Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The structures of novel antimicrobial antibiotics, amythiamicins A, B and C, were elucidated by chemical degradations and NMR spectral analyses. The main frame from C-1 to C-41 of these antibiotics was the same as that of amythiamicin D. Amino acid autoanalyses of amythiamicins A, B and C showed that these have another one mole of serine and proline in comparison with amythiamicin D. Stereochemistries of both amino acids were determined to be L by chiral HPLC. These seryl-prolyl residues in amythiamicins A, B and C are attached at C-41 through an oxazoline ring, amide and ester bond, respectively.
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PMID:Novel antibiotics, amythiamicins. III. Structure elucidations of amythiamicins A, B and C. 796 Nov 66

The G protein alpha-subunit G alpha 2 is essential to the developmental program of Dictyostelium. G alpha 2 is transiently phosphorylated on a serine residue(s) following stimulation with extracellular cAMP (Gundersen, R. E., and Devreotes, P.N. (1990) Science 248, 591-593). To aid in defining the function of alpha-subunit phosphorylation, we identified the site of G alpha 2 phosphorylation. Comparison of the isoelectric points (pI) of the phosphorylated and nonphosphorylated forms indicated that a single mole of phosphate is added to G alpha 2. Cleavage at tryptophan residues and immunoprecipitation with a specific peptide antibody localized the phosphorylated serine in the N-terminal 119 residues. Analysis of a series of G alpha 1 and G alpha 2 chimeras further confined the site between amino acids 33 and 215. Site-directed mutagenesis of serines between amino acids 33 and 119 produced two mutants that were not phosphorylated, S45A and S113A. Ser113 was identified as the site by sequential Edman degradation of 32P-radiolabeled G alpha 2 digested with endoproteinase Glu-C. We have expressed the G alpha 2 mutants S113A, S113I, S113T, and S113D in a G alpha 2 null cell line to examine the function of phosphorylation.
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PMID:Serine 113 is the site of receptor-mediated phosphorylation of the Dictyostelium G protein alpha-subunit G alpha 2. 806 9

The pH titration of nine amino acids (glycine, proline, valine, serine, glutamine, tryptophan, arginine, histidine and aspartic acid) in presence of urea in the concentration range 1-8 mole dm-3 has been performed. The results support suppression of the first dissociation constant (K1) of the amino acids and acceptance of H+ ions by the amide forming uronium ion (UH+). The second dissociation constant (K2) of the amino acids is affected relatively weakly by urea. Quantitative evaluation of different species existing in solution and the degree of dissociation of the acids as well as the degree of binding of H+ ion to the amide have been made. It has been found that the polarity of the aqueous-urea medium does not straight forwardly correlate with the altered pK1 of the amino acids. Urea can also affect the pH-titration behaviour of gelatin with an increase of the intrinsic pK of the acidic groups of the protein by 0.45 unit.
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PMID:Interaction of amino acids and gelatin with urea. 814 76

Ligand binding to the serine receptor of Escherichia coli has been studied using isothermal titration calorimetry. Bacterial inner membranes enriched in the serine receptor (Tsr) were titrated as sonicated membrane samples and after solubilization in octyl beta-D-glucopyranoside (OG) to determine the number of moles of ligand bound per mole of receptor (n), the binding constant (Ka), and the enthalpy of binding (delta H) of serine to the receptor. The n value for serine binding to OG-solubilized Tsr protein (n = 0.5) was consistent with one molecule of serine binding to a receptor dimer, but in sonicated inner membrane samples, the n value was smaller (n approximately equal to 0.25), indicating that not all of the binding sites were accessible to added serine. At 7 and 27 degrees C, the values for Ka and delta H were equivalent for the membrane and OG-solubilized samples and were found to be 4.7 x 10(4) M-1 and -15 kcal/mol, and 3.6 x 10(4) M-1 and -18 kcal/mol, respectively. The influence of covalent modification at the sites of methylation on the affinity of the receptor for serine was also investigated, and found to have only a modest effect. The property of half-site saturation is suggestive of models for transmembrane signaling where the receptor subunit interactions are modulated by ligand binding.
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PMID:The serine receptor of bacterial chemotaxis exhibits half-site saturation for serine binding. 820 92

Histidine ammonia-lyase (histidase) from Pseudomonas putida was irreversibly inactivated by L-cysteine at pH 10.5 in the presence of oxygen. Inactivation was accompanied by the formation of a new uv-absorbing species centered around 340 nm. L-[35S]cysteine labeling experiments revealed that 4 mol of L-cysteine was bound per mole of enzyme tetramer upon complete modification. However, the radiolabel was dissociated from the protein under denaturing conditions without loss of the 340-nm absorbance. Prior inactivation of histidase by cyanide, borohydride, or bisulfite precluded the formation of the 340-nm species in subsequent L-cysteine modification experiments. This suggests a common target site for modification of histidase by all of these reagents. Based on its strong absorbance at 340 nm an octapeptide was isolated from L-cysteine-inactivated histidase following trypsin and staphylococcal V8 protease digestion. Electrospray MS/MS revealed that this peptide (Gly138-SerValGlyAlaSerGlyAsp145) contained an unidentified modification of mass 184 Da located on Ser143. This peptide and the serine residue are conserved in all histidases and phenylalanine ammonia-lyases for which the amino acid sequence is available. Ser143 represents the binding site for an electrophilic cofactor required for histidase activity.
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PMID:Identification of Ser143 as the site of modification in the active site of histidine ammonia-lyase. 823 49

Arachidonyl trifluoromethyl ketone (AACOCF3) is a slow- and tight-binding inhibitor of the human cytosolic phospholipase A2 (cPLA2) [Street et al. (1993) Biochemistry 32, 5935]. 19F and 13C NMR experiments have been carried out to elucidate the structure of the cPLA2.AACOCF3 complex. One mole of AACOCF3 per mole of enzyme is tightly bound in the active site while excess molar equivalents of the inhibitor associate loosely and nonspecifically with hydrophobic regions of the protein. Incubation of the cPLA2.AACOCF3 complex with a 10-fold molar excess of a structurally related inhibitor allows the slow dissociation of the enzyme-inhibitor complex to be followed with 19F NMR. These results establish that the bound inhibitor is in slow exchange with the free ligand and that inhibition of the cPLA2 by AACOCF3 is not due to irreversible modification of the protein. AACOCF3 labeled with 13C at the carbonyl position was used to determine the nature of the bound inhibitor species. A comparison of the 13C NMR chemical shift value obtained from labeled enzyme-inhibitor complex (delta c 101.0 ppm) with the chemical shift values obtained from model compounds suggests that the enzyme-bound inhibitor species is a charged hemiketal. These results are very similar to those obtained previously with alpha-chymotrypsin and a peptidyl trifluoromethyl ketone inhibitor [Liang, T.-C., & Abeles, R. H. (1987) Biochemistry 26, 7603] and, by analogy with the serine proteases, a structural model for the cPLA2.AACOCF3 complex is proposed.
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PMID:NMR structural studies of the tight complex between a trifluoromethyl ketone inhibitor and the 85-kDa human phospholipase A2. 825 73

Thrombospondin, a glycoprotein of three identical disulfide-bonded subunits, is a constituent of platelet alpha-granules and a variety of normal and transformed cells and binds to cell surfaces and becomes incorporated into extracellular matrix. It has been implicated in processes such as wound healing and tumor growth and metastasis. In addition, thrombospondin was shown recently to be an inhibitor of the fibrinolytic enzyme, plasmin. In the cause of studying the effects of thrombospondin on other serine proteinases, we found that thrombospondin binds neutrophil elastase in an active-site-dependent manner and competitively inhibits the activity of the enzyme. In a competitive binding assay, neutrophil elastase bound to thrombospondin with a dissociation constant of 17 +/- 7 nM, expressed per mole of thrombospondin trimer, or 52 +/- 20 nM, expressed per mole of thrombospondin subunit. In kinetic studies of the inhibition of the amidolytic activity of neutrophil elastase by thrombospondin, 2.7 +/- 0.3 mol of elastase interacted with 1 mol of thrombospondin trimer with a site-binding constant of 57 +/- 13 nM. Lower limits for the on rate constant of 5 x 10(6) M-1 s-1 and off rate constant of 0.27 s-1 were established. Affinity of binding of neutrophil elastase to thrombospondin was sensitive to ionic strength and calcium ions. Thrombospondin was cleaved by neutrophil elastase, but the site(s) of the limited cleavage are independent of the competitive inhibition of elastase activity by thrombospondin. Neutrophil elastase inactivated with phenylmethylsulfonyl fluoride did not compete with active elastase for binding to thrombospondin, implying that a functional active site is important for the interaction of elastase with thrombospondin. Thrombospondin protected fibronectin from cleavage by neutrophil elastase. In summary, the binding of neutrophil elastase to thrombospondin is tight, reversible, and close enough to the active site of elastase to exclude small synthetic tripeptidyl p-nitroanilide substrates and macromolecular protein substrates. Two potential reactive centers that may be involved in binding elastase have been identified in the calcium-binding type 3 domains of thrombospondin. Neutrophil elastase is the enzyme primarily responsible for degrading and solubilizing connective tissue during inflammatory processes. These findings suggest a previously unsuspected mechanism for regulation of elastase activity at inflammatory sites.
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PMID:Thrombospondin is a tight-binding competitive inhibitor of neutrophil elastase. 846 50

The function of the uterine smooth muscle in gestation and parturition is affected by a variety of hormones and biomolecules, some of which alter the intracellular levels of cAMP and Ca2+. Since the activity of smooth muscle MLCK has been shown to be modulated by phosphorylation, the effect of this modification of pregnant sheep myometrium (psm) MLCK by the catalytic subunit of cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) was studied. In contrast to other smooth muscle MLCK reported, PKA incorporates 2.0-2.2 moles phosphate into a mole of psm MLCK both in the presence and absence of Ca(2+)-calmodulin. Modification of serine residues inhibited the activity of the enzyme. PKC also incorporated 2.0-2.1 moles of phosphate per mole psmMLCK under both conditions but had no effect on the MLCK activity. Sequential phosphorylation by PKC and PKA incorporated 3.8-4.1 moles phosphate suggesting that the amino acid residues modified by the two kinases are different. Phosphoamino acid analysis of the MLCK revealed that PKC phosphorylated serine and threonine residues. The double reciprocal plots of the enzyme activity and calmodulin concentrations showed that the Vmax of the reaction is not altered by phosphorylation by PKA but the calmodulin concentration require for half-maximal activation is increased about 4-fold. Only 10 out of 17 monoclonal antibodies to various regions of the turkey gizzard MLCK cross-reacted with psmMLCK suggesting structural differences between these enzymes. Comparison of the deduced amino acid sequence of the cDNA encoding the C-terminal half of the psmMLCK molecule showed that while cgMLCK and psmMLCK are highly homologous, a number of nonconservative substitutions are present, particularly near the PKA phosphorylation site B (S828).
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PMID:Phosphorylation and partial sequence of pregnant sheep myometrium myosin light chain kinase. 856 50

We report the successful one-step separation of tissue kallikrein from the salivary glands of an insectivore, the Eastern Atlantic mole (Scalopus aquaticus) by perfusion chromatography. Purified mole salivary kallikrein was characterized as a 30-kDa serine proteinase with a pI of 5.3 and a pH optimum of 9.0. It was readily recognized by human tissue kallikrein antibody in immunoblot analyses. It preferentially hydrolyzes fluorogenic peptidyl substrates with arginyl residues, rather than lysyl residues at the P1 substrate recognition site, indicating that it is like other mammalian kallikreins. Mole kallikrein efficiently releases kinin from low molecular weight human, dog, and bovine kininogen substrates with specific activities similar to that of human tissue kallikrein. Steady state kinetics performed with the synthetic tripeptidyl substrates, Phe-Phe-Arg-, Pro-Phe-Arg, and Val-Leu-Arg-7-amino-4-methylcoumarin, gave K(m) values for mole kallikrein of 3.3, 46.1, and 2.8 microM, respectively, and specificity constants, kcat/K(m), of 3818, 165, and 8714 s-1 pM-1, respectively. Mole kallikrein, when compared with human and rat tissue kallikreins, more closely resembles human kallikrein based on immunoreactivity and kininogenase activity. Mole kallikrein appears to be a member of a single gene or small multigene family. S. aquaticus is recommended for studying the evolution of mammalian proteins and may offer advantages over rodent models for biomedical research.
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PMID:Purification and characterization of salivary kallikrein from an insectivore (Scalopus aquaticus): substrate specificities, immunoreactivity, and kinetic analyses. 861 26

Two 29 kDa subunits of the multicatalytic proteinase (proteasome) complex, the C8 and C9 components, are phosphorylated in vivo and can be phosphorylated in vitro by casein kinase II (CKII). The major phosphate acceptor is the C8 subunit being phosphorylated in serine, both in vivo and in vitro. The phosphopeptides generated by Glu-C endoprotease digestion from the in vivo 29 kDa labeled subunit and from the in vitro phosphorylation of the recombinant C8 subunit with CKII are identical, suggesting that CKII is likely responsible for the in vivo phosphorylation of the C8 subunit. The in vitro stoichiometry of phosphorylation of the proteasome complex and the recombinant C9 and C8 subunits by CKII is 2-2.5, 0.2, and 2 mol of phosphate per mole, respectively. Several C8 protein constructs allow the location of the CKII phosphorylation sites to be the COOH terminal portion of the protein, and direct mutational analyses show that Ser-243 and Ser-250 are the residues of the C8 subunit phosphorylated by CKII. The in vitro phosphorylation of the proteasome by CKII does not affect its proteolytic activity (on proteins or fluorogenic synthetic peptides), therefore suggesting its involvement in the interaction of the proteasome with other cellular proteins, i.e. in the formation of the 26S complex and/or in the interaction with the nuclear translocation machinery.
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PMID:Phosphorylation of C8 and C9 subunits of the multicatalytic proteinase by casein kinase II and identification of the C8 phosphorylation sites by direct mutagenesis. 861 99


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