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)

We have shown previously that acetaldehyde forms stable covalent adducts with tubulin, resulting in impaired microtubule formation. The present study explored the mechanism responsible for impaired microtubule formation caused by the substoichiometric stable binding of acetaldehyde to tubulin. The free tubulin dimer was much more reactive with acetaldehyde than microtubules, binding more than twice as much aldehyde. The dimer also formed nearly twice as many stable adducts on its alpha-chain as on its beta-chain, whereas microtubules exhibited an equal distribution of adducts between the two subunits. These data confirm that the alpha-chain of free tubulin, but not microtubules, has an accessible highly reactive lysine (HRL) residue that is a preferential target of acetaldehyde binding. Adduct formation with the HRL residue also correlated with impaired tubulin polymerization, and only 0.08 moles of acetaldehyde bound per mole of HRL was required for complete inhibition; however, adducts with other lysine residues (bulk adducts) did not affect assembly. Adducts to microtubule-associated proteins (MAPs) also impaired the assembly of tubulin, but were much less effective than HRL adducts. In a copolymerization assay, HRL-adducted tubulin, in addition to being itself assembly incompetent, also interfered with polymerization of normal (unadducted) tubulin. Bulk adducts did not alter assembly and were incorporated normally into the growing polymer. When tubulin was cleaved by the proteolytic enzyme, subtilisin, microtubule formation could readily take place in the absence of MAPs. In this polymerization system, HRL adducts, but not bulk adducts, still markedly inhibited assembly. When low concentrations of acetaldehyde (50 microM) were used to generate HRL adducts, an adduct on only 1 out of 20 tubulin molecules was sufficient to totally block polymerization. These findings indicate that substoichiometric amounts of acetaldehyde bound to HRL of tubulin can markedly inhibit microtubule formation via direct interference of dimer-dimer interactions, and further suggest that low concentrations of acetaldehyde could generate sufficient amounts of HRL adducts in cellular systems to alter microtubule formation and function.
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PMID:Substoichiometric inhibition of microtubule formation by acetaldehyde-tubulin adducts. 163 40

Treatment of murine leukemia virus reverse transcriptase (MuLV RT) with 4-(oxoacetyl)-phenoxyacetic acid (OAPA) results in the loss of DNA polymerase as well as template-primer binding activity but has no effect on the RT-associated RNase-H activity. Binding stoichiometry revealed that approximately 3 mol of OAPA bound per mole of enzyme, when complete enzyme activation occurred. However, in the presence of template-primer, OAPA does not abolish polymerase activity and 2 mol of OAPA remains bound to 1 mol of enzyme. This observation suggests that only one OAPA reactive site is responsible for the loss of polymerase activity. This site was located on a single tryptic peptide by comparing the maps of the native enzyme and the enzyme treated with OAPA in the presence and absence of template-primer. The appearance of a new peptide peak eluting at 125 min from a C-18 reverse-phase column was consistently noted in the tryptic digest of enzyme treated with OAPA. This peak was absent in tryptic peptides made from the control enzyme or the enzyme protein that was treated with OAPA in the presence of activated DNA or synthetic template-primers. Amino acid composition and sequence analyses of this peptide revealed that it spanned residues 312-342 in the primary amino acid sequence of MuLV RT. Since this peptide does not contain arginine residues and Lys-329 exhibited resistance to tryptic digestion, we conclude that Lys-329 is the target of OAPA action.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Lysine-329 of murine leukemia virus reverse transcriptase: possible involvement in the template-primer binding function. 169 96

In recent years, many studies have suggested a direct role for alpha 2-macroglobulin (alpha 2M), a plasma proteinase inhibitor, in growth factor regulation. When coincubated in the presence of either trypsin, pancreatic elastase, human neutrophil elastase, or plasmin, 125I-insulin rapidly formed a complex with alpha 2M which was greater than 80% covalent. The covalent binding was stable to reduction but abolished by competition with beta-aminopropionitrile. Neither native alpha 2M nor alpha 2M pretreated with proteinase or methylamine incorporated 125I-insulin. Experiments utilizing alpha 2M cross-linked with cis-dichlorodiammineplatinum(II) indicated that 125I-insulin must be present during alpha 2M conformational change to covalently bind. A maximum stoichiometry of 4 mol of insulin bound per mole of alpha 2M and the short half-life of the alpha 2M intermediate capable of covalent incorporation were consistent with thiol ester involvement. Protein sequence analysis of unlabeled insulin-alpha 2M complexes, together with results of beta-aminopropionitrile competition, confirmed that insulin incorporation occurs via the same gamma-glutamyl amide linkage responsible for covalent proteinase and methylamine binding to alpha 2M. Although intact insulin apparently incorporated through its sole lysine residue on the B chain, we found that isolated A chain also bound covalently to alpha 2M. Phenyl isothiocyanate derivatization of the N-terminus had no effect on A-chain binding, supporting the possibility of heretofore unreported gamma-glutamyl ester linkages to alpha 2M.
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PMID:Mechanism of insulin incorporation into alpha 2-macroglobulin: implications for the study of peptide and growth factor binding. 170 57

We studied the binding of peptides containing five basic residues to membranes containing acidic lipids. The peptides have five arginine or lysine residues and zero, one, or two alanines between the basic groups. The vesicles were formed from mixtures of a zwitterionic lipid, phosphatidylcholine, and an acidic lipid, either phosphatidylserine or phosphatidylglycerol. Measuring the binding using equilibrium dialysis, ultrafiltration, and electrophoretic mobility techniques, we found that all peptides bind to the membranes with a sigmoidal dependence on the mole fraction of acidic lipid. The sigmoidal dependence (Hill coefficient greater than 1 or apparent cooperativity) is due to both electrostatics and reduction of dimensionality and can be described by a simple model that combines Gouy-Chapman-Stern theory with mass action formalism. The adjustable parameter in this model is the microscopic association constant k between a basic residue and an acidic lipid (1 less than k less than 10 M-1). The addition of alanine residues decreases the affinity of the peptides for the membranes; two alanines inserted between the basic residues reduces k 2-fold. Equivalently, the affinity of the peptide for the membrane decreases 10-fold, probably due to a combination of local electrostatic effects and the increased loss of entropy that may occur when the more massive alanine-containing peptides bind to the membrane. The arginine peptides bind more strongly than the lysine peptides: k for an arginine residue is 2-fold higher than for a lysine residue. Our results imply that a cluster of arginine and lysine residues with interspersed electrically neutral amino acids can bind a significant fraction of a cytoplasmic protein to the plasma membrane if the cluster contains more than five basic residues.
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PMID:Binding of basic peptides to acidic lipids in membranes: effects of inserting alanine(s) between the basic residues. 173 30

The reaction between the three Bowman-Birk proteinase inhibitors isolated from fenugreek seeds (TFI-B2, TFI-N2 and TFI-A8) and the human and bovine proteinases was investigated by studying the complexes formed and their properties. TFI-B2, the Lys-Leu trypsin chymotrypsin inhibitor, can bind 1.9 mol human trypsin (HT), 1.3 mol bovine trypsin (BT) and/or 0.4 mol human (HCT) or bovine (BCT) chymotrypsin per mole of inhibitor. HT was bound at the two reactive sites and BT mainly at the lysine-containing trypsin-reactive site, whereas HCT and BCT were only bound at the leucine-containing chymotrypsin-reactive site. TFI-N2, the Arg-Leu trypsin chymotrypsin inhibitor, could bind 1 mol BT and BCT, but 1.3 mol HT and 1.2 mol HCT per mole of inhibitor. In addition to the usual binding, the human enzymes could also be bound at the respective "wrong" reactive site. TFI-A8, the Arg-Arg trypsin inhibitor, binds 2 mol HT or BT per mole of inhibitor at the two trypsin-reactive sites, whereas HCT and BCT (about 0.2 mol/mol) are bound to one of the two "wrong" reactive sites.
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PMID:Inhibitors of human and bovine trypsin and chymotrypsin in fenugreek (Trigonella foenum-graecum L.) seeds. Reaction with the human and bovine proteinases. 176 94

Natural and synthetic peptides that contain detectable intramolecular alpha-helical structure in aqueous solution have been used to evaluate the helical propensities for the common amino acids. Experimental spectroscopic data must be fit to a model of the helix-coil transition in order to determine quantitative stability constants for each amino acid. We present here a statistical mechanical description of helix formation in peptides or protein fragments that takes into account multiple internal conformations, heterogeneity in the stabilizing effects of different side chains, and specific side-chain-side-chain interactions. The model enables one to calculate values of [theta]222 for a given peptide using the length dependence of the helix signal computed by a quantum mechanical treatment of the n pi * transition that dominates the 222-nm band. In addition, the helical probability at any residue in the chain is readily computed, and should prove useful as nmr spectral data become available. The free energy of specific side-chain interactions, including ion pair formation, can be evaluated. Application of the analysis to experimental data on a pair of isomeric peptides, only one of which contains ion pairs, indicates that forming a single glutamate-lysine ion pair stabilizes the alpha-helix by 0.50 kcal/mole in 10 mM sodium ion and pH 7. A survey of the CD data measured for a variety of model peptides is presented, indicating that a single set of s values and sigma constant can account for some but not all of the available results.
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PMID:The helix-coil transition in heterogeneous peptides with specific side-chain interactions: theory and comparison with CD spectral data. 181 7

Interfacial catalysis in the scooting mode by phospholipase A2 (PLA2) from pancreas and venoms (18 different preparations) was examined on vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol under the conditions where the rates of transbilayer and intervesicle exchanges of the enzyme, substrate, and the products of hydrolysis were negligible on the time scale (less than 30 min) on which all the substrate molecules on the outer monolayer of the target vesicles were hydrolyzed. The reaction progress curves for all PLA2s exhibited no latency period (less than 3 s). When the vesicle to PLA2 ratio in the reaction mixture was high so that according to the Poissonian distribution model at most one enzyme was bound to a vesicle, the extent of hydrolysis increased linearly with the amount of enzyme in the reaction mixture. However, the extent of hydrolysis per enzyme, NS, remained the same for all PLA2s, and it corresponded to the size of the target vesicles determined by independent methods. Similarly, the initial rate of hydrolysis increased linearly with the enzyme concentration, and the slope of the log-log plot was one under the conditions of one or more enzyme per vesicle. Such observations showed that monomeric PLA2 is fully catalytically active at the interface. This conclusion was supported by the absence of intermolecular resonance energy transfer from tryptophan-3 donor in the native PLA2 to the anthraniloyl acceptor in An87-PLA2, the catalytically active derivative of PLA2 with an anthraniloyl fluorophore on lysine 87. In this system, intermolecular resonance energy transfer was seen only when the donor-acceptor molecules were "crowded" at a high surface density with a relatively low lipid to protein mole ratio. On the basis of these results, it was concluded that secretory PLA2s from venoms and pancreas are fully catalytically active as monomers. Additional studies reported here showed that acylation of PLA2 was not necessary for catalysis or binding to the interface and that the binding of the substrate to the active site of PLA2 was not necessary for the binding of the enzyme to the interface.
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PMID:Interfacial catalysis by phospholipase A2: monomeric enzyme is fully catalytically active at the bilayer interface. 185 41

There are clusters of basic amino acids on many cytoplasmic proteins that bind transiently to membranes (e.g., protein kinase C) as well as on the cytoplasmic domain of many intrinsic membrane proteins (e.g., glycophorin). To explore the possibility that these basic residues bind electrostatically to monovalent acidic lipids, we studied the binding of the peptides Lysn and Argn (n = 1-5) to bilayer membranes containing phosphatidylserine (PS) or phosphatidylglycerol (PG). We made electrophoretic mobility measurements using multilamellar vesicles, fluorescence and equilibrium binding measurements using large unilamellar vesicles, and surface potential measurements using monolayers. None of the peptides bound to vesicles formed from the zwitterionic lipid phosphatidylcholine (PC) but all bound to vesicles formed from PC/PS or PC/PG mixtures. None of the peptides exhibited specificity between PS and PG. Each lysine residue that was added to Lys2 decreased by one order of magnitude the concentration of peptide required to reverse the charge on the vesicle; equivalently it increased by one order of magnitude the binding affinity of the peptides for the PS vesicles. The simplest explanation is that each added lysine binds independently to a separate PS with a microscopic association constant of 10 M-1 or a free energy of approximately 1.4 kcal/mol. Similar, but not identical, results were obtained with the Argn peptides. A simple theoretical model combines the Gouy-Chapman theory (which accounts for the nonspecific electrostatic accumulation of the peptides in the aqueous diffuse double layer adjacent to the membrane) with mass action equations (which account for the binding of the peptides to greater than 1 PS). This model can account qualitatively for the dependence of binding on both the number of basic residues in the peptides and the mole fraction of PS in the membrane.
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PMID:Binding of peptides with basic residues to membranes containing acidic phospholipids. 188 32

In order to identify the essential reactive amino acid residues of 5-enolpyruvylshikimate-3-phosphate synthase, the reaction of the enzyme with its substrate analogue bromopyruvate was investigated. Incubation of the enzyme with bromopyruvate resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo-first-order and saturation kinetics with a Kinact of 28 microM and a maximum rate constant of 0.31 min-1. The inactivation was prevented by preincubation of the enzyme with the substrates shikimate 3-phosphate, 5-enolpyruvylshikimate 3-phosphate or by the combination of shikimate 3-phosphate plus glyphosate (N-phosphonomethylglycine), an inhibitor of the enzyme. Addition of sodium [3H]borohydride to the reaction mixture had no effect on the rate of inactivation but resulted in the incorporation of 3H label to the modified enzyme. Upon 90% inactivation, approximately 1 mol of bromo[14C]pyruvate was incorporated per mole of enzyme modified in the absence or presence of sodium borohydride. When the enzyme was incubated with bromopyruvate in the presence of sodium [3H]borohydride, approximately 1 mol of 3H label was found to be associated per mole of the modified enzyme. Tryptic digestion of these labeled proteins followed by reverse phase chromatographic separation resulted in the isolation of three radioactive peptides. Analyses of these three peptides indicated that bromopyruvate inactivated the enzyme by modifying Cys-408 and Lys-411, which are conserved in all enzyme sequences studied to date.
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PMID:5-Enolpyruvylshikimate-3-phosphate synthase from Escherichia coli--the substrate analogue bromopyruvate inactivates the enzyme by modifying Cys-408 and Lys-411. 189 81

Uridine di- and triphosphopyridoxals were used to probe the substrate-binding site in potato tuber UDP-glucose pyrophosphorylase (EC 2.7.7.9). The enzyme was rapidly inactivated in time- and dose-dependent manners when incubated with either reagent followed by reduction with sodium borohydride. The inactivations were almost completely retarded by UDP-Glc and UTP but only slightly by alpha-D-glucose 1-phosphate. The complete inactivation corresponded to the incorporation of about 0.9-1.0 mol of either reagent per mole of enzyme monomer. Both reagents appear to bind specifically to the UDP-Glc-(UTP)-binding site. Structural studies of the labeled enzymes revealed that the two reagents modified the identical set of five lysyl residues (Lys-263, Lys-329, Lys-367, Lys-409, and Lys-410), in which Lys-367 was most prominently modified. The ratios of the amounts of labels incorporated into these residues were similar for the two reagents. Furthermore, linear relationships were observed between the residual activities and the amounts of incorporation into each lysyl residue. We conclude that the five lysyl residues are located at or near the UDP-Glc(UTP)-binding site of potato tuber UDP-Glc pyrophosphorylase and that the modification of these residues occurs in a mutually exclusive manner, leading to the inactivation of the enzyme.
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PMID:Identification of lysyl residues located at the substrate-binding site in UDP-glucose pyrophosphorylase from potato tuber: affinity labeling with uridine di- and triphosphopyridoxals. 190 67


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