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)

Binding of a tetra peptide, lysyl tryptophenyl glycyl lysine O-ter butyl ester (KWGK) with duplex forms of G, C containing polynucleotides, Poly [d(G-C)], Poly [d(G-5M C)], Poly (dG), Poly (dC) and E.coli DNA were studied under low salt conditions using UV absorption, fluorescence, and circular dichroic (C.D) spectroscopy. On addition of the peptide (upto a P/N mole ratio of 0.5), the Poly [d(G-5M C)] under low salt (1 mM Na Cl) conditions, was converted from Z to B-form as shown by the inversion of C.D spectra. The two binding constants (K1 and K2) were determined from fluorescence spectroscopy of which K2 estimates the intercalation of the tryptophenyl side chain between the base pairs of DNA and K1 estimates the electrostatic interactions between the lysyl side chains and phosphate groups. The strength of intercalation is: Z-form of Poly [d(G-5M C)] >> B form of Poly [d(G-5M C)] >> E.Coli DNA > Poly (dG).Poly (dC). This means that peptide seems to have strong preference for Z compared to B-form and for alternating over non-alternating G, C Sequences. This suggests that tryptophan intercalation may act as a discriminating factor in recognizing Z and B-forms and may have a potential role in Protein-Nucleic acid interactions that are important for transcription.
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PMID:Tryptophan intercalation in G, C containing polynucleotides: Z to B conversion of poly [d(G-5M C)] in low salt induced by a tetra peptide. 887 59

Mellitin, a cationic amphiphilic peptide, has an apparent activating effect on interfacial catalysis by phospholipase A2 (PLA2) of bee venom on zwitterionic vesicles of 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine (POPC) and on anionic vesicles of 1,2-dimyristoylglycero-sn-3-phosphomethanol (DMPM), as well as on covesicles of POPC/DMPM (3:7). On the other hand, mellitin-induced increase in the rate of pig pancreatic PLA2 is seen only on anionic vesicles. Interfacial kinetic protocols and spectroscopic methods show that the activation is due to enhanced substrate replenishment resulting from intervesicle exchange of zwitterionic or anionic phospholipids through vesicle-vesicle contacts established by mellitin. It is shown that as the hydrolysis on POPC vesicles progresses, due to a high propensity of bee PLA2 for binding to the product containing zwitterionic vesicles, most of the enzyme in the reaction mixture is trapped on few vesicles that are initially hydrolyzed, and thus reaction ceases. Under these conditions, mellitin promotes substrate replenishment by direct exchange of the products of hydrolysis from the enzyme-containing vesicles with the substrate present in excess vesicles which have not been hydrolyzed. Pig PLA2 has poor affinity for POPC vesicles, and the affinity is only modestly higher in the presence of low mole fractions of the products of hydrolysis; therefore, the enzyme is not trapped on those vesicles. Biophysical studies confirm that the phospholipid exchange occurs through stable intervesicle contacts formed by low mole fractions of mellitin, without transbilayer movement of phospholipids or fusion of vesicles. At high mole fraction (> 1.5%) mellitin induces leakage in POPC vesicles and does not form additional contacts. In POPC/DMPM vesicles, the contacts are formed even at high mole fractions of mellitin. Changes in intrinsic tryptophan fluorescence of mellitin indicate that bound mellitin exists in at least two different functional forms depending on the lipid composition and on the lipid:peptide ratio. A model is proposed to accommodate amphiphilic mellitin as a transmembrane channel or an intervesicle contact.
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PMID:Synergism between mellitin and phospholipase A2 from bee venom: apparent activation by intervesicle exchange of phospholipids. 909 18

The folding and stability of maltose binding protein (MBP) have been investigated as a function of pH and temperature by intrinsic tryptophan fluorescence, far- and near-UV circular dichroism, and high-sensitivity differential scanning calorimetric measurements. MBP is a monomeric, two-domain protein containing 370 amino acids. The protein is stable in the pH range of 4-10.5 at 25 degrees C. The protein exhibits reversible, two-state, thermal and guanidine hydrochloride-mediated denaturation at neutral pH. The thermostability of MBP is maximal at pH 6, with a Tm of 64.9 degrees C and a deltaHm of 259.7 kcal mol(-1). The linear dependence of deltaHm on Tm was used to estimate a value of deltaCp of 7.9 kcal mol(-1) K(-1) or 21.3 cal (mol of residue)(-1) K(-1). These values are higher than the corresponding deltaCp's for most globular proteins studied to date. However, the extrapolated values of deltaH and deltaS (per mole of residue) at 110 degrees C are similar to those of other globular proteins. These data have been used to show that the temperature at which a protein undergoes cold denaturation depends primarily on the deltaCp (per mol of residue) and that this temperature increases with an increase in deltaCp. The predicted decrease in stability of MBP at low temperatures was experimentally confirmed by carrying out denaturant-mediated unfolding studies at neutral pH at 2 and 28 degrees C.
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PMID:Thermodynamic characterization of the reversible, two-state unfolding of maltose binding protein, a large two-domain protein. 912 24

Lignin peroxidases (LiP) from the white-rot fungus Phanerochaete chrysosporium oxidize veratryl alcohol (VA) by two electrons to veratryl aldehyde, although the VA cation radical (VA.+) is an intermediate [Khindaria, A., et al. (1995) Biochemistry 34, 6020-6025]. It was speculated, on the basis of kinetic evidence, that VA*+ can form a catalytic complex with LiP compound II. We have used low-temperature EPR to provide direct evidence for the formation of the complex. The EPR spectrum of VA*+ obtained at 4 K was explained by a model for coupling between the oxoferryl moiety of the heme (S = 1) and VA.+ (S = 1/2) similar to the model proposed for an oxyferryl and a porphyrin pi cation radical of horseradish peroxidase. The coupling constant suggested that VA.+ was equally ferro- and antiferromagnetically coupled to the oxoferryl moiety. The spectrum was simulated with g perpendicular only marginally greater than g parallel. This was surprising since the only other known organic radical coupled to the heme iron in a peroxidase is the tryptophan cation radical in cytochrome c peroxidase which exhibits a g tensor with g parallel greater than g perpendicular. Spin concentration analysis suggested that the 1 mol of VA*+ was coupled to the oxoferryl moiety per mole of enzyme. The VA.+ signal decayed with a first-order decay constant of 1.76 s-1, in close agreement with the earlier published decay constant of 1.85 s-1 from room-temperature EPR studies. The exchange coupling between VA.+ and the oxoferryl moiety strongly advocates calling this species (VA.+ and LiP compound II) a catalytic complex.
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PMID:Detection and characterization of the lignin peroxidase compound II-veratryl alcohol cation radical complex. 936 91

Carboxymethylcellulase (CMCase) was extracted and purified from an angiosperm parasite Cuscuta reflexa free from beta-glucosidase and other enzyme activities. The molecular mass and Stokes' radius of the purified enzyme are 144 kDa and 44 A, respectively. The diffusion coefficient and frictional ratio of the enzyme were 5.15 x 10(-7) cm2/sec and 1.27. The SDS-PAGE revealed homotetrameric nature of the enzyme with a subunit molecular mass of 35 +/- 1 kDa. Titration against DTNB and NBS revealed 19 sulfhydryl groups and 8 tryptophan groups, respectively, per mole of the enzyme. A sharp pH optimum at 5.0 was obtained. Cuscuta CMCase activity is unique amongst plant endoglucanases in being stimulated by Mg2+ and Mn2+ ions and by various thiols. Reaction product analysis, mode of enzyme action and substrate specificity test suggest the endo- nature of the purified CMCase. The enzyme showed K(m) value of 26 +/- 1 mg/ml for carboxymethylcellulose (sodium salt).
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PMID:Physico-chemical and functional characterization of a high molecular weight carboxymethylcellulase from Cuscuta reflexa. 949 45

A cysteine protease inhibitor exhibiting antifungal activity from pearl millet seeds has been purified to homogeneity by ammonium sulphate precipitation and chromatographic procedures involving CM- sephadex and SP-sepharose cation exchange columns. The molecular characterization has revealed its molecular mass as 24 kD and isoelectric point 9.8. The amino acid composition data shows presence of high content of serine and glycine (34 residues/mole) and absence of tryptophan. The inhibitor exhibits potent antifungal activity against Trichoderma reesei, a dead wood fungus with minimum inhibitory dose to inhibit mycelial growth or spore germination is as low as 1 microgram/ml (250 ng/disc). In addition to Trichoderma reesei, the antifungal activity is observed against some important phytopathogenic fungi, namely, Claviceps, Helminthosporium, Curvularia, Alternaria and Fusarium species. To the best of our knowledge, a cysteine protease inhibitor as an antifungal protein is reported for the first time from a plant system.
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PMID:Cysteine protease inhibitor from pearl millet: a new class of antifungal protein. 961 Mar 68

Treatment of spinach leaf ferredoxin-dependent nitrite reductase with N-bromosuccinimide (NBS), under conditions where slightly less than 1 mol of tryptophan is modified per mole of nitrite reductase, inhibits the catalytic activity of the enzyme by ca. 80% without any effect on substrate binding or other enzyme properties. Complex formation between nitrite reductase and ferredoxin completely protects the enzyme against this inhibition. Transient kinetic measurements show that the second-order rate constant for reduction of NBS-modified nitrite reductase by reduced ferredoxin is approximately four-fold larger than that observed for the native, unmodified enzyme. Also, reduction of NBS-modified nitrite reductase by the 5-deazariboflavin radical shows a different kinetic pattern than that observed with the native enzyme, suggesting that tryptophan modification increases access of the radical to the low-potential [4Fe-4S] cluster of the enzyme, decreases the accessibility to the siroheme group of the enzyme, or both. The tryptophan that is modified has been identified as the absolutely conserved W92. A methionine, M73, that is also modified by NBS, has been identified. The ferredoxin-binding site on spinach nitrite reductase thus appears to include W92 and perhaps M73, in addition to the previously identified R375, R556, and K436.
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PMID:A conserved tryptophan at the ferredoxin-binding site of ferredoxin:nitrite oxidoreductase. 963 2

ATP binding to the large tumor (T) antigen encoded by the simian virus 40 (SV40) genome plays an essential role in the replication of viral DNA [Fanning, E., and Knippers, R. (1992) Annu. Rev. Biochem. 61, 55-85]. To better explore the functions of T antigen during the replication process, we have studied the interactions of T antigen with fluorescent 3'(2')-O-(2,4,6-trinitrophenyl) (TNP) adenine nucleotide analogues. Binding of TNP-ATP and TNP-ADP was accompanied by an 8-fold fluorescence enhancement and a concomitant blue shift (11 nm) of the maximal emission wavelength; the intrinsic protein tryptophan fluorescence was quenched maximally by 50%. Both signals were utilized to characterize the nucleotide binding activity of T antigen. TNP-ATP and TNP-ADP bound to the ATP binding site with dissociation constants of 0.35 microM and 2.6 microM. TNP substitution enhanced the affinity of ADP for T antigen by approximately 11-fold. The binding stoichiometry was 1 mol of TNP nucleotide per mole of monomer T antigen. The binding of TNP-ATP was more temperature dependent than that of TNP-ADP. The enthalpy change contributed nearly half of the energy for TNP-ATP binding, whereas binding of TNP-ADP was primarily entropy driven. Both TNP-ATP and TNP-ADP were strong inhibitors of the T antigen ATPase activity, confirming the high affinities of the TNP nucleotides for the ATP binding site. Like the parent nucleotides, they also induced T antigen hexamer formation. Using the TNP nucleotides as fluorescent probes, we have measured the affinity of various nucleotides and analogues for T antigen. The results indicate that the nucleotide binding specificity of T antigen was similar to that of the prokaryotic helicases Dna B and Rep, suggesting closely related ATP binding sites in the three DNA helicases.
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PMID:Characterization of the nucleotide binding properties of SV40 T antigen using fluorescent 3'(2')-O-(2,4,6-trinitrophenyl)adenine nucleotide analogues. 979 94

The ArsA ATPase is the catalytic subunit of a novel arsenite pump, with two nucleotide-binding consensus sequences in the N- and C-terminal halves of the protein. The single tryptophan-containing Trp159 ArsA was used to elucidate the elementary steps of the ATPase mechanism by fluorescence stopped-flow experiments. The binding and hydrolysis of MgATP is a multistep process with a minimal kinetic mechanism (Mechanism 1). A notable feature of the reaction is that MgATP binding induces a slow transient increase in fluorescence of ArsA, which is independent of the ATP concentration, indicative of the build-up of a pre-steady state intermediate. This finding, coupled with a phosphate burst, implies that the steady-state intermediate builds up subsequent to product release. We propose that the rate-limiting step is an isomerization between different conformational forms of ArsA. kcat is faster than the phosphate burst, indicating that both nucleotide binding sites of ArsA are catalytic. Consistent with this interpretation, approximately 2 mol of phosphate are released per mole of ArsA during the phosphate burst.
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PMID:The ATPase mechanism of ArsA, the catalytic subunit of the arsenite pump. 1034 68

1. Genes related to trp (transient receptor potential) are proposed to encode store-operated channels. We examined the ionic permeation of recombinant channels formed by stable and transient expression of the TRP homologue bCCE1 in Chinese hamster ovary (CHO) cells (CHO(CCE1)) and rat basophilic leukaemia (RBL) cells, respectively. 2. Store-operated currents were activated in CHO(CCE1) cells by internal dialysis of IP3 under strong buffering of intracellular Ca2+. The action of IP3 was mimicked by thapsigargin but not by IP4. 3. With extracellular Ca2+, Na+ and Mg2+, the store-operated currents of CHO(CCE1) rectified inwardly in the presence of internal Cs+. Outward currents were not detected below +80 mV. Identical currents were recorded with external Ba2+ and also with no external Na+ and Mg2+. In the absence of external Mg2+, the inward currents showed an anomalous mole fraction behaviour between Ca2+ and Na+. Half-maximal inhibition of Na+ currents was observed with approximately 100 nM and full block with 2-5 microM external Ca2+. 4. In the parental CHO(-) cells, IP3 dialysis evoked inward currents that also displayed anomalous mole fraction behaviour between Ca2+ and Na+. However, half-maximal block of Na+ currents required 5 times higher Ca2+ concentrations in CHO(-) cells. Additionally, the density of Ca2+ and Na+ currents at -80 mV was 5 and 2 times larger in CHO(CCE1) cells, respectively. 5. In RBL cells, dialysis of IP3 evoked store-operated currents that showed 1.4-fold larger densities at -80 mV in cells expressing bCCE1. 6. The enhanced density of store-operated currents in CHO(CCE1) cells and in bCCE1-transfected RBL cells probably reflects the phenotype of CCE1. These results suggest a highly selective permeation of Ca2+ through recombinant channels formed by CCE1 either alone or in combination with endogenous channel proteins.
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PMID:Phenotype of a recombinant store-operated channel: highly selective permeation of Ca2+. 1042 2


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