Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Sequences of 47 members of the Zn-containing alcohol dehydrogenase (ADH) family were aligned progressively, and an evolutionary tree with detailed branch order and branch lengths was produced. The alignment shows that only 9 amino acid residues (of 374 in the horse liver ADH sequence) are conserved in this family; these include eight Gly and one Val with structural roles. Three residues that bind the catalytic Zn and modulate its electrostatic environment are conserved in 45 members. Asp 223, which determines specificity for NAD, is found in all but the two NADP-dependent enzymes, which have Gly or Ala. Ser or Thr 48, which makes a hydrogen bond to the substrate, is present in 46 members. The four Cys ligands for the structural zinc are conserved except in zeta-crystallin, the sorbitol dehydrogenases, and two bacterial enzymes. Analysis of the evolutionary tree gives estimates of the times of divergence for different animal ADHs. The human class II (pi) and class III (chi) ADHs probably diverged about 630 million years ago, and the newly identified human ADH6 appeared about 520 million years ago, implying that these classes of enzymes may exist or have existed in all vertebrates. The human class I ADH isoenzymes (alpha, beta, and gamma) diverged about 80 million years ago, suggesting that these isoenzymes may exist or have existed in all primates. Analysis of branch lengths shows that these plant ADHs are more conserved than the animal ones and that class III ADHs are more conserved than class I ADHs. The rate of acceptance of point mutations (PAM units) shows that selection pressure has existed for ADHs, implying that these enzymes play definite metabolic roles.
J Mol Evol 1992 Jun
PMID:Progressive sequence alignment and molecular evolution of the Zn-containing alcohol dehydrogenase family. 159 44

The conserved asparagine 111 of ribulose-1,5-bisphosphate carboxylase/oxygenase from the photosynthetic bacteria Rhodospirillum rubrum was identified as a candidate for a side-chain that might be involved in the carboxylase/oxygenase specificity. It was replaced by site-directed mutagenesis with aspartic acid, leucine, glutamine or glycine residues. The mutant enzymes exhibit a very low carboxylase activity compared with the wild-type enzyme. The values of Km(RuBP) and kcat for Asn111----Gly, the most active mutant, are 420 microM and 0.034 s-1, compared with 13 microM and 3.0 s-1 for wild-type. The mutation of Asn111----Gly causes a more than tenfold decrease in the CO2/O2 specificity factor, tau, tau Asn111----Gly = 0.56 and tau wild-type = 6.7. This is the first reported change in rubisco specificity by a single site-directed mutation alone and suggests a target for future protein engineering studies.
J Mol Biol 1992 Jun 05
PMID:Mutation of asparagine 111 of rubisco from Rhodospirillum rubrum alters the carboxylase/oxygenase specificity. 160 88

The decrease in conformational stability, delta(delta G), has been measured for 72 aliphatic side-chain mutants from four proteins in which a larger side-chain is replaced by a smaller side-chain so that steric effects are minimal. When these delta(delta G) values are corrected to the same accessibility, namely 100% buried, then the following -delta(delta G) values per -CH2- group (in kcal/mol) are obtained: Ile----Val (1.26), Ala (1.26), Gly (1.26); Leu----Ala (1.16), Gly (1.21); Val----Ala (1.23), Gly (1.53). The average of these values is 1.27(+/- 0.07) kcal/mol. The 72 individual values range from 0 to 2.4 kcal/mol with an average value of 1.27(+/- 0.51) (standard deviation) kcal/mol. When the delta Gtr values from n-octanol to water are corrected for the difference in volume between the solutes and the solvents, the average value for the same substitutions is 1.25(+/- 0.05) kcal/mol. This suggests that proteins gain 1.3(+/- 0.5) kcal/mol in stability for each -CH2- group buried in folding, and, furthermore, that the volume corrected delta Gtr values for n-octanol for the amino acid side-chains provide good estimates of the contribution of the hydrophobic effect to globular protein stability.
J Mol Biol 1992 Jul 05
PMID:Contribution of the hydrophobic effect to globular protein stability. 161 60

Peptidyl diazomethane (PDAM) derivatives, a class of irreversible inhibitors for cysteine proteinase, were screened for the ability to impair Trypanosoma cruzi invasion and intracellular development in primary cultures of heart muscle cells (HMC). T. cruzi GP57/51, a purified cysteinyl proteinase, and the substrate Z-Phe-Arg-NHMec were used to determine inhibition rate constants (k'+2) by continuous kinetic assays. The k'+2 values ranged from 25,400 to 2,800. The best inhibitors of GP57/51 had bulky hydrophobic residues in the P1 position (in addition to P2), the S1 sub-site specificity of the enzyme being thus similar to mammalian cathepsin L. The effects of these PDAM on parasite infectivity were then investigated. The ability to invade HMC was markedly impaired when trypomastigotes were briefly exposed to 10 microM of Z-(S-Bzl)Cys-Phe-CHN2. Striking effects were observed when PDAM were added to HMC cultures that had been previously infected with trypomastigotes: Z-(S-Bzl)Cys-Phe-CHN2 with an IC50 of 0.4 microM, and less markedly Z-Phe-Phe-CHN2 and Z-Tyr-Phe-CHN2 (or Z-Phe-Tyr-CHN2) blocked amastigote replication as well as their transformation into trypomastigotes, thereby arresting intracellular development. Bz-Phe-Gly-CHN2, in contrast, failed to display antiparasite activity. Direct characterization of the target cysteinyl proteinase was sought, by incubating viable amastigotes or infected HMC with Z-[125I]Tyr-Phe-CHN2. Affinity labeling implicated GP57/51 as the major cysteinyl proteinase target for this probe. We propose that T. cruzi intracellular development is critically dependent on GP57/51 (cruzipain). Selective inhibitors for this cysteinyl proteinase may have therapeutic potential.
Mol Biochem Parasitol 1992 Jun
PMID:Inhibitors of the major cysteinyl proteinase (GP57/51) impair host cell invasion and arrest the intracellular development of Trypanosoma cruzi in vitro. 162 Jan 57

Many adhesive proteins present in extracellular matrices and in blood contain the tetrapeptide sequence -Arg-Gly-Asp-Ser- (or RGDS) at their cell recognition site. Since this sequence, or similar ones, was found in many proteins involved in major biological mechanisms, conformational investigations were performed on the RGDS fragment. A preliminary review of available crystal structures indicates that the RxDy sequences exhibit 3 well-defined structural patterns: one corresponding to a strong interaction between the Arg and Asp ionic side chains which are only about 4 A apart, one with the ions separated by about 8 A, and another in which the side chains are further apart (about 11 A). The conformational behaviour of the isolated RGDS fragment was next tackled using sequential building, Monte Carlo and molecular dynamics computational techniques. Analysis of the RGDS sequence conformational possibilities, as simulated in vacuum and in water solution, indicates that they can be classified into several conformational classes, which correspond roughly to the behaviour of the RGDS fragment as observed in protein matrices. This suggests the possibility of understanding the biological role of the RGDS or parent sequences in recognition processes.
J Comput Aided Mol Des 1992 Apr
PMID:Computational simulations of the conformational behaviour of the adhesive proteins RGDS fragment. 162 55

An extensive exploration of the conformational hypersurface of Met-enkephalin has been carried out, in order to characterize different low-energy conformational domains accessible to this pentapeptide. The search strategy used consisted of two steps. First, systematic nested rotations were performed using the ECEPP potential. Ninety-two low-energy structures were found and minimized using the CHARMm potential. High and low-temperature molecular dynamics trajectories were then computed for the lowest energy structures in an interative fashion until no lower energy conformers could be found. The same search strategy was used in these studies simulating three different environments, a distance-dependent dielectric epsilon = r, and two constant dielectrics epsilon = 10 and epsilon = 80. The lowest energy structure found in a distance-dependent dielectric is a Gly-Gly beta-II'-type turn. All other structures found for epsilon = r within 10 kcal/mol of this lowest energy structure are also bends. In the more polar environments, the density of conformational states is significantly larger compared to the apolar media. Moreover, fewer hydrogen bonds are formed in the more polar environments, which increases the flexibility of the peptide and results in less structured conformers. Comparisons are made with previous calculations and experimental results.
J Comput Aided Mol Des 1992 Apr
PMID:Characterization of low-energy conformational domains for Met-enkephalin. 162 57

A mouse monoclonal, anti-idiotypic, anti-opioid receptor antibody (Ab2-AOR) has been generated from monoclonal anti-morphine antibodies (Ab1). Hybridoma culture supernatants were screened by a solid phase radioimmunoassay (RIA), based on their competition with radiolabelled morphine for Ab1. One of the Ab2s that gave a positive RIA also competed at rat brain opioid receptors with tritiated opioid ligands dihydromorphine (DHM), naloxone, etorphine, Tyr-D-Ala-Gly-Phe-D-Leu (DADLE), Tyr-D-Ala-Gly-NMe-Phe-Gly-ol (DAMGE) and Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE). SDS-PAGE revealed Ab2-AOR to be highly purified after successive affinity and protein A-Sepharose chromatography. Ab2-AOR at concentrations of 10-100 nM competed with both mu- and delta-selective specific ligands for brain opioid receptors. Less than 13 micrograms/ml Ab2-AOR completely inhibited specific opioid radioligand binding to both soluble and membrane-bound opioid receptors. To demonstrate its anti-delta receptor activity further, a double-antibody ELISA procedure was developed that is based on the binding of Ab2-AOR to immobilized NG 108-15 cells (which contain only delta opioid receptors). Dose-dependent, opioid peptide- and opiate alkaloid-competitive binding of Ab2-AOR-containing ascites fluid to NG 108-15 cells was observed. A mu opioid agonist effect was demonstrated for Ab2-AOR, in that it decreased by 70% [3H]thymidine incorporation into DNA of fetal brain cell aggregates. This agonist-like action of Ab2-AOR was blocked by naltrexone. The antibody bound specifically to brain tissue sections and the presence of diprenorphine blocked this interaction. Hence, an Ab2 with mu and delta specificity has been characterized.
Brain Res Mol Brain Res 1991 Mar
PMID:A monoclonal anti-idiotypic antibody to mu and delta opioid receptors. 164 33

Rhizobium fredii strain USDA257 does not nodulate soybean (Glycine max (L.) Merr.) cultivar McCall. Mutant 257DH5, which contains a Tn5 insert in the bacterial chromosome, forms nodules on this cultivar, but acetylene-reduction activity is absent. We have sequenced the region corresponding to the site of Tn5 insertion in this mutant and find that it lies within a 1176bp open reading frame that we designate nolC. nolC encodes a protein of deduced molecular weight 43564. Nucleotide sequences homologous to nolC are present in several other Rhizobium strains, as well as Agrobacterium tumefaciens, but not in Pseudomonas syringae pathovar glycinea. nolC lacks significant sequence homology with known genes that function in nodulation, but is 61% homologous to dnaJ, an Escherichia coli gene that encodes a 41 kDa heat-shock protein. Both R. fredii USDA257 and mutant 257DH5 produce heat-shock proteins of 78, 70, 22, and 16kDa. A 4.3kb EcoRI-HindIII subclone containing nolC expresses a single 43kDa polypeptide in mini-cells. A longer, 9.4kb EcoRI fragment expresses both the 43kDa polypeptide and a 78kDa polypeptide that corresponds in size to that of the largest heat-shock protein. Thus, although nolC has strong sequence homology to dnaJ and appears to be linked to another heat-shock gene, it does not directly function in the heat-shock response.
Mol Microbiol 1991 Mar
PMID:nolC, a Rhizobium fredii gene involved in cultivar-specific nodulation of soybean, shares homology with a heat-shock gene. 164 77

The specific binding of the selective mu-, delta-, and kappa-opioid ligands [3H][D-Ala2,MePhe4,Gly-ol5]enkephalin ([3H] DAGOL), [3H][D-Pen2,D-Pen5]enkephalin ([3H]DPDPE), and [3H]U69593, respectively, to crude membranes of the guinea pig and rat whole kidney, kidney cortex, and kidney medulla was investigated. In addition, the distribution of specific 3H-opioid binding sites in the guinea pig and rat kidney was visualized by autoradiography. Homogenate binding and autoradiography demonstrated the absence of mu- and kappa-opioid binding sites in the guinea pig kidney. No opioid binding sites were demonstrable in the rat kidney. In the guinea pig whole kidney, cortex, and medulla, saturation studies demonstrated that [3H]DPDPE bound with high affinity (KD = 2.6-3.5 nM) to an apparently homogeneous population of binding sites (Bmax = 8.4-30 fmol/mg of protein). Competition studies using several opioid compounds confirmed the nature of the delta-opioid binding site. Autoradiography experiments demonstrated that specific [3H]DPDPE binding sites were distributed radially in regions of the inner and outer medulla and at the corticomedullary junction of the guinea pig kidney. Computer-assisted image analysis of saturation data yielded KD values (4.5-5.0 nM) that were in good agreement with those obtained from the homogenate binding studies. Further investigation of the delta-opioid binding site in medulla homogenates, using agonist ([3H]DPDPE) and antagonist ([3H]diprenorphine) binding in the presence of Na+, Mg2+, and nucleotides, suggested that the delta-opioid site is linked to a second messenger system via a GTP-binding protein. Further studies are required to establish the precise localization of the delta binding site in the guinea pig kidney and to determine the nature of the second messenger linked to the GTP-binding protein in the medulla.
Mol Pharmacol 1991 Jul
PMID:Opioid binding sites in the guinea pig and rat kidney: radioligand homogenate binding and autoradiography. 164 66

The receptor for colony-stimulating factor-1 (CSF-1) is a receptor protein-tyrosine kinase. To study the possible function of CSF-1 receptor autophosphorylation, two autophosphorylation sites, Tyr-706, located in the kinase insert, and Tyr-807, a residue conserved in all protein-tyrosine kinases, were changed independently to either phenylalanine or glycine. Wild-type and mutant receptors were stably expressed in Rat-2 cells. In response to CSF-1, cells expressing Phe- or Gly-706 mutant receptors showed increased growth rate and altered cell morphology. Both the Phe- and Gly-706 mutant receptors associated with and phosphorylated phosphatidylinositol-3 kinase at levels comparable with those of wild-type receptors. However, these mutant receptors differed subtly from each other and from the wild-type receptor in their ability to induce different aspects of the response to CSF-1. The Phe-706 mutant receptor was most strongly affected in its ability to increase growth rate or elevate the levels of c-fos and NGF1A mRNAs, whereas the Gly-706 mutant receptor was most markedly affected in its ability to induce a change in cell morphology or increase the levels of c-jun and NGF1A mRNAs. These findings indicate that Tyr-706 itself, or this region of the receptor, may be important for interaction of the CSF-1 receptor with different signalling pathways. Gly-807 mutant receptors lacked protein-tyrosine kinase activity, failed to respond to CSF-1, and were defective in biosynthetic processing. Phe-807 mutant receptors had 40 to 60% reduced protein-tyrosine kinase activity in vitro. Although cells expressing Phe-807 receptors were able to respond to CSF-1, the changes in growth rate and cell morphology were significantly less than seen with wild-type receptors, and the induction of early response genes was also slightly lower than for the wild-type receptor. In contrast, Phe-807 receptors were equivalent to wild-type receptors when tested for their ability to interact with phosphatidylinositol-3 kinase. These findings indicate that phosphorylation of Tyr-807 may be important for full activation of the receptor.
Mol Cell Biol 1991 Sep
PMID:Tyrosine 706 and 807 phosphorylation site mutants in the murine colony-stimulating factor-1 receptor are unaffected in their ability to bind or phosphorylate phosphatidylinositol-3 kinase but show differential defects in their ability to induce early response gene transcription. 165 61


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