Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Isolectin II (LOL II) isolated from the seeds of Lathyrus ochrus has been crystallized in the presence of the N2 fragment (18,500 Da) isolated from human lactotransferrin, which contains an N-acetyllactosamine type biantennary glycan linked to Asn137. This is the first example of a legume lectin crystallized with an N-glycosylprotein. Crystals of the LOL II-N2 complex belong to the tetragonal space group (P4(1)2(1)2 or the enantiomorph) with cell dimensions: a = b = 63.5 A, c = 251.9 A. They diffract well up to at least 3.5 A resolution and more weakly up to 2.8 A resolution. Assuming one functional half-entity in the asymmetric unit, an alpha, beta monomer complexed to one N2 fragment (24,500 Da + 18,500 Da) would give a Vm of 2.95 A3/Da and a solvent content of approximately 58%. SDS/polyacrylamide gels of the dissolved crystals show the presence of both the LOL II and N2 fragment.
J Mol Biol 1992 Oct 05
PMID:Crystallization and preliminary X-ray diffraction study of Lathyrus ochrus isolectin II complexed to the human lactotransferrin N2 fragment. 140 96

The role of GTP hydrolysis in microtubule dynamics has been reinvestigated using an analogue of GTP, guanylyl-(alpha, beta)-methylene-diphosphonate (GMPCPP). This analogue binds to the tubulin exchangeable nucleotide binding site (E-site) with an affinity four to eightfold lower than GTP and promotes the polymerization of normal microtubules. The polymerization rate of microtubules with GMPCPP-tubulin is very similar to that of GTP-tubulin. However, in contrast to microtubules polymerized with GTP, GMPCPP-microtubules do not depolymerize rapidly after isothermal dilution. The depolymerization rate of GMPCPP-microtubules is 0.1 s-1 compared with 500 s-1 for GDP-microtubules. GMPCPP also completely suppresses dynamic instability. Contrary to previous work, we find that the beta--gamma bond of GMPCPP is hydrolyzed extremely slowly after incorporation into the microtubule lattice, with a rate constant of 4 x 10(-7) s-1. Because GMPCPP hydrolysis is negligible over the course of a polymerization experiment, it can be used to test the role of hydrolysis in microtubule dynamics. Our results provide strong new evidence for the idea that GTP hydrolysis by tubulin is not required for normal polymerization but is essential for depolymerization and thus for dynamic instability. Because GMPCPP strongly promotes spontaneous nucleation of microtubules, we propose that GTP hydrolysis by tubulin also plays the important biological role of inhibiting spontaneous microtubule nucleation.
Mol Biol Cell 1992 Oct
PMID:Role of GTP hydrolysis in microtubule dynamics: information from a slowly hydrolyzable analogue, GMPCPP. 2302 82

A number of studies have demonstrated the activation of phospholipase C-mediated hydrolysis of phosphatidylcholine (PC-PLC) both by growth factors and by the product of the ras oncogene, p21ras. Evidence has been presented indicating that the stimulation of this phospholipid degradative pathway is sufficient to activate mitogenesis in fibroblasts as well as that it is sufficient and necessary for induction of maturation in Xenopus laevis oocytes. However, the mechanism whereby PC-PLC transduces mitogenic signals triggered by growth factors or oncogenes remains to be elucidated. In this study, data are presented that show the involvement of protein kinase C zeta subspecies in the channelling of the mitogenic signal activated by insulin-p21ras-PC-PLC in Xenopus oocytes as well as the lack of a critical role of protein kinase C isotypes alpha, beta, gamma, delta, and epsilon in these pathways.
Mol Cell Biol 1992 Sep
PMID:Evidence for a role of protein kinase C zeta subspecies in maturation of Xenopus laevis oocytes. 150 83

Two membrane-bound nitrate reductases, NRA and NRZ, exist in Escherichia coli. Both isoenzymes are composed of three structural subunits, alpha, beta, and gamma encoded by narG/narZ, narH/narY and narI/narV, respectively. The genes are in transcription units which also contain a fourth gene encoding a polypeptide, delta, which is not part of the final enzyme. A strain which is devoid of, or does not express, the nar genes, was used to investigate the role of the delta and gamma polypeptides in the formation and/or processing of the nitrate reductase. When only the alpha and beta polypeptides are produced, an (alpha beta) complex exists which is inactive and soluble. When the alpha, beta and delta polypeptides are produced, the (alpha beta) complex is active with artificial donors such as benzyl viologen but is soluble. When the alpha, beta and gamma polypeptides are produced, the (alpha beta) complex is inactive but partially binds the membrane. It was concluded that the gamma polypeptide is involved in the binding of the (alpha beta) complex to the membrane while the delta polypeptide is indispensable for the (alpha beta) nitrate reductase activity. The activation by the delta polypeptide does not seem to involve the insertion of the redox centres of the enzyme since the purified inactive (alpha beta) complex was shown to contain the four iron-sulphur centres and the molybdenum cofactor, which are normally present in the native purified enzyme. The extreme sensitivity of this inactive complex to thermal denaturation or tryptic treatment favours the idea that the delta polypeptide promotes the correct assembly of the alpha and beta subunits. Although this corresponds to the definition of a chaperone protein this possibility has been rejected. In this study we have also demonstrated that the delta or gamma polypeptide encoded by one nar operon can be substituted successfully for by its respective counterpart from the other nar operon to give an active membrane bound heterologous nitrate reductase enzyme.
Mol Microbiol 1992 Jan
PMID:Involvement of the narJ or narW gene product in the formation of active nitrate reductase in Escherichia coli. 154 6

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 beta-alkyl substituted acrolein congeners crotonaldehyde, trans-2-pentenal, trans-2-hexenal, 2,4-hexadienal, and trans-2-heptenal were clearly mutagenic in a slightly modified preincubation Ames test with Salmonella typhimurium TA100 with and without S9 mix using a threefold bacterial cell density and a 90-min preincubation time, whereas trans-cis-2,6-nonadienal did not show any mutagenic activity. The greatest impediment to adequate mutagenicity testing of these compounds is their toxicity toward bacteria. Within the congener family tested, toxicity increases as a function of both chain length and lipophilicity, and it becomes more and more difficult to demonstrate mutagenicity. Mutagenicity decreases with increasing chain length. This effect may be explained by increasing toxicity. The effect of S9 mix seems to be mostly nonenzymatic detoxication by nonspecific scavanger protection of bacterial cytotoxicity. No indication could be found that bioactivation plays a role in S9-mediated reduction of bacterial cytotoxicity. Although positive mutagenic outcomes could be obtained with the SOS chromotest for other alpha, beta-unsaturated carbonyl compounds, these acrolein congeners were not genotoxic in this test, most probably because they are toxic for the Escherichia coli bacteria PQ37 and PQ243.
Environ Mol Mutagen 1992
PMID:Mutagenicity of beta-alkyl substituted acrolein congeners in the Salmonella typhimurium strain TA100 and genotoxicity testing in the SOS chromotest. 160 Sep 62

Macrophage cytocidal activation requires the sequential impingement on the macrophage of a priming stimulus (interferon [IFN] alpha, beta, or gamma) and a triggering stimulus (such as polyinosinic acid:polycytidylic acid [poly [I:C]] or bacterial lipopolysaccharide). The mechanism of progression from the IFN-primed state to the cytocidal state is poorly understood. By quantifying the level of expression of a gene product (complement component factor B [Bf]) associated with cytocidal activation and through the use of phenotypically distinct populations of macrophages (unprimed and IFN-primed), we have investigated the functional necessity of changes in intracellular concentration of free calcium ions ([Ca2+]i) in signaling the transition from the primed to the cytocidal state. Elevating the [Ca2+]i by incubation of unprimed macrophages with the calcium ionophore, ionomycin, failed to induce the expression of Bf. By contrast, Bf was expressed at high levels when IFN-primed macrophages were exposed to ionomycin, suggesting that priming induced within the macrophages the capacity to respond to a nonspecific change in [Ca2+]i. Quantification of the [Ca2+]i in response to exposure to ionomycin revealed an initial transient elevation, followed by a secondary sustained component. No differences in these changes were observed between unprimed and IFN-primed macrophages. We therefore questioned if changes in [Ca2+]i were also implicated in the transition between the primed and the cytocidal state using the ligand, poly [I:C]. In contrast to ionomycin, incubation of IFN-primed macrophages with poly [I:C] did not sustain measurable increases in [Ca2+]i, yet fully stimulated the transition from the IFN primed to the cytocidal state. However, incubation of IFN-primed macrophages with poly [I:C] in the presence of 1) a Ca2+/ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffer calculated to clamp the extracellular concentration of free calcium ions to a value approximately equal to the resting [Ca2+]i; 2) the calcium channel blocker verapamil; or 3) the intracellular Ca2+ antagonists (W-7, W-13, and TMB-8) substantially inhibited the induction of Bf. Collectively, these data support the following conclusions. First, that changes in [Ca2+]i comprise an important element in the induction of progression from the IFN-primed to the cytocidal state. Second, the failure to detect global changes in [Ca2+]i in response to the ligand, poly [I:C], suggests that changes in [Ca2+]i or Ca2+ movement may occur in either a spatially restricted or in an asynchronous cyclical fashion and are not detected by population fluorescence measurements. Third, the source of the relevant Ca2+ is extracellular. Fourth, our findings suggest that priming influences macrophage functional responses at a locus that is distal to the changes in [Ca2+]i, thereby potentially allowing signaling processes to be utilized to initiate different cellular responses.
Mol Biol Cell 1992 Mar
PMID:Transmembrane-mediated changes in [Ca2+] are involved in the signaling pathway leading to macrophage cytocidal differentiation: implications of localized changes in intracellular [Ca2+] and of interferon priming on Ca2+ utilization. 162 33

The induction of T cell proliferation and differentiation into mature effector cells is dependent on two principal exogenous signals that are provided by the antigen or mitogen and IL2. The enzyme protein kinase C (PKC) has a major role in the antigen-receptor signalling pathway in T cells, but appears not to be involved in signalling via the IL2-receptor (IL2-R). Since both pathways trigger a series of sequentially coordinated transcriptional events in which numerous genes are activated, we tested whether a T cell mitogen acting via the TCR/CD3 complex, and IL2, affect the expression of the conventional, Ca(2+)-dependent, PKC genes (alpha, beta and gamma) in T cells. Stimulation of human peripheral blood lymphocytes or an enriched population of human T cells with phytohemagglutinin resulted in augmented mRNA levels of PKC alpha and PKC beta, but not PKC gamma-gene. The response peaked at 24-48 hr when a 3-5-fold increase was observed. Stimulation of IL2-R alpha-expressing T cells with human recombinant IL2 induced cell proliferation and transcription of the IL2-R alpha gene (greater than 100-fold), but did not change mRNA levels of PKC alpha or PKC beta genes. The results suggest that stimulation of human T cells with mitogens acting via the TCR/CD3 complex, that involve activation of PKC, is accompanied also by a late activation of selected PKC genes. By contrast, agonists such as IL2, that operate via a different signalling pathway, do not modify the expression of any of the known conventional PKC genes.
Mol Immunol
PMID:Mitogen-induced human T cell proliferation is associated with increased expression of selected PKC genes. 163 62

gamma-Aminobutyric acid (GABA)-activated Cl- currents in neonatal rat cortical neurons and in cultured cells engineered for the expression of specific molecular forms of the GABAA receptor alpha, beta, and gamma subunits, were recorded with the patch-clamp technique in the whole-cell configuration. The effects of various allosteric modulators of GABAA receptors were determined. Diazepam and clonazepam showed greater efficacy as positive modulators of GABA-elicited currents in alpha 2 beta 1 gamma 2 or alpha 3 beta 1 gamma 2 receptors than in alpha 1 beta 1 gamma 2 or alpha 5 beta 1 gamma 2 receptors or in cortical neurons. Alpidem was more efficacious at alpha 1 beta 1 gamma 2 or alpha 2 beta 1 gamma 2 receptors than at alpha 1 beta 1 gamma 2 or alpha 5 beta 1 gamma 2 receptors or in cortical neurons. Conversely, zolpidem was equally efficacious for all these receptors except for alpha 5 beta 1 gamma 2. Both imidazopyridines (alpidem and zolpidem) were virtually ineffective at modulating the GABA response of alpha 5 beta 1 gamma 2 receptors and in almost all the receptors assembled from alpha 1, alpha 2, alpha 3 or alpha 5 subunits together with beta 1 and gamma 1 subunits. The beta-carboline derivatives methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) and methyl-beta-carboline-3-carboxylate (beta-CCM) elicited a positive allosteric modulation of alpha 1 beta 1 gamma 1 or alpha 2 beta 1 gamma 1 receptors, whereas they acted as negative allosteric modulators at nearly all other receptors tested, as they do in cortical neurons. Although the positive allosteric modulation by beta-carbolines never exceeded a doubling of the GABA response, DMCM was more efficacious at alpha 1 beta 1 gamma 1 receptors and beta-CCM was more efficacious at alpha 2 beta 1 gamma 1 receptors. DMCM was inactive at alpha 3 beta 1 gamma 1 receptors, whereas beta-CCM was virtually inactive at alpha 5 beta 1 gamma 1 receptors. The benzodiazepine 4'-chlorodiazepam, which is a negative modulator resistent to flumazenil inhibition, acted at all the various GABAA receptors that contained a gamma subunit.
Mol Pharmacol 1991 Jun
PMID:Influence of recombinant gamma-aminobutyric acid-A receptor subunit composition on the action of allosteric modulators of gamma-aminobutyric acid-gated Cl- currents. 164 44

The identification of hormone response elements in the promoter regions of hormonally regulated genes has revealed a striking similarity between the estrogen response element (ERE) and a palindromic thyroid hormone response element (TRE) derived from the GH gene promoter. In addition, this TRE was described as a strong retinoic acid receptor response element for all three subtypes: alpha, beta, and gamma. We show here that the TRE in the absence of thyroid hormone receptor (TR) behaves similarly to imperfect EREs, which can synergize to mediate a strong estrogen-dependent activation of transcription. However, in the presence of TR, but the absence of T3, activation of the TRE constructs by estrogen receptor (ER) is inhibited. In vitro, ER and TR were found to bind to the TRE in the absence and presence of their respective ligands; however, TRs form a more stable complex with the TRE than does ER. To examine whether repression of ER activity on the TRE constructs by TR was due to heterodimer formation, we employed truncated TR mutants (tTR) that lacked the DNA-binding domain, but contained the ligand-binding/dimerization domain. The tTRs were shown to be efficient inhibitors of TR, but not of ER. Thus, inhibition of ER activity on TREs by TRs does not result from heterodimer formation. We discuss a mechanism in which TRs, in the absence of thyroid hormone, control TRE activation by related receptors by preventing their access to the TRE. This mechanism can greatly enhance the fidelity of the ligand-specific response from a TRE.
Mol Endocrinol 1991 Mar
PMID:Thyroid hormone receptors repress estrogen receptor activation of a TRE. 165 92


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