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1.
The milk-clotting activity of Mucor-rennin obtained from Mucor pusillus Lindt, was not changed by the addition of DFP in the reaction mixture. This finding suggested the probable absence of a serine residue at the active center of the enzyme. Sulfhydryl reagents such as Nekelgon, N-ethyl maleimide, PCMB failed to influence the milk-clotting reaction, indicating that a. reactive sulfhydryl group is not required for the enzymatic activity. The activity was inhibited when Mucor-rennin was treated with iodine at pH higher than 5.0. It was shown that 131I2 was incorporated into the enzyme. When Mucor-rennin was photooxidized in the presence of methylene blue, the milk-clotting activity was inactivated. In this case, tyrosine, tryptophan, and histidine residues in the enzyme were oxidized. Among these amino acids, the histidine residue was more rapidly oxidized than other amino acids. A parallel relation was observed between the decrease of the amount of histidine residue and the inactivation of the enzyme. From these results, it is concluded that the histidine residue present in Mucor-rennin has a relation to the active center of this enzyme. 相似文献
2.
The site of hemolytic activity of a toxin isolated from Aspergillus fumigatus designated Asp-hemolysin was determined by photooxidation techniques. The hemolytic activity of this toxin was strongly inhibited by photooxidation with methylene blue, rose bengal, riboflavin, or eosin G as a sensitizer, whereas crystal violet, hematoxylin, naphthol yellow S, bromothymol blue, methyl orange, and cresol red had no effect. pH dependence of the inactivation with methylene blue was observed in the narrow range of pH values from 7.0 to 8.0, like that of the inactivation with rose bengal or riboflavin. The histidine, cysteine, methionine, tryptophan, and tyrosine content of methylene blue-photooxidized Asp-hemolysin was significantly decreased, while other amino acids were not affected. The hemolytic activity of the toxin was lost more slowly than the histidine residue, being maintained at about 50% even at the time when the histidine residue was completely lost after 30 min. Photooxidation of Asp-hemolysin in the presence of rose bengal also caused a decrease in histidine, methionine, and threonine content. These findings suggest that residues of cysteine, methionine, threonine, tryptophan, and/or tyrosine but not histidine may play an important role through stereostructure in the manifestation of the hemolytic activity of Asp-hemolysin. 相似文献
3.
N I Churakova N A Kravchenko E P Serebriakov E D Kaverzneva 《Biokhimii?a (Moscow, Russia)》1976,41(5):822-826
One out of six trytophan residues in two lysozyme modification, obtained under lysozyme photooxidation in the presence of methylene blue, is found to be oxidized to N'-formylkinurenine (in one modification) and to kinurenine (in the other modification). The transition of one modification into another via detaching of N'-formyl group by soft acid hydrolysis has shown that one and the same tryptophan residue is oxidized in both products, Possible mechanism of tryptophan oxidation to the products mentioned is discu-sed on the basis of the hypothesis on signlet mechanism of lysozyme photooxidation in the presence of methylene blue. 相似文献
4.
A systematic study was made of the photochemical action of methylene blue on amino acids.Tyrosine, tryptophan, histidine, methionine, and cystine were highly reactive during the photoöxidation; the rest of the amino acids acted sluggishly or not at all.In tyrosine, tryptophan, and histidine, the entire oxygen uptake and CO2 evolution were due to the cyclic nucleus, involving rupture of the rings.During the photochemical action of methylene blue on tyrosine, tryptophan, and methionine, intermediary oxidizing agents were formed; in methionine this was shown to be H2O2.The photoöxidation of methionine resulted in the formation of methionine sulfoxide as an end product.Iodometric titration and measurement of ultraviolet absorption during irradiation of methionine indicate the formation of an intermediary dehydrogenation product which appears to differ from Lavine's dehydromethionine.Cystine was photoöxidized, probably beyond the cysteic acid stage.Peptide bonds did not participate in the photochemical action of methylene blue.Methylation of the α-amino group of lysine to the corresponding secondary and tertiary compounds produced increased reactivity in the photoöxidation. 相似文献
5.
《Biochimica et Biophysica Acta.Protein Structure》1976,420(2):265-278
Of the three tyrosine residues available for nitration by tetranitromethane in hemerythrin, nitration of tyrosine residue 70 has no effect on dissociation of octomers to monomers, but nitration of tyrosines 18 and/or 67 results in dissociation to monomers. The latter data suggests these residues are important for subunit association. The reactive sulfhydryl, the modification of which produces dissociation, was protected as a mixed disulfide during the nitration but was regenerated for analysis of the state of association. Residue 70 can be selectively modified because of its exposed position and perhaps because of its slightly lower pKa of 6.9, compared to 7.3 as an average of all nitrotyrosines in a completely nitrated hemerythrin.Solvent perturbation studies in 20% Me2SO indicate that 3 tyrosines, in agreement with the nitration results, and 2 tryptophan residues are exposed; however, oxidation at a 2-fold molar excess of N-bromosuccinimide oxidizes three tryptophan whereas a 3.5-fold excess oxidizes all four, but results in a rapid active site destruction. Photo-oxidation with methylene blue results in oxidation of only two tryptophan residues. These data have been interpreted to indicate that two tryptophans are free and two are involved in subunit association.Photo-oxidation with methylene blue results in the destruction of three histidines but no decrease in active site absorption. Histidine modification with diethyloxydiformate shows that three histidines react with no change in active site absorption. These results indicate that four histidines are unreactive toward these modifying agents and are therefore either buried or are ligands to the iron. 相似文献
6.
Hiroko Takesada Mamoru Nakanishi Akiko Y. Hirakawa Masamichi Tsuboi 《Biopolymers》1976,15(10):1929-1938
Effects of deuteration on the Raman spectrum of a tryptophan residue have been examined. The 1386 cm?1 line of deuterated tryptophan residue has been found to be useful for tracing the hydrogen-deuterium exchange reaction of this residue in a protein. An examination on bovine α-lactalbumin at pH 6.4 and at 20°C indicates that two of the four tryptophan residues exchange with a rate constant much greater than 9 × 10?4 sec?1, while the other two exchange with a rate constant of 4 × 10?5 sec?1. The latter two have been assigned to Trp 28 and Trp 108 of this protein. The kinetics of hydrogen-deuterium exchange reaction of completely “free” tryptophan residue have been examined by a proton magnetic resonance study on tryptophan itself. By taking the result of this examination into account, the chance of exposure to the solvent for Trp 28 or Trp 108 has been estimated to be 3 × 10?6 at pH 6.4 and at 20°C. 相似文献
7.
EGON JOSEFSSON 《Physiologia plantarum》1972,27(2):236-239
dl -Tryptophan(methylene)-14C and indole-3-acetaldehyde oxime(methylene)-14C were supplied to cut shoots of 7-day-old plants of Sinapis alba L. Although both compounds were effective as precursors of 3-indolylmethylglucosinolate, the incorporation of the aldoxime radioactivity was more effective than the incorporation of the amino acid radioactivity. This, together with other information, suggests that indole-3-acetaldehyde oxime is an intermediate in the biosynthesis of 3-indolylmethylglucosinolate from tryptophan. 相似文献
8.
Kamaluddin Mala Nath Sushama W. Deopujari 《Origins of life and evolution of the biosphere》1989,19(2):119-132
Dehydrogenation of ascorbic acid and reduced nicotinamide adenine dinucleotide (NADH) with methylene blue using complexes of the type [Fe(II)(CN)5 (L)]
n– (wheren=3 or 4; L=glycine, histidine, imidazole, and triglycine) as catalyst have been studied at pH 9.18. Similar kinetic behavior was observed for the dehydrogenation of ascorbic acid as well as for NADH; both reactions showed first order dependency on the substrates. First order dependence was observed only at lower concentrations of methylene blue; at higher concentrations of methylene blue, the reactions were independent of methylene blue. The order with respect to catalyst varied between 0.3–0.5. A tentative mechanism which conforms to the observed kinetics has been proposed. It is believed that on the primitive earth when the reducing potential of the atmosphere was not high enough, lower oxidation state iron complexes like [Fe(II)(CN)5(L)]
n– might have been involved in dehydrogenase-type activity. 相似文献
9.
Human erythrocytes were shown previously to catalyze the oxyhemoglobin-requiring hydroxylation of aniline, and the reaction was stimulated apparently preferentially by NADPH in the presence of methylene blue (K. S. Blisard and J. J. Mieyal,J. Biol. Chem.254, 5104, 1979). The current study provides a further characterization of the involvement of the NADPH-dependent electron transport system in this reaction. In accordance with the role of NADPH, the hydroxylase activity of erythrocytes or hemolysates from individuals with glucose-6-phosphate dehydrogenase deficiency (i.e., with diminished capacity to form NADPH) displayed decreased responses to glucose or glucose 6-phosphate, respectively, in the presence of methylene blue in comparison to samples from normal adults; maximal activity could be restored by direct addition of NADPH to the deficient hemolysates. Kinetic studies of the methylene blue-stimulated aniline hydroxylase activity of normal hemolysates revealed a biphasic dependence on NADPH concentrations: a plateau was observed at relatively low concentrations (KmNADPH ~ 20 μm), whereas saturation was not achieved at the higher concentrations of NADPH. The latter low efficiency phase (i.e., at the higher concentrations of NADPH) could be ascribed to a direct transfer of electrons from NADPH to methylene blue to hemoglobin. The high efficiency phase suggested involvement of the NADPH-dependent methemoglobin reductase; accordingly 2′-AMP, an analog of NADP+, effectively inhibited this reaction, but the pattern was noncompetitive. This behavior is suggestive of a mechanism by which both NADPH and methylene blue are substrates for the reductase and interact with it in a sequential fashion. The kinetic patterns observed for variation in NADPH concentration at several fixed concentrations of methylene blue, and vice versa, are consistent with this interpretation. 相似文献
10.
The absorption band at 260 mμ of NAD+ shifts to 360 mg by interaction with GAPDH or its analogues. Two explanations have been given on this red shift; one is an addition of such nucleophilic residue as sulfhydryl group in the enzyme to the position four in nicotinamide nucleus of NAD+, and the other is the charge transfer from such aromatic amino acid as tryptophan to NAD+. In the present paper, possibility of the charge transfer from indole residue to NAD+ was investigated quantum chemically. Taking into account of the electric field due to the charges in the enzyme, the absorption band of the NAD+-enzyme complex at 360 mμ was explained as a charge transfer from indole nucleus to NAD+. The blue shift of the absorption band of NADH at 340 mμ was also explained by taking into account of the electric field and this supported the proposition of Kosower (1962a).Stacking of adenine nucleus with indole nucleus in the NAD+-enzyme complex was suggested from the NMR spectroscopic data. Our molecular orbital calculations predicted that the effects of adenine on spectral shifts were not significant. 相似文献
11.
Methylene blue added to suspensions of activated staphylococci in amounts sufficient to furnish 1 x 105 molecules of dye/bacterium inactivates the phage precursor content of the cells without causing cell death when the mixtures are exposed to strong light of 4000–8000 Å. There is a lag phase of approximately 15 minutes in the photodynamic inactivation of phage precursor by methylene blue. This delay seems to be due to a primary reaction between the cell and methylene blue after the completion of which exposure to light brings about the inactivation of precursor quite promptly. 相似文献
12.
Physico chemical changes of ovalbumin illuminatied in the presence of methylene blue were examined. Solubility of ovalbumin was remarkably reduced, but its extents were varied with the value of pH, that of ionic strength and illumination time. Illumination brought about aggregation of protein molecules which was revealed on the ultra centrifugal patterns. Electrophoretical patterns showed that three peaks characteristic of native ovalbumin went into one peak after 24 hr and into two peaks after 48 hr. After an illumination for 6 hr, titration curves showed that bound protons decreased below pH 8.0 and increased over pH 8.0. The spectra of illuminated ovalbumin were displaced upward and the absorption maximum shifted toward the longer region of wave length. 相似文献
13.
《Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology》1988,952(2):266-271
The spin-labeled tryptophan was used as a structural probe of hemoglobin contact sites. The ESR spectral data indicated that the probe exhibits weak binding to hemoglobin with a dissociation constant of 3.2 · 10−5 and 4.0 mol bound per hemoglobin tetramer. The spectrum suggested that the bound tryptophan was ‘partially immobilized’ with a correlation time reflecting the environment of the tryptophan binding site of 8.2 ns. The topology of the contact sites was investigated by using dual spin-label methodology in which spin-labeled tryptophan and (2H,15N) substituted and deuterated maleimide spin label [2H-15N]MSL covalently-bound to Cys-ß93 residue were used. The ESR spectral data suggested that the tryptophan binding sites were located within 8–10Åof the nitroxide free radical of spin-labeled hemoglobin. The environment of the contact sites is discussed. 相似文献
14.
Nobuyuki Yamasaki Tomomitsu Hatakeyama Hideki Ohba Gunki Funatsu 《Bioscience, biotechnology, and biochemistry》2013,77(8):2227-2232
Chemical modification of tryptophan residues in ricin E was investigated with regard to saccharide-binding. Two out of ten tryptophan residues in ricin E were modified with N- bromosuccinimide at pH 4.5 in the absence of specific saccharide accompanied by a marked decrease in the cytoagglutinating activity. Such a loss of the cytoagglutinating activity was found to be principally due to the oxidation of one tryptophan residue per B-chain. In the presence of lactose, one tryptophan residue/mol was protected from the modification with retention of a fairly high cytoagglutinating activity. However, G a IN Ac did not show such a protective effect. The binding of lactose to ricin E altered the environment of the tryptophan residue at the low affinity binding site of ricin E, leading to a blue shift of the fluorescence spectrum and an UV-difference spectrum with a maximum at 290 nm and a trough at 300 nm. The ability to generate such spectroscopic changes induced by lactose was retained in the derivative in which one tryptophan residue/mol was oxidized in the presence of lactose, but not in the derivative in which two tryptophan residues/mol were oxidized in the absence of lactose. Based on these results, it is suggested that one of the two surface-localized tryptophan residues is responsible for saccharide binding at the low affinity binding site of ricin E, which can bind lactose but lacks the ability to bind GalNAc. 相似文献
15.
The rotational correlation time of melittin, obtained from the nanosecond anisotropy of the emission from its single tryptophan residue, has been found to increase considerably in phosphate solution relative to that in aqueous solution, consistent with protein aggregation. The steady-state fluorescence spectra as well as the absorption spectra in phosphate solution exhibit a very good degree of similarity with those of the protein bound to egg phosphatidylcholine (PC) and distearoylphosphatidylcholine (DSPC) bilayer liposomes. The value of the second-order rate constant for dynamic quenching, , by acrylamide in 0.5 M phosphate solution is comparable to those for the protein-phospholipids complexes (1·109 and 0.7·109 M?1·s?1 for egg PC and DSPC, respectively). Similarities are also found in the nanosecond properties. There is a much stronger and quite similar dependence of the fluorescence spectra on time in the nanosecond range and of the fluorescence decay times on the emission wavelength in both cases as compared to the case in aqueous solution. These observations support the notion that melittin binds to the phospholipids in an aggregated form. The results suggest that the reduction in the values of bound melittin relative to that in aqueous solution and the blue shift of the fluorescence spectrum (from 352 to 337 nm) are brought about by shielding of the tryptophan residue from the solvent through a combination of protein aggregation and enhancement of its α-helical content (suggested by published CD data). The magnitude of the values for bound melittin, however, is still relatively high implying the occurrence of rather frequent encounters between the tryptophan residue and the hydrophilic acrylamide molecules. Thus, the residue is found not to penetrate deep into the phospholipid bilayer. 相似文献
16.
Masafumi Mukamoto Ryota Kimura Mudenda B. Hang'ombe Tomoko Kohda Shunji Kozaki 《Microbiology and immunology》2013,57(3):163-169
Clostridium septicum alpha‐toxin has a unique tryptophan‐rich region (302NGYSEWDWKWV312) that consists of 11 amino acid residues near the C‐terminus. Using mutant toxins, the contribution of individual amino acids in the tryptophan‐rich region to cytotoxicity and binding to glycosylphosphatidylinositol (GPI)‐anchored proteins was examined. For retention of maximum cytotoxic activity, W307 and W311 are essential residues and residue 309 has to be hydrophobic and possess an aromatic side chain, such as tryptophan or phenylalanine. When residue 308, which lies between tryptophans (W307 and W309) is changed from an acidic to a basic amino acid, the cytotoxic activity of the mutant is reduced to less than that of the wild type. It was shown by a toxin overlay assay that the cytotoxic activity of each mutant toxin correlates closely with affinity to GPI‐anchored proteins. These findings indicate that the WDW_W sequence in the tryptophan‐rich region plays an important role in the cytotoxic mechanism of alpha‐toxin, especially in the binding to GPI‐anchored proteins as cell receptors. 相似文献
17.
Of the three tyrosine residues available for nitration by tetranitromethane in hemerythrin, nitration of tyrosine residue 70 has no effect on dissociation of octomers to monomers, but nitration of tyrosines 18 and/or 67 results in dissociation to monomers. The latter data suggests these residues are important for subunit association. The reactive sulfhydryl, the modification of which produces dissociation, was protected as a mixed disulfide during the nitration but was regenerated for analysis of the state of association. Residue 70 can be selectively modified because of its exposed position and perhaps because of its slightly lower pk of 6.9, compared to 7.3 as an average of all nitrotyrosines in a completely nitrated hemerythrin. Solvent perturbation studies in 20% Me2SO indicate that 3 tyrosines, in agreement with the nitration results, and 2 tryptophan residues are exposed; however, oxidation at a 2-fold molar excess of N-bromosuccinimide oxidizes three tryptophan whereas a 3.5-fold excess oxidizes all four, but results in a rapid active site destruction. Photo-oxidation with methylene blue results in oxidation of only two tryptophan residues. These data have been interpreted to indicate that two tryptophans are free and two are involved in subunit association. Photo-oxidation with methylene blue results in the destruction of three histidines but no decrease in active site absorption. Histidine modification with diethyloxydiformate shows that three histidines react with no change in active site absorption. These results indicate that four histidines are unreactive toward these modifying agents and are therefore either buried or are ligands to the iron. 相似文献
18.
Kamal N Chowdhury S Madan T Sharma D Attreyi M Haq W Katti SB Kumar A Sarma PU 《Molecular and cellular biochemistry》2005,275(1-2):223-231
The role of tryptophan (Trp17) in immunoreactivity of P1, the diagnostically relevant peptide from a major allergen/antigen of Aspergillus fumigatus, was evaluated by chemically modifying tryptophanyl residue of P1. In BIAcore kinetic studies, unmodified P1 showed a 100-fold higher binding with ABPA (Allergic Bronchopulmonary Aspergillosis) patients’ IgG [KD (equilibrium dissociation constant) = 2.74 e−8 ± 0.13 M] than the controls’ IgG (KD = 2.97 e−6± 0.14 M), whereas chemically-modified P1 showed similar binding [KD patients’ IgG = 3.25 e−7± 0.16 M, KD controls’ IgG = 3.86 e−7± 0.19 M] indicating loss of specific immunoreactivity of P1 on tryptophan modification. Modified P1 showed loss of specific binding to IgE and IgG antibodies of ABPA patients in ELISA (Enzyme-Linked Immunosorbent Assay). The study infers that tryptophan residue (Trp17) is essential for immunoreactivity of P1. 相似文献
19.
《生物化学与生物物理学报:生物膜》2002,1558(2):222-237
Reaction of the melanotropin hormone analogs [Nle4,D-Phe7]-α-MSH and [Nle4,D-Phe7]-α-MSH(4-10), which were extended at their N-terminus by a thiol-functionalized spacer arm, with preformed liposomes containing thiol-reactive (phospho)lipid derivatives resulted in the aggregation of the vesicles and in a partial leakage of their inner contents. This aggregation/leakage effect, which was only observed when the peptides were covalently conjugated to the surface of the liposomes, was correlated with the fusion of the vesicles as demonstrated by the observed decrease in resonance energy transfer between probes in a membrane lipid mixing assay. A limited fusion was confirmed by monitoring the mixing of the liposome inner contents (formation of 1-aminonaphthalene-3,6,8-trisulfonic acid/p-xylene bis(pyridinium bromide) complex). The membrane-active properties of the peptides could be correlated with changes in the fluorescence emission spectra of their tryptophan residue, which suggested that after their covalent binding to the outer surface of the liposomes they can partition within the core of the bilayers. A blue shift of 10 nm was observed for [Nle4,D-Phe7]-α-MSH which was correlated with an increase in fluorescence anisotropy and with changes in the accessibility of the coupled peptide as assessed by the quenching of fluorescence of its tryptophan residue by iodide (Stern-Volmer plots). These results should be related to the previously described capacity of α-MSH, and analogs, to interact with membranes and with the favored conformation of these peptides which, via a β-turn, segregate their central hydrophobic residues into a domain that could insert into membranes and, as shown here, trigger their destabilization. 相似文献
20.
Papain [EC 3.4.22.2] was photooxidized using methylene blue as a sensitizer. The photooxidzed enzyme lost its caseinolytic activity and had significantly decreased histidine and tryptophan contents. The tyrosine content was the same before and after the photooxidation. The SH content of the photooxidized enzyme, as determined after reduction with dithiothreitol, was also unchanged. The loss of histidine was always slower than the loss of enzymatic activity, being less than one residue per molecule even when the enzymatic activity was completely lost. However, the inactivation and the oxidation of a histidine residue were pH-dependent in a similar fashion in the pH range of 5.0-8.0, the pH profiles conforming to theoretical titration curves with apparent pKa values of 6.6 and 6.7, respectively. The fact that the ionization of a histidine residue in papain has a normal imidazole pKa value is entirely in accord with the finding for stem bromelain [EC 3.4.22.4] (Murachi, T., Tsudzuki, T., & Okumura, K. (1975) Biochemistry 14, 249-255), and is of great significance in relation to the mechanism of catalysis by these enzymes. 相似文献