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: C30B5 .7
413,019 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1 The stimulatory effects of neurotensin (NT) and several NT fragments were evaluated in two pharmacological preparations: rat stomach strips and isolated spontaneously beating atria of guinea-pigs.2 In rat stomach strips, NT elicited a dose-dependent contractile effect in concentrations varying between 1.3 x 10(-9) and 5.4 x 10(-7) M.3 The contractile effect of NT (1.3 and 5.4 x 10(-8) M) in this tissue was not modified by atropine (3.4 x 10(-7) M), methysergide (2.0 x 10(-6) M), a mixture of cimetidine (8.0 x 10(-6) M) and diphenhydramine (7.8 x 10(-6) M), indomethacin (1.4 x 10(-5) M), 8-Leu-angiotensin II (1.0 x 10(-6) M), glucagon (2.0 x 10(-6) M) or somatostatin (3.0 x 10(-7) M).4 Rat stomach strips desensitized by bradykinin (6.1 x 10(-6) M) or substance P (7.4 x 10(-6) M) maintained their sensitivities to NT (1.3 and 5.4 x 10(-8) M).5 In guinea-pig atria, NT produced a dose-dependent positive inotropic action in concentrations varying between 5.4 x 10(-10) and 2.7 x 10(-7) M.6 The inotropic effect of NT (2.7 x 10(-9) M) was not influenced by methysergide (2.8 x 10(-6) M), atropine (3.4 x 10(-7) M), practolol (1.5 x 10(-5) M), 8-Leu-angiotensin II (1.0 x 10(-6) M), or indomethacin (1.4 x 10(-5) M), but it was reduced by 37% by cimetidine (4.0 x 10(-5) and 2.0 x 10(-4) M). A combination of cimetidine (4.0 x 10(-5) M) and diphenhydramine (3.9 x 10(-6) M) did not produce a greater inhibition of NT than cimetidine alone.7 Atria desensitized by bradykinin (6.1 x 10(-6) M) or glucagon (2.0 x 10(-6) M) maintained their sensitivities to NT (2.7 x 10(-9) M). Substance P was inactive both as an agonist or antagonist of NT.8 These results suggest the existence of specific NT receptors in rat stomach strips and guinea-pig atria.9 The data derived from our structure-activity study suggest that the minimum structure required for the full stimulation of NT receptors in these two preparations is H-Arg(9)-Pro(10)-Tyr(11)-Ile(12)-Leu(13)-OH. The sequence PyroGlu(1)-Leu(2)-Tyr(3)-Glu(4)-Asn(5)-Lys(6)-Pro(7)-Arg(8)- and the amino acids Ile(12) and Leu(13) appear to contribute mainly to the affinity or binding of NT to its receptor. The chemical groups responsible for the full activation (intrinsic activity) of NT receptors seem to be located in the sequence -Arg(9)-Pro(10)-Tyr(11).
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PMID:The stimulatory effects of neurotensin and related peptides in rat stomach strips and guinea-pig atria. 735 2

Many studies have been reported on the reaction of formaldehyde (FA) with amino acids or proteins, and FA is assumed to react with the alpha-amino group as well as some of the side chain groups. In most of these investigations a large excess of FA relative to amino acids or proteins was employed. In the present study, however, we carried out the reaction with a smaller excess of FA in order to clarify the reactivity, firstly with the alpha-amino groups, and secondly with specific side chain groups. No evidence for so-called Schiff base (-N = CH2) formation was obtained in the reaction with the alpha-amino groups, but the formation of an acid-labile N-hydroxymethyl compound as a major product was suggested by NMR and IR at greater than pH 9.2. There was no indication, however, of the presence of such a product below pH 9.2, and the amount of N-hydroxymethyl product increased in parallel with the reaction pH. The higher the reaction pH (greater than or equal to 9.2), the greater the consumption of FA, up to 2 mol/mol Ala. In addition, the larger the excess of FA, the smaller the amount of free amino group remaining (at pH 9.7). In the assignment of IR spectra, discrete absorption bands of the alpha-carboxyl group of Ala were observed, which reflected ionization states of the alpha-amino group, and these were utilized for analysis of the reaction mechanism. Furthermore, among amino acids with side chain groups, His, Trp, and Arg showed high reactivity and Asn showed moderate reactivity. The products were relatively stable and were purified and subjected to instrumental analyses. Sixteen other amino acids including Tyr and Lys did not yield stable products. The products from Arg were unique because of the non-involvement of the amino group, and were reversibly converted to the original Arg upon acid hydrolysis. The products from His, Trp and Asn all involved amino or amide nitrogen forming cyclic ring structures with methylene derived from FA. The chemical structures of these products were determined on the basis of elemental analyses, MS and NMR.
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PMID:Reevaluation of the reaction of formaldehyde at low concentration with amino acids. 739 Sep 97

The DNA sequence of a 24.7 kb region covering the left arm of chromosome XIV from Saccharomyces cerevisiae was determined. This region contains 17 open reading frames (ORFs) which code for proteins of more than 100 amino acids. Five ORFs correspond to the KRE1, ATP11, DAL82, RFA2 and MCK1 loci, described previously. Two ORFs present high similarity to known proteins: NO345 with the hexose transporter family, and NO351 with the yeast chorismate mutase/prephenate dehydratase enzyme encoded by PHA2. Six ORFs show limited similarity with known proteins or some specific features: NO339 presents 11 potential transmembrane domains. NO343, which is internal to NO345, presents a putative signal sequence and a potential transmembrane domain. NO348 shows similarity with YCW2, TUP1 and SEC13. NO364 reveals a signature for a pyridoxal-phosphate attachment site. Finally, NO384 and NO388 present a biased amino acid composition, being rich in Asn or Glu/Lys/Arg, respectively. Four other ORFs (NO342, NO376, NO381 and NO397) show no similarity to proteins within the databases screened.
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PMID:Sequencing analysis of a 24.7 kb fragment of yeast chromosome XIV identifies six known genes, a new member of the hexose transporter family and ten new open reading frames. 750 83

An endo-acting proline-specific oligopeptidase (prolyl oligopeptidase [POPase], EC 3.4.21.26) was purified to homogeneity from the Triton X-100 extracts of cells of Treponema denticola ATCC 35405 (a human oral spirochete) by a procedure that comprised five successive fast protein liquid chromatography steps. The POPase is a cell-associated 75- to 77-kDa protein with an isoelectric point of ca. 6.5. The enzyme hydrolyzed (optimum pH 6.5) the Pro-pNA bond in carbobenzoxy-Gly-Pro-p-nitroanilide (Z-Gly-Pro-pNA) and bonds at the carboxyl side of proline in several human bioactive peptides, such as bradykinin, substance P, neurotensin, angiotensins, oxytocin, vasopressin, and human endothelin fragment 22-38. The minimum hydrolyzable peptide size was tetrapeptide P3P2P1P'1, while the maximum substrate size was ca. 3 kDa. An imino acid residue in position P1 was absolutely necessary. The hydrolysis of Z-Gly-Pro-pNA was potently inhibited by the following, with the Ki(app) (in micromolar) in parentheses: insulin B-chain (0.7), human endothelin-1 (0.5), neuropeptide Y (1.7), substance P (32.0), T-kinin (4.0), neurotensin (5.0), and bradykinin (16.0). Chemical modification and inhibition studies suggest that the POPase is a serine endopeptidase whose activity depends on the catalytic triad of COOH ... Ser ... His but not on a metal. The amino acid sequence around the putative active-site serine is Gly-Gly-Ser-Asn-Pro-Gly. The enzyme is suggested to contain a reactive cysteinyl residue near the active site. Amino acid residues 4 to 24 of the first 24 N-terminal residues showed a homology of 71% with the POPase precursor from Flavobacterium meningosepticum and considerable homology with the Aeromonas hydrophila POPase. The ready hydrolysis of human bioactive peptides at bonds involving an imino acid residue suggests that enzymes like POPase may contribute to the chronicity of periodontal infections by participating in the peptidolytic processing of those peptides.
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PMID:An endo-acting proline-specific oligopeptidase from Treponema denticola ATCC 35405: evidence of hydrolysis of human bioactive peptides. 752 1

Infection with virus variants exhibiting changes in the peptide sequences defining immunodominant determinants that abolish recognition by antiviral cytotoxic T cells (CTL) presents a considerable challenge to the antiviral T-cell immune system and may enable some viruses to persist in hosts. The potential importance of such variants with respect to mechanisms of viral persistence and disease pathogenesis was assessed by infecting adult mice with variants of lymphocytic choriomeningitis virus (LCMV) strain WE. These variants were selected in vivo or in vitro for resistance to lysis by CD8+ H-2b-restricted antiviral CTL. The majority of anti-LCMV CTL in infected H-2b mice recognize epitopes defined by residues 32 to 42 and 275 to 289 (epitopes 32-42 and 275-289) of the LCMV glycoprotein or 397 to 407 of the viral nucleoprotein. The 8.7 variant exhibits a change in the epitope 32-42 (Val-35-->Leu), and variant CL1.2 exhibits a change in the epitope 275-289 (Asn-280-->Asp) of the wild-type LCMV-WE. The double-mutated 8.7-B23 variant had the variation of 8.7 and an additional change located in the epitope 275-289 (Asn-280-->Ser). The 8.7 variant peptide with unchanged anchor positions bound efficiently to H-2Db and H-2Kb molecules but induced only a very weak CTL response. CTL epitope 275-289 of CL1.2 and 8.7-B23 altered at predicted anchor residues were unable to bind Db molecules and were also not recognized by antiviral CTL. Infection of C57BL/6 mice (H-2b) with the variants exhibiting mutations of one of the CTL epitopes, i.e., 8.7 or CL1.2, induced CTL responses specific for the unmutated epitopes comparable with those induced by infection with WE, and these responses were sufficient to eliminate virus from the host. In contrast, infection with the double-mutated variant 8.7-B23 induced CTL activity that was reduced by a factor of about 50-fold compared with wild-type LCMV. Consequently, high doses (10(7) PFU intravenously) of this virus were eliminated slowly and only by about day 100 after infection. 8.7-B23 failed to cause lethal lymphocytic choriomeningitis after intracerebral infection with a dose of > 10(4) PFU in C57BL/6 mice (but not in mice of nonselecting H-2d haplotype); with the other variants or wild-type LCMV, doses greater than 10(6) to 10(7) PFU were necessary to avoid lethal choriomeningitis.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Immunobiology of cytotoxic T-cell escape mutants of lymphocytic choriomeningitis virus. 753 51

The gene coding for the 3-phosphoglycerate kinase (EC 2.7.2.3) of Pyrococcus woesei was cloned and sequenced. The gene sequence comprises 1230 bp coding for a polypeptide with the theoretical M(r) of 46,195. The deduced protein sequence exhibits a high similarity (46.1% and 46.6% identity) to the other known archaeal 3-phosphoglycerate kinases of Methanobacterium bryantii and Methanothermus fervidus [Fabry, S., Heppner, P., Dietmaier, W. & Hensel, R. (1990) Gene 91, 19-25]. By comparing the 3-phosphoglycerate kinase sequences of the mesophilic and the two thermophilic Archaea, trends in thermoadaptation were confirmed that could be deduced from comparisons of glyceraldehyde-3-phosphate dehydrogenase sequences from the same organisms [Zwickl, P., Fabry, S., Bogedain, C., Haas, A. & Hensel, R. (1990) J. Bacteriol. 172, 4329-4338]. With increasing temperature the average hydrophobicity and the portion of aromatic residues increases, whereas the chain flexibility as well as the content in chemically labile residues (Asn, Cys) decreases. To study the phenotypic properties of the 3-phosphoglycerate kinases from thermophilic Archaea in more detail, the 3-phosphoglycerate kinase genes from P. woesei and M. fervidus were expressed in Escherichia coli. Comparisons of kinetic and molecular properties of the enzymes from the original organisms and from E. coli indicate that the proteins expressed in the mesophilic host are folded correctly. Besides their higher thermostability according to their origin from hyperthermophilic organisms, both enzymes differ from their bacterial and eucaryotic homologues mainly in two respects. (a) The 3-phosphoglycerate kinases from P. woesei and M. fervidus are homomeric dimers in their native state contrary to all other known 3-phosphoglycerate kinases, which are monomers including the enzyme from the mesophilic Archaeum M. bryantii. (b) Monovalent cations are essential for the activity of both archaeal enzymes with K+ being significantly more efficient than Na+. For the P. woesei enzyme, non-cooperative K+ binding with an apparent Kd (K+) of 88 mM could be determined by kinetic analysis, whereas for the M. fervidus 3-phosphoglycerate kinase the K+ binding is rather complex: from the fitting of the saturation data, non-cooperative binding sites with low selectivity for K+ and Na+ (apparent Kd = 270 mM) and at least three cooperative and highly specific K+ binding sites/subunit are deduced. At the optimum growth temperature of P. woesei (100 degrees C) and M. fervidus (83 degrees C), the 3-phosphoglycerate kinases show half-lives of inactivation of only 28 min and 44 min, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Dimeric 3-phosphoglycerate kinases from hyperthermophilic Archaea. Cloning, sequencing and expression of the 3-phosphoglycerate kinase gene of Pyrococcus woesei in Escherichia coli and characterization of the protein. Structural and functional comparison with the 3-phosphoglycerate kinase of Methanothermus fervidus. 758 50

Plasma protein S is a cofactor of activated protein C (APC) in the regulation of the blood coagulation system. Rat protein S homogeneously purified from plasma showed cofactor activity for rat APC, but not for human APC when the APC cofactor activity was assayed using protein S- and C4b-binding protein (C4BP)-depleted human plasma. Rat plasma protein S was separated by gel chromatography into two forms, a free form and a form complexed with C4BP. Rat protein S forms complexes with rat and human C4BP in a solid-phase model with apparent dissociation constants (Kds) of 6.7 x 10(-8) and 1.2 x 10(-8) M, respectively, in the presence of 5 mM Ca2+. Human protein S also forms a complex with solid-phase human and rat C4BP with Kds of 6.3 x 10(-9) and 2.7 x 10(-8) M, respectively. Human C4BP strongly inhibited the APC cofactor activity of both human and rat protein S, whereas rat C4BP was only weakly inhibitory. The degree of the inhibitory activity of C4BP appears to depend on the affinity between protein S and C4BP. In order to evaluate the structure-function relationship of the rat protein S, the complete cDNA sequence of rat protein S was determined. This cDNA of 3,315 bp was composed of a 103-bp 5'-noncoding region, a 2,028-bp coding region that encodes a preprosequence of 41 amino acids, a mature protein S of 634 amino acids and a stop codon, and a 1,184-bp 3'-noncoding region. The rat mature protein S consisted of domains with distinct functions similar to those of human protein S, and with two potential Asn-linked glycosylation sites. The amino acid sequence of the mature form of rat protein S showed 80.4, 78.7, and 79.7% identity with those of human, bovine, and rabbit mature protein S, respectively. These findings suggest that despite the species-specificity of the APC cofactor activity of rat protein S, it is structurally very similar to human protein S. Expression of rat protein S mRNA (approximately 3.5 kb) was demonstrated by RNA blot analysis not only in the liver, but also in the lung, spleen, testis, and uterus of rats.
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PMID:Molecular cloning and functional characterization of rat plasma protein S. 760 28

Despite extensive sequence similarity between the N- and C-terminal halves of the Type I isozyme of mammalian hexokinase (ATP:D-hexose 6-phosphotransferase; EC 2.7.1.1), they are functionally distinct, the C-terminal half being responsible for catalysis and the N-terminal half thought to play a regulatory role. We have examined the effects of several site-directed mutations on kinetic and regulatory properties of the rat Type I isozyme. Mutation of the C-terminal residues, Asp 532 to Asn, Arg 539 to Met, and Gly 896 or Gly 898 to Val, resulted in drastic loss of catalytic activity (< 10% of wild-type enzyme), consistent with previous suggestions that these residues are involved in binding of ATP. Mutation of the corresponding residues in the N-terminal half of the enzyme caused much less marked (> 50% of wild type), but significant, effects on activity which are presumed to result from subtle effects on conformation of the enzyme. Mutation of Lys 899 to Met resulted in an approximately 50% decrease in specific activity and an approximately fivefold increase in the Km for ATP, consistent with the view that Lys 899 participates in binding of ATP through electrostatic interactions with the phosphate sidechain. Cys residues corresponding to Cys 158 and Cys 606 of Type I hexokinase are found in other hexokinases that exhibit marked sensitivity to inhibition by the product, glucose 6-phosphate (Glc-6-P), but analogous residues are not found in hexokinases insensitive to Glc-6-P. However, this correlation appears to be coincidental since neither the mutation of Cys 158 or Cys 606 to Ala nor any of the other mutations examined abolished sensitivity of Type I hexokinase to inhibition by the Glc-6-P analog 1,5-anhydroglucitol-6-P or to antagonism of this inhibition by P(i).
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PMID:Residues putatively involved in binding of ATP and glucose 6-phosphate to a mammalian hexokinase: site-directed mutation at analogous positions in the N- and C-terminal halves of the type I isozyme. 764 67

The complex formed between hen egg white lysozyme (HEL) and the monoclonal antibody HyHEL-10 Fab fragment has an interface composed of van der Waals interactions, hydrogen bonds, and a single ion pair. The antibody overlaps part of the active site cleft. Putative critical residues within the epitope region of HEL, identified from the x-ray crystallographic structure of the complex, were replaced by site-directed mutagenesis to probe their relative importance in determining affinity of the antibody for HEL. Twenty single mutations of HEL at three contact residues (Arg-21HEL, Asp-101HEL, and Gly-102HEL) and at a partially buried residue (Asn-19HEL) in the epitope were made, and the effects on the free energies of dissociation were measured. A correlation between increased amino acid side-chain volume and reduced affinity for HELs with mutations at position 101 was observed. The D101GHEL mutant is bound to HyHEL-10 as tightly as wild-type enzyme, but the delta delta Gdissoc is increased by about 2.2 kcal (9.2 kJ)/mol for the larger residues in this position. HEL variants with lysine or histidine replacements for arginine at position 21 are bound 1.4-2.7 times more tightly than those with neutral or negatively charged amino acids in this position. These exhibit 1/40 the affinity for HyHEL-10 Fab compared with wild type. There is no side-chain volume correlation with delta delta Gdissoc at position 21. Although Gly-102HEL and Asn-19HEL are in the epitope, replacements at these positions have no effect on the affinity of HEL for the antibody.
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PMID:High-resolution mapping of the HyHEL-10 epitope of chicken lysozyme by site-directed mutagenesis. 768 15

Effects of mutations on hydrogen exchange kinetics, structure, and stability suggest that the slow exchange core is a key element in protein folding. Single amino acid variants of bovine pancreatic trypsin inhibitor (BPTI) have been made with glycine or alanine replacement of residues Tyr 35, Gly 37, Asn 43, and Asn 44. The crystal structures of Y35G and N43G are reported [Housset, D., Kim, K.-S., Fuchs, J., & Woodward, C. (1991) J. Mol. Biol. 220, 757-770; Danishefsky, A. T., Housset, D., Kim, K.-S., Tao, F., Fuchs, J., Woodward, C., & Wlodawer, A. (1993) Protein Sci. 2, 577-587; Kim, K.-S., Tao, F., Fuchs, J. A., Danishefsky, A. T., Housset, D., Wlodawer, A., & Woodward, C. (1993a) Protein Sci. 2, 588-596]. NMR chemical shifts indicate few changes from the wild type (WT) in G37A and N44G. Stabilities of the four mutants were measured by calorimetry and by hydrogen exchange. Values of delta delta(WT-->mut), the difference in delta G of folding/unfolding between the wild type and mutant, estimated by both methods are in good agreement and are in the range 4.7-6.0 kcal/mol. There is no general correlation between stability and hydrogen exchange rates at pH 3.5 and 30 degrees C. Exchange occurs by two parallel pathways, one involving small noncooperative fluctuations of the native state, and the other involving cooperative, global unfolding. In the mutant proteins, the rates for exchange by the unfolding mechanism are accelerated by a factor corresponding to the increase in the unfolding/folding equilibrium constant.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Hydrogen exchange identifies native-state motional domains important in protein folding. 769 May 87


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