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)

The specificities of methionine aminopeptidase and amino-terminal acetylation in the yeast Saccharomyces cerevisiae were investigated in vivo by sequencing a series of altered iso-1-cytochrome c. Twenty iso-1-cytochromes c, each having a different penultimate residue in the sequence Met-Xaa-Phe-Leu-, were created by transforming yeast directly with synthetic oligonucleotides. The degree of methionine cleavage and amino-terminal acetylation was estimated from the levels of pertinent peptides separated by high performance liquid chromatography. The results confirmed our earlier hypothesis (Sherman, F., Stewart, J. W., and Tsunasawa, S. (1985) BioEssays 3, 27-31) that methionine is completely removed from penultimate residues having radii of gyration of 1.29 A or less (glycine, alanine, serine, cysteine, threonine, proline, and valine). However, only partial cleavage occurred in the sequences Met-Thr-Pro-Leu- and Met-Val-Pro-Leu-, demonstrating that proline at the third position inhibits methionine cleavage when the penultimate residue has an intermediate radius of gyration. Acetylation of the retained amino-terminal methionine occurred completely with the Ac-Met-Glu-Phe-Leu- and Ac-Met-Asp-Phe-Leu- sequences and partially with the Ac-Met-Asn-Phe-Leu-sequence. Although the consensus for acetylation of the retained amino-terminal methionine is not completely known, these results and the results of published sequences indicated that Ac-Met-Glu- and Ac-Met-Asp- (methionine followed by an acidic residue) is sufficient for amino-terminal acetylation in eukaryotes but not in prokaryotes.
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PMID:The specificities of yeast methionine aminopeptidase and acetylation of amino-terminal methionine in vivo. Processing of altered iso-1-cytochromes c created by oligonucleotide transformation. 217 47

Although kinins have been reported to affect cerebral vascular tone and permeability, their actions are not potentiated by angiotensin converting enzyme inhibitors. To investigate cerebral vascular kinin metabolism, porcine cerebral microvessels were isolated by differential sieving and centrifugation and characterized by microscopic examination and marker enzyme enrichment. Purified microvessels contained a membrane-bound carboxypeptidase which hydrolyzed the C-terminal Phe-Arg bond of both kallidin and bradykinin. Hydrolysis was optimal at pH 7.0, was activated more than 300% by 0.1 mM CoCl2, and was inhibited by o-phenanthroline and the carboxypeptidase N (EC 3.4.17.3) inhibitor DL-2-mercaptomethyl-3-guanidino-ethylthiopropanoic acid (MERGETPA) (IC50 = 2 microM). Conversely, inhibitors of angiotensin I converting enzyme (captopril), neutral endopeptidase (phosphoramidon), post proline cleaving enzyme (Z-Pro-prolinal), dipeptidyl(amino)peptidase IV (diprotin A) and amino-peptidase M (amastatin) had no effect. When the rates of C-terminal hydrolysis of kallidin by detergent-solubilized cerebral microvasculature were determined over a range of substrate concentrations (16.6 to 250 microM), the Km and Vmax values obtained were 26.0 +/- 3.0 microM and 14.7 +/- 1.3 nmol/min/ml (N = 4) respectively. These data suggest that a cerebral microvascular carboxypeptidase may play a role in vivo in modulating the effects of kinins on cerebral blood flow and permeability and in preventing circulating kinins from crossing the blood-brain barrier.
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PMID:Kallidin and bradykinin metabolism by isolated cerebral microvessels. 339 72

A heat stable microtubule-associated protein of Mr 190,000 (190-kDa MAP) has been purified from bovine adrenal cortex (Murofushi, H., Kotani, S., Aizawa, H., Hisanaga, S., Hirokawa, N., and Sakai, H. (1986) J. Cell Biol. 103, 1911-1919). Limited chymotryptic digestion of 190-kDa MAP produced a fragment of Mr 27,000 (27-kDa fragment), which bound to microtubules reconstituted in the presence of taxol. This fragment was purified with the aid of cosedimentation with microtubules. The purified 27-kDa fragment showed an ability to stimulate tubulin polymerization in the absence of taxol. Electron microscopic observation of microtubules reconstituted from purified 27-kDa fragment and tubulin revealed that the microtubules were in the form of thick bundles and that lateral projections which can be seen in microtubules reconstituted from intact 190-kDa MAP and tubulin were not observed. These results indicate that 27-kDa fragment includes or is a part of microtubule-binding domain of 190-kDa MAP and that this fragment is active in stimulating microtubule assembly. Amino acid analysis revealed that the 27-kDa fragment was rich in lysine, proline, and alanine, the sum of these three being about 45% of the total amino acids and that the contents of methionine, tyrosine, phenylalanine, and histidine were very low. These data suggest that the microtubule binding domain of the 190-kDa MAP comprises an unique structure.
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PMID:Limited chymotryptic digestion of bovine adrenal 190,000-Mr microtubule-associated protein and preparation of a 27,000-Mr fragment which stimulates microtubule assembly. 381 66

A methionine aminopeptidase (MAP) found in rat liver microsomes behaves as membrane-bound enzyme. Triton-solubilized MAP when chromatographed on DEAE-cellulose columns was separated from other microsomal arylamidases. The enzyme hydrolyzes N-terminal methionine from methionyl-lysyl-bradykinin (Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) being then characterized as a typical aminopeptidase. It also shows preferential arylamidase activity upon Met-2-naphthylamide. MAP was activated by 2-mercaptoethanol and inhibited by p-hydroxymercuribenzoate. Contrarily to other well characterized aminopeptidases, MAP was not affected by EDTA, puromycin or bestatin. Altogether these data suggest that MAP is a unique microsomal enzyme distinct from other previously described aminopeptidases. It could be involved in the removal of methionine from nascent peptides during protein synthesis.
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PMID:Microsomal methionine aminopeptidase: properties of the detergent-solubilized enzyme. 393 47

Captopril (Cap) 20 mg.kg-1.d-1 and clonidine (Clo) 300 micrograms.kg-1.d-1 were given po to SHR from their parents mating day to 24 wk of age, with untreated, age-matched SHR and WKY as controls. Stress cardiac function was assessed by the development of LVdp/dtmax in response to incremental pressure load (phenylephrine, Phe) and volume load (dextran, Dex). The slope of delta HR/delta MAP relationship curve was used as an index of baroceptor sensitivity. Results showed that both Cap and Clo caused decreases in BP in SHR. Cap not only reduced markedly the left ventricular mass/body weight (LVM/BW), (mg.g-1, 2.7 +/- 0.4 vs SHR 3.5 +/- 0.3, P < 0.01), but also normalized the LVdp/dtmax, -LVdp/dtmax, T value, and the stress cardiac function. Clo neither decreased the LVM/BW (mg.g-1, 3.4 +/- 0.5 vs SHR 3.5 +/- 0.3, P > 0.05), nor improved the resting and stress cardiac function. The results suggested that attenuation of the left ventricular hypertrophy (LVH) is beneficial not only to the resting but also stress cardiac performance.
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PMID:Effects of captopril and clonidine on resting and stress cardiac performance of left ventricle in spontaneously hypertensive rats. 770 47

Deletion of the SLT2 gene of Saccharomyces cerevisiae, which codes for a homologue of MAP (mitogen-activated) protein kinases, causes an autolytic lethal phenotype in cells grown at 37 degrees C. The gene encodes domains characteristic of protein kinases, which include a lysine (at position 54) that lies 19 residues from a glycine-rich cluster, considered to be the putative ATP binding site. The ability of three mutant alleles of SLT2 generated by site-directed mutagenesis, namely E54 (glutamic acid), R54 (arginine) and F54 (phenylalanine), to complement slt2 mutants was tested. All three failed to complement the autolytic phenotype and were unable to restore growth and viability of cells. A strain obtained by transplacement of slt2-F54 also behaved as a thermosensitive autolytic mutant. By immunoprecipitation with polyclonal antibodies raised against Slt2 protein expressed in Escherichia coli, it was possible to confirm that alteration of the lysine-54 residue did not affect the stability of the protein, thus allowing us to conclude that activity of the Slt2 protein kinase is critically required for growth and morphogenesis of S. cerevisiae at 37 degrees C. A significant fraction of the mutant cell population lysed at 24 degrees C and the cells displayed a characteristic alteration of the surface consisting of a typical depression in an area of the cell wall. At 37 degrees C, the cell surface was clearly disorganized.
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PMID:Activity of the yeast MAP kinase homologue Slt2 is critically required for cell integrity at 37 degrees C. 823 2

Inhibitors of ornithine decarboxylase (ODC), such as alpha-difluoromethylornithine (DFMO), may influence the cytotoxicity of anti-tumour agents that interact with DNA. Intracellular levels of putrescine and spermidine were markedly reduced by ODC inhibitors while the level of spermine, which is the main polyamine in nuclei, was unchanged. By combining a novel inhibitor of ODC, such as (2R, 5R)-6-heptyne-2,5-diamine (MDL 72.175, MAP), with an inhibitor of S-adenosylmethionine decarboxylase (SAMDC), such as 5'-[[(Z)-4-aminobut-2-enyl]methylamino]-5'-deoxyadenosine (MDL 73.811, AbeAdo), spermine was selectively depleted in a human ovarian cancer cell line OVCAR-3 (i.e. spermine became almost undetectable whereas the levels of spermidine and putrescine were not affected). The depletion of spermine blocked DNA synthesis with a consequent accumulation of cells in the G1 phase of the cell cycle. Pretreatment with MAP plus AbeAdo did not change the cytotoxicity of alkylating agents, such as L-phenylalanine mustard (L-PAM), 1,4-bis(2'-chloroethyl)-1,4-diazabicyclo-[2.2.1] heptane diperchlorate (DABIS), 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), cis-diamminedichloroplatinum (II) (cis-DDP), N-deformyl-N-[4-N-N,N-bis (2-chloroethylamino)benzoyl] (tallimustine) or CC-1065, whereas it markedly reduced the cytotoxicity of DNA topoisomerase II inhibitors, such as doxorubicin (DX) and 4'-demethylepipodophyllotoxin-5-(4,6-O)-ethylidene- beta-D-glycopyranoside (VP-16). The addition of spermine before drug treatment restored the sensitivity to the DNA topoisomerase II inhibitors, thus indicating that the reduced effect was related to the intracellular spermine level. The reason for the reduction in cytotoxicity is unclear, but it does not appear to be related to a cell cycle effect or to a decrease in the intracellular level of DNA topoisomerase II. Drugs that modify polyamine biosynthesis are under early clinical development as potential new anti-tumour agents. These findings illustrate the need for caution in combining such drugs with DNA topoisomerase II inhibitors.
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PMID:Treatment with inhibitors of polyamine biosynthesis, which selectively lower intracellular spermine, does not affect the activity of alkylating agents but antagonizes the cytotoxicity of DNA topoisomerase II inhibitors. 908 39

Proline dipeptidase (prolidase) was purified from cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography. The enzyme is a homodimer (39.4 kDa per subunit) and as purified contains one cobalt atom per subunit. Its catalytic activity also required the addition of Co2+ ions (Kd, 0.24 mM), indicating that the enzyme has a second metal ion binding site. Co2+ could be replaced by Mn2+ (resulting in a 25% decrease in activity) but not by Mg2+, Ca2+, Fe2+, Zn2+, Cu2+, or Ni2+. The prolidase exhibited a narrow substrate specificity and hydrolyzed only dipeptides with proline at the C terminus and a nonpolar amino acid (Met, Leu, Val, Phe, or Ala) at the N terminus. Optimal prolidase activity with Met-Pro as the substrate occurred at a pH of 7.0 and a temperature of 100 degrees C. The N-terminal amino acid sequence of the purified prolidase was used to identify in the P. furiosus genome database a putative prolidase-encoding gene with a product corresponding to 349 amino acids. This gene was expressed in Escherichia coli and the recombinant protein was purified. Its properties, including molecular mass, metal ion dependence, pH and temperature optima, substrate specificity, and thermostability, were indistinguishable from those of the native prolidase from P. furiosus. Furthermore, the Km values for the substrate Met-Pro were comparable for the native and recombinant forms, although the recombinant enzyme exhibited a twofold greater Vmax value than the native protein. The amino acid sequence of P. furiosus prolidase has significant similarity with those of prolidases from mesophilic organisms, but the enzyme differs from them in its substrate specificity, thermostability, metal dependency, and response to inhibitors. The P. furiosus enzyme appears to be the second Co-containing member (after methionine aminopeptidase) of the binuclear N-terminal exopeptidase family.
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PMID:Characterization of native and recombinant forms of an unusual cobalt-dependent proline dipeptidase (prolidase) from the hyperthermophilic archaeon Pyrococcus furiosus. 973 78

Raf is a key serine-threonine protein kinase which participates in the transmission of growth, anti-apoptotic and differentiation messages. These signals can be initiated after receptor ligation and are transmitted to members of the MAP kinase cascade that subsequently activate transcription factors controlling gene expression. Raf is a member of a multigene family which includes: Raf-1, A-Raf and B-Raf. The roles that individual Raf kinases play in the regulation of normal and malignant hematopoietic cell growth are not clear. The following studies show that all three Raf kinases are functionally present in certain human hematopoietic cells, and their aberrant expression can result in abrogation of cytokine dependency. Cytokine-dependent TF-1 cells were infected with retroviruses encoding amino-terminal deleted (delta) A-Raf, B-Raf and Raf-1 proteins. These Raf proteins were conditionally inducible as they were fused to the hormone-binding domain of the estrogen receptor (ER). A hierarchy in the abilities of Raf-containing retroviruses to abrogate cytokine dependency was observed as deltaA-Raf:ER was 20- to 200-fold more efficient than either deltaRaf-1:ER or deltaB-Raf:ER, respectively. This result was unexpected as A-Raf is an intrinsically weaker kinase than either Raf-1 or B-Raf. The activated Raf proteins induced downstream MEK and MAP (ERK1 and ERK2) kinase activities in the cells which proliferated in response to Raf activation. Furthermore, a functional MEK signaling pathway was necessary as treatment of the cells with a MEK1-inhibitor suppressed Raf-mediated proliferation. To determine whether the regulatory phosphorylation residues contained in the modified Raf oncoproteins were necessary for transformation, they were altered by site-directed mutagenesis. Substitution of the regulatory phosphorylation tyrosine residues with phenylalanine in either A-Raf or Raf-1 reduced the capacity of these oncoproteins to abrogate cytokine dependency. In contrast, changing the critical aspartic acid residues of B-Raf to either tyrosine or phenylalanine increased the frequency of estradiol-responsive cells. Thus, the amino acids present in the regulatory residues modulated the capability of Raf proteins to abrogate the cytokine dependency of TF-1 cells. Differences in the levels of Raf and downstream kinase activities were observed between cytokine-dependent and estradiol-responsive deltaRaf:ER-infected cells as estradiol-responsive cells usually expressed more Raf and MEK activity than GM-CSF-dependent, deltaRaf:ER-infected cells. Abrogation of cytokine dependency by the activated deltaRaf:ER proteins was associated with autocrine growth factor synthesis which was sufficient to promote the growth of uninfected TF-1 cells. In summary, these observations indicate that the aberrant expression of certain activated deltaRaf:ER oncoproteins can alter the cytokine dependency of human hematopoietic TF-1 cells. These cells will be useful in evaluating the roles of the individual Raf oncoproteins in signal transduction, cell cycle progression, autocrine transformation, regulation of apoptosis and differentiation. Moreover, these Raf-infected cells may be important in evaluating the efficacy of novel anticancer drugs designed to inhibit Raf and downstream signal transduction molecules.
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PMID:Differential abilities of activated Raf oncoproteins to abrogate cytokine dependency, prevent apoptosis and induce autocrine growth factor synthesis in human hematopoietic cells. 984 21

An apparent conservative mutation, Leu to Val, at the second residue of the rat liver mitochondrial aldehyde dehydrogenase (ALDH) presequence resulted in a precursor protein that was not imported into mitochondria. Additional mutants were made to substitute various amino acids with nonpolar side chains for Leu2. The Ile, Phe, and Trp mutants were imported to an extent similar to that of the native precursor, but the Ala mutant was imported only about one-fourth as well. It was shown that the N-terminal methionine was removed from the L2V mutant in a reaction catalyzed by methionine aminopeptidase. The N-terminal methionine of native pALDH and the other mutant presequences was blocked, presumably by acetylation. Because of the difference in co-translational modification, the L2V mutant sustained a significant loss in the available hydrophobic surface of the presequence. Import competence was restored to the L2V mutant when it was translated using a system that did not remove Met1. The removal of an Arg-Gly-Pro helix linker segment (residues 11-14) from the L2V mutant, which shifted three leucine residues toward the N-terminus, also restored import competence. These results lead to the conclusion that a minimum amount of hydrophobic surface area near the N-termini of mitochondrial presequences is an essential property to determine their ability to be imported. As a result, both electrostatic and hydrophobic components must be considered when trying to understand the interactions between precursor proteins and proteins of the mitochondrial import apparatus.
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PMID:The loss in hydrophobic surface area resulting from a Leu to Val mutation at the N-terminus of the aldehyde dehydrogenase presequence prevents import of the protein into mitochondria. 1021 35


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