial aminopeptidases: properties and functions. 870 9

A full-length recombinant human apolipoprotein C-II (ApoC-II) has been successfully expressed in Escherichia coli using the T7 expression system. The recombinant ApoC-II. which was expressed intracellularly in the inclusion bodies, was solubilized with 8 M urea and purified using Sephadex G-75 gel permeation chromatography. Four liters of the bacterial culture yielded 16-20 mg of purified recombinant ApoC-II. Sequencing and mass spectrometric analyses indicated that the isolated recombinant ApoC-II contained predominantly (64%) the native form with threonine as the N-terminus, but also contained a minor (36%) molecular form of ApoC-II with an additional methionine at the N-terminus (Met-ApoC-II). Analysis of the recombinant ApoC-II by tryptic digestion and high performance liquid chromatography-electrospray mass spectrometry provides additional conclusive evidence that, with the exception of the N-terminus of Met-ApoC-II, the expressed ApoC-II has the expected peptide sequence. However, this extra N-terminal methionine residue can be excised by further in vitro treatment with methionine aminopeptidase. The purified recombinant ApoC-II was found to be competent in the activation of bovine milk lipoprotein lipase. Thus, the recombinant ApoC-II prepared from E. coli may have a pharmacological application for the treatment of patients with genetic hypertriglyceridemia caused by ApoC-II deficiency.
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PMID:Isolation and characterization of recombinant human apolipoprotein C-II expressed in Escherichia coli. 876 43

Previously, we cloned a human cDNA encoding a protein which has a 92% amino acid sequence identity to a rat initiation factor-2 associated protein (p67). Rat p67 plays an important role in translational regulation by preventing the phosphorylation of the alpha subunit of initiation factor-2. Interestingly, several lines of indirect evidence suggested that this protein may also function as a methionine aminopeptidase (MetAP). To test this hypothesis, we expressed the human cDNA in a baculovirus system, purified it to homogeneity and characterized it. Using 13 different peptide substrates, we found that the human p67 has a similar substrate specificity with other MetAPs. Kinetic analyses revealed that the Kcat/K(m) values of the human MetAP on two representative substrates are similar to those of yeast and porcine MetAPs. Furthermore, we found that this enzyme, like other MetAPs, is also a cobalt-dependent metalloenzyme.
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PMID:Evidence that the human homologue of a rat initiation factor-2 associated protein (p67) is a methionine aminopeptidase. 885 18


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