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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)

Authentic soluble human beta-globin chains were produced in Escherichia coli using an expression plasmid (pHE2beta) containing full-length cDNAs coding for human beta-globin chain and methionine aminopeptidase. Spectral properties of the purified beta-globin were identical to those of authentic beta-globin. Soluble beta-globin showed low (16 kDa) and high molecular mass (32 kDa) forms that could be separated by gel filtration chromatography. SDS-polyacrylamide gel electrophoresis and electrospray mass spectrometry revealed the 32-kDa species was dimeric beta-globin formed by an intermolecular disulfide bond, while the 16-kDa species was authentic monomeric beta-globin. Monomeric forms of beta-globin, like authentic native beta-globin, formed tetrameric hemoglobin (Hb) A (alpha2beta2) in vitro upon incubation with alpha-globin, while dimeric forms did not. When beta-globin dimers, however, were converted to monomers by incubation with dithiothreitol, the beta-globin chain monomers assembled with alpha-globin and formed hemoglobin tetramers. alpha-Globin was more thermally unstable than beta-globin, while assembled tetramers promoted higher stability. Disulfide-bonded beta-globin dimers showed a slight increase in thermal stability compared with beta-globin; however, dimers were still more unstable than tetrameric Hb A. These results indicate that presence of alpha chains favors assembly with beta-globin, beta-beta dimers cannot bind alpha chains, and that Hb A tetramer formation results in the most thermally stable species.
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PMID:Expression of soluble human beta-globin chains in bacteria and assembly in vitro with alpha-globin chains. 890 Jan 44

Selection of the proper start codon for the synthesis of a polypeptide by the Escherichia coli translation initiation apparatus involves several macromolecular components. These macromolecules interact in a specific and concerted manner to yield the translation initiation complex. This review focuses on recent data concerning the properties of the initiator tRNA and of enzymes and factors involved in the translation initiation process. The three initiation factors, as well as methionyl-tRNA synthetase and methionyl-tRNA(f)Met formyltransferase are described. In addition, the tRNA recognition properties of EF-Tu and peptidyl-tRNA hydrolase are considered. Finally, peptide deformylase and methionine aminopeptidase, which catalyze the amino terminal maturation of nascent polypeptides, can also be associated to the translation initiation process.
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PMID:Molecular recognition governing the initiation of translation in Escherichia coli. A review. 895 98

Chinese hamster dihydrofolate reductase (ch-DHFR) was overexpressed in Escherichia coli DH5 alpha under the transcriptional control of PRPL promoters regulated by temperature-sensitive repressors. The desired recombinant product is soluble and constitutes about 30% of the total soluble proteins of the bacterial cell. With repeated cycles of freezing and thawing as a first step, the purification of the recombinant ch-DHFR to homogeneity requires only one further step, gel filtration on a Sephadex G-75 column with 85-90% enzyme recovery, two to three times higher than that obtained with the commonly used affinity chromatography on a methotrexate-Sepharose column. The purified enzyme migrates as a single protein band on SDS-polyacrylamide gel electrophoresis with approximate mass of 23 kDa, in accord with that calculated from the DNA sequence. The initiation methionine residue at the N-terminus of the enzyme is completely removed by E. coli methionine aminopeptidase as judged by amino-terminal analysis. The steady-state kinetic parameters, dissociation constants for binary complexes of dihydrofolate, NADPH, and methotrexate with ch-DHFR, and the inhibitor constant of methotrexate have also been determined. The enzyme is activated about 4-fold in 3 M urea and about 2.5-fold in 0.5 M guanidine hydrochloride.
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PMID:Soluble expression in Escherichia coli, one-step purification, and characterization of Chinese hamster dihydrofolate reductase. 905 90

The inhibition of new blood vessel formation (angiogenesis) is an effective means of limiting both the size and metastasis of solid tumors. The leading anti-angiogenic compound, TNP-470, has proven to be effective in in vitro and in animal model studies, and is currently being tested in phase III antitumor clinical trials. Despite many detailed pharmacological studies, little is known of the molecular mode of action of TNP-470. Using a derivative of the TNP-470 parent compound, the fungal metabolite, fumagillin, we have purified a mammalian protein that is selectively and covalently bound by this natural product. This fumagillin binding protein was found to be a metalloprotease, methionine aminopeptidase (MetAP-2), that is highly conserved between human and Saccharomyces cerevisiae. In the absence of MetAP-1, a distantly related methionine aminopeptidase, MetAP-2 function is essential for vegetative growth in yeast. We demonstrate that fumagillin selectively inhibits the S. cerevisiae MetAP-2 protein in vivo. The binding is highly specific as judged by the failure of fumagillin to inhibit MetAP-1 in vivo. Hence, these results identify MetAP-2 as an important target of study in the analysis of the potent biological activities of fumagillin.
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PMID:The anti-angiogenic agent fumagillin covalently binds and inhibits the methionine aminopeptidase, MetAP-2. 917 76

A gene for a methionine aminopeptidase (MAP; EC 3.4.11.18), which catalyzes the removal of amino-terminal methionine from the growing peptide chain on the ribosome, has been cloned from the hyperthermophilic Archaeon, Pyrococcus furiosus, by a novel method effectively using its cosmid protein library, sequenced and expressed in Escherichia coli. The DNA sequence encodes a protein containing 295 amino acid residues with methionine at the N-terminus. From protein analyses of the recombinant protein expressed in E. coli, by using both amino acid sequence analysis from the N-terminus by automated Edman degradation and analyses of molecular masses of the peptides generated by two enzymatic cleavages performed independently, digestions with lysylendopeptidase and Endoproteinase Asp-N, with ionspray mass spectrometry, the primary structure of the protein has been elucidated to be completely identical with that deduced from its DNA sequence. Comparison of the amino acid sequence of P. furiosus MAP (P.f. MAP) with those of other MAPs from Eukarya and Bacteria showed that the protein has a high degree of sequence homology in the stretches surrounding the five cobalt-binding residues fully preserved in all of MAPs determined so far, but P.f. MAP belongs to Type II because it has an extra long insertion of about 60 amino acid residues between the fourth and fifth cobalt-binding ligands, similar to MAPs from human and rat, and to Met-AP2 from Saccharomyces cerevisiae, in comparison to Type I MAPs from Bacteria. Therefore, P.f. MAP seems to be rather close to those from Eukarya, although it is distinct in lacking the N-terminal extension of about 90-150 residues universally found in MAPs from Eukarya. These findings suggest that P.f. MAP is evolutionally located at the Eukarya-Bacteria boundary. The enzyme expressed in E. coli exhibits a considerable thermostability, with a half-life of approximately 4.5 h at 90 degrees C and an optimum temperature of around 90 degrees C.
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PMID:Methionine aminopeptidase from the hyperthermophilic Archaeon Pyrococcus furiosus: molecular cloning and overexpression in Escherichia coli of the gene, and characteristics of the enzyme. 939 90

A hemoglobin expression system in Escherichia coli is described. In order to produce authentic human hemoglobin, we need to co-express both methionine aminopeptidase and globin genes under the control of a strong promoter. We have constructed three plasmids, pHE2, pHE4 and pHE7, for the expression of human normal adult hemoglobin and a plasmid, pHE9, for the expression of human fetal hemoglobin, in high yields. The globin genes can be derived from either synthetic genes or human globin cDNAs. The extra amino-terminal methionine residues of the expressed globins can be removed by the co-expressed methionine aminopeptidase. The heme is inserted correctly into the expressed alpha-globin from our expression plasmids. A fraction (approximately 25%) of the heme is not inserted correctly into the expressed beta- or gamma-globin. However, the incorrectly inserted hemes can be converted into the correct conformation by carrying out a simple oxidation-reduction process on the purified hemoglobin molecule. We have investigated the functional properties of the expressed hemoglobins by measuring their oxygen-binding properties and their structural features by obtaining their 1H-NMR spectra. Our results show that authentic human normal adult and fetal hemoglobins can be produced from our expression plasmids in E. coli and in high yields. Our expression system allows us to design and to produce any recombinant hemoglobins needed for our research on the structure-function relationship in hemoglobin.
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PMID:Production of human normal adult and fetal hemoglobins in Escherichia coli. 946 74

When hen egg-white lysozyme was produced in Escherichia coli, it possessed an extra methionine residue at the N-terminus (Met(-1)-lysozyme). The Met(-1)-lysozyme showed a decreased refolding yield and solubility compared with the native hen egg-white lysozyme, as the methionine is a hydrophobic amino acid. A Met(-2)Pro(-1) or Met(-2)Ser(-1) sequence was introduced at the N-terminus of hen egg-white lysozyme. The methionine residue in these hen egg-white lysozymes was completely removed by methionine aminopeptidase, as expected, since the penultimate residue was proline or serine. From the analyses of solubility, stability and refolding yield, it was found that an extra Ser residue attached to the N-terminus of hen egg-white lysozyme (Ser(-1)-lysozyme) showed closer characteristics to the native hen egg-white lysozyme than did Met(-1) or an extra Pro residue attached to the N-terminus of hen egg-white lysozyme (Pro(-1)-lysozyme). Moreover, the tertiary conformation of Ser(-1)-lysozyme examined by NMR spectroscopy and its activity were almost identical with those of native hen egg-white lysozyme.
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PMID:Improvement of the refolding yield and solubility of hen egg-white lysozyme by altering the Met residue attached to its N-terminus to Ser. 951 23

The structure of the proline-specific aminopeptidase (EC 3.4.11.9) from Escherichia coli has been solved and refined for crystals of the native enzyme at a 2.0-A resolution, for a dipeptide-inhibited complex at 2.3-A resolution, and for a low-pH inactive form at 2.7-A resolution. The protein crystallizes as a tetramer, more correctly a dimer of dimers, at both high and low pH, consistent with observations from analytical ultracentrifuge studies that show that the protein is a tetramer under physiological conditions. The monomer folds into two domains. The active site, in the larger C-terminal domain, contains a dinuclear manganese center in which a bridging water molecule or hydroxide ion appears poised to act as the nucleophile in the attack on the scissile peptide bond of Xaa-Pro. The metal-binding residues are located in a single subunit, but the residues surrounding the active site are contributed by three subunits. The fold of the protein resembles that of creatine amidinohydrolase (creatinase, not a metalloenzyme). The C-terminal catalytic domain is also similar to the single-domain enzyme methionine aminopeptidase that has a dinuclear cobalt center.
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PMID:Structure and mechanism of a proline-specific aminopeptidase from Escherichia coli. 952 Mar 90

The thermostability of methionine aminopeptidase from a hyperthermophile P. furiosus (PfMAP) was extremely high: the denaturation temperature was 106.2 degreesC at pH 10.2. To explore the contribution of electrostatic interaction to the superior thermostability of PfMAP, the thermostability of PfMAP was examined by differential scanning calorimetry (DSC) in various salt concentrations in the acidic region far from the isoelectric point of PfMAP. (1) In 20 mM glycine buffer, the DSC curve of PfMAP exhibited a single peak. Transition temperatures (Tm) were lowered with decreasing pH from 4 to 3. The heat denaturation of PfMAP was not reversible. (2) Denaturation enthalpy (DeltaH) measured at different pHs linearly correlated with Tm up to 102 degreesC, suggesting that the denaturation heat capacity (DeltaCp) for PfMAP is constant up to 100 degreesC. DeltaCp was estimated to be 0.82 J K-1 g-1. (3) In the presence of 10-100 mM KCl at pH 3.2, two peaks appeared on the DSC curves. The first peak shifted to lower temperatures with increasing concentration of KCl and, oppositely, the second one to higher temperatures. It was found that the first and second peaks originated from the heat denaturation of the native form of PfMAP and the melting of the non-native associated form having molten globule-like structure, respectively, judged from the CD spectra and ultracentrifugation analyses. This indicates the following: first, the attractive electrostatic interaction is an important factor in stabilizing the native form of PfMAP; second, the presence of KCl stimulates the formation of the molten globule-like state of PfMAP and stabilizes it. (4) In a comparison of the sequence and crystal structure of PfMAP, which has been recently determined (1xgs.pdb), with those of MAP from Escherichia coli (EcMAP), it was predicted that the extra four short-range ion pairs less than 3 A involved in PfMAP are crucial candidates as determinants for the superior thermostability of PfMAP.
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PMID:Electrostatic stabilization in methionine aminopeptidase from hyperthermophile Pyrococcus furiosus. 955 28

Preparations of rHMfA (recombinant histone A from Methanothermus fervidus) synthesized in E. coli by the heterologous expression of the hmfA gene were found to contain a mixture of rHMfA molecules, approximately 40% that retained the N-terminal formyl-methionyl residue (f-met-rHMfA), approximately 50% that lacked the formyl moiety but retained the methionyl residue (met-rHMfA), and only approximately 10% that had lost both components of the protein synthesis initiating amino acid residue and therefore had the same N-terminal sequence as native HMfA molecules synthesized in Mt. fervidus. Expression of the hmfA gene in E. coli cells grown in the presence of trimethoprim and thymidine, coupled with the concurrent over-expression of a methionine aminopeptidase-encoding map gene, has been shown to overcome this N-terminal heterogeneity problem and to result in rHMfA preparations in which > 85% of the molecules have the fully processed, native N-terminal sequence. This procedure should be generally useful for ensuring N-terminal processing of recombinant proteins synthesized in E. coli.
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PMID:Improved N-terminal processing of recombinant proteins synthesized in Escherichia coli. 963 92


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