共查询到20条相似文献,搜索用时 8 毫秒
1.
《Comparative biochemistry and physiology. B, Comparative biochemistry》1993,104(3):743-746
- 1.1. Ovine placental lactogen was modified by reaction with o-nitrophenylsulfenyl chloride. Fluorescence measurements indicated that one of the two tryptophan residues of the molecule had reacted. Besides, there was some reagent not covalently bound.
- 2.2. The reagent was covalently bound to Trp-150. No evidence of modification of Trp-90 was found.
- 3.3. Binding capacity to lactogenic as well as somatogenic receptors was diminished but not abolished upon modification, indicating that absolute molecular integrity of Trp-150 is not required for binding.
- 4.4. This behavior is similar to that of the tryptophan residues of ovine prolactin.
2.
The modification of alpha-chymotrysin with phenacyl bromide has been reinvestigated over a wide pH range. Evidence is presented that indicates that the nature of the phenacyl-modified enzymes prepared by this reaction is dependent upon the pH of the reaction medium. The phenacyl alpha-chymotrypsin produced at low pH is most probably the Met-192 phenacylsulfonium salt, as proposed earlier, since it readily undergoes dealkylation using 2-mercaptoethanol. However, the phenacyl-enzyme prepared at neutral pH possesses a much reduced enzymatic activity and does not react with 2-mercaptoethanol to regenerate native alpha-chymotrypsin. In addition, incubation of the Met-192 phenacyl sulfonium enzyme at neutral pH causes a smooth irreversible change to the new phenacyl-enzyme as monitored by changes in enzymatic activity, susceptibility to dealkylation using 2-mercaptoethanol, and ultraviolet difference absorption spectral properties. The stoichiometries of both the low and neutral pH modification reactions have been determined, using [carbonyl-14C]phyenacyl bromide, to be 1 phenacyl group/enzyme molecule. In efforts to obtain information about the nature and mechanism of formation of the phenacyl alpha-chymotrypsin produced at neutral pH, alkylation reactions of modified alpha-chymotrypsins produced by His-57 functionalization with tosylphenylalanine chloromethyl ketone and by Met-192 oxidation to the sulfoxide have been investigated. The combined results of these studies have been initially interpreted in terms of a neutral pH, phenacyl bromide modification resulting in formation of a new modified enzyme via the Met-192 sulfonium salt. 相似文献
3.
S Nakamura H Ogata K Takeo A Kuwahara R Suzuno 《Hoppe-Seyler's Zeitschrift für physiologische Chemie》1975,356(6):677-692
Bovine alpha2-globulin contains a protein which increases the activity of bovine alpha-chymotrypsin against synthetic substrates. The active protein fraction migrates slowly on polyacrylamide gel electrophoresis, so it was named slow alpha2-globulin (Salpha2). The fraction was isolated from bovine serum and purified. Its sedimentation constant S20 was 18.5 S. It was thus identified with the alpha2-macroglobulin (alpha2M). By kinetic studies, the dissociation constant of the alpha-chymotrypsin-alpha2 M complex was calculated to be of the order of 10(-7) l/mol. The purified alpha2 M was shown to bind alpha-chymotrypsin at a definite rate. If the binding ratio was assumed to be 1:2, the molecular weight was calculated to be about 8 X 10(5). 相似文献
4.
The role of several active site residues of alpha-chymotrypsin in the prototypical refolding transition between active and inactive forms of this enzyme is examined using chemical modification. Oxidation of Met-192 to the sulfoxide results in a derivative which remains entirely in an active state from pH 6 to 9. The derivative becomes inactive only at high pH with pKa = 10.3, delta H0 = 9.5 kcal and delta S0 = -15 eu., indicating the sulfoxide group supplies about 2.1 kcal of active state stabilization relative to the unoxidized methionine side chain. The refolding transition of N-methyl-His-57-alpha-chymotrypsin, in which a nitrogen of the "charge relay" histidine is methylated, displays one ionization process with an apparent pKa of 9.45. The absence of an additional ionization process with a pKa near 7 provides evidence that one of the ionizations in the six state mechanism which describes this transition in alpha-chymotrypsin is linked to the charge relay system. We also demonstrate, using alpha-chymotrypsin, Met-192-sulfoxide-alpha-chymotrypsin and N-methyl-His-57-alpha-chymotrypsin, that the 230 nm circular dichroism band is a quantitative probe of the active-inactive equilibrium, although the chromophore or chromophores responsible for this and another very large negative band at 202 nm have not been identified. Circular dichroism was used to observe the active-inactive equilibrium in methan sulfonyl-alpha-chymotrypsin and phenylmethane sulfonyl-alpha-chymotrypsin. The enhanced stability of the active state of these derivatives relative to alpha-chymotrypsin can be rationalized in terms of steric effects in the substrate side chain binding site. 相似文献
5.
6.
7.
S Hori 《Biochimica et biophysica acta》1975,384(1):58-68
Partially purified tryptophan-5-monooxygenase (L-tryptophan, tetrahydropteridine: oxygen oxidoreductase (5-hydroxylating) EC 1.14.16.4)from bovine pineal gland was activated by preincubation with sulfhydryl agents such as dithiothreitol, L-cysteine, cysteamine, L-cysteine ethylester, N-acetyl-L-cysteine, 2-mercaptoethanol and reduced glutathione, at alkaline pH (optimum pH equals 8.5). Dithiothreitol was the most effective of these, leading to approximately 50-fold activation of the enzyme after preincubation. Fe-2+ or other reducing agents such as borohydride, dithionite and ascorbate facilitated the velocity of the activation in the presence of sulfhydryl agents. In the absence of sulfhydryl agents, no activation was observed even in the presence of Fe-2+ or other reducing agents, suggesting an obligatory role or sulhydryl agents during the activation. The relative velocity and full extent of the activation were dependent on the concentrations of both the sulfhydryl agent and the enzyme in the activation mixture. The kinetic analysis of the activation indicated that the sulfhydryl agent reacts with more than 2 sites in the enzyme; one type of site is reduced by sulfhydryl agents, Fe-2+ or other reducing agents and the other specifically modified by a sulfhydryl agent. The activated enzyme did not require any exogenous Fe-2+ for its catalytic activity, but some roles of iron maybe exist in its catalytic reaction. The optimum pH for catalytic reaction of the activated enzyme was approximately 6.5. The apparent Km for L-tryptophan and pteridine cofactor, tetrahydro-pteridine (2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropterin), of the activated enzyme were 30 and 35 muM respectively. 相似文献
8.
9.
G Oshima 《International journal of biological macromolecules》1989,11(1):43-48
Although loss of chymotrypsin activity in dilute solution deviated from first order kinetics at low enzyme concentration, it displayed first order kinetics at concentrations more than 4 nM. First order rate constants varied with the ratio of surface area to volume, with the kind of vessel containing the enzyme, and with the particular test material (DS, polybrene, lecithin or BSA) coating the vessel. The reaction was saturable at lower chymotrypsin concentrations in glass than in polypropylene tubes, while less enzyme was lost at high concentrations. All these facts showed that loss of enzyme activity is incompletely, but markedly, due to a solid surface-catalysed reaction. Intrinsic fluorescence of native chymotrypsin at pH values 8 and 3, and of active site-blocked enzyme, decreased with time at 37 degrees C. No extensive autolysis of chymotrypsin was observed during the time-dependent loss of enzyme activity. Therefore, the apparent loss of chymotrypsin activity in dilute solution was mainly due to an irreversible conformational change of the molecules, as associated with the solid-surface-catalysed reaction. 相似文献
10.
11.
12.
XC Sarcoma, Vero and Aedes aegypti plasma membranes have been studied in viable cells and in purified membrane of XC Sarcoma cells by the spin label method. The temperature dependence of the order parameter of fatty acid spin labels is found to be linear in all three cells and membrane and shows no evidence of a lipid phase transition. The order parameter of the fatty acid labels substituted at the 5-position is shown to increase as a function of the cholesterol: phospholipid molar ratio in cells that have been studied to date. Cells attached to their growing surface are studied for the first time by electron paramagnetic resonance spectroscopy (EPR). The resulting spectra are anisotropic due to the non-spherical shape of the cells and show that these labels orient preferentially perpendicular to the cell surface. The viscosity of the extracted XC cell membrane is estimated to be 2.5 P from rotational correlation time measurements of the spin label 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO). 相似文献
13.
Protein stabilization via hydrophilization. Covalent modification of trypsin and alpha-chymotrypsin 总被引:1,自引:0,他引:1
V V Mozhaev V A Siksnis N S Melik-Nubarov N Z Galkantaite G J Denis E P Butkus N M Zaslavsky BYuMestechkina K Martinek 《European journal of biochemistry》1988,173(1):147-154
This paper experimentally verifies the idea presented earlier that the contact of nonpolar clusters located on the surface of protein molecules with water destabilizes proteins. It is demonstrated that protein stabilization can be achieved by artificial hydrophilization of the surface area of protein globules by chemical modification. Two experimental systems are studied for the verification of the hydrophilization approach. The surface tyrosine residues of trypsin are transformed to aminotyrosines using a two-step modification procedure: nitration by tetranitromethane followed by reduction with sodium dithionite. The modified enzyme is much more stable against irreversible thermoinactivation: the stabilizing effect increases with the number of aminotyrosine residues in trypsin and the modified enzyme can become even 100 times more stable than the native one. Alpha-chymotrypsin is covalently modified by treatment with anhydrides or chloroanhydrides of aromatic carboxylic acids. As a result, different numbers of additional carboxylic groups (up to five depending on the structure of the modifying reagent) are introduced into each Lys residue modified. Acylation of all available amino groups of alpha-chymotrypsin by cyclic anhydrides of pyromellitic and mellitic acids results in a substantial hydrophilization of the protein as estimated by partitioning in an aqueous Ficoll-400/Dextran-70 biphasic system. These modified enzyme preparations are extremely stable against irreversible thermal inactivation at elevated temperatures (65-98 degrees C); their thermostability is practically equal to the stability of proteolytic enzymes from extremely thermophilic bacteria, the most stable proteinases known to date. 相似文献
14.
Jan Willem Erisman James N. Galloway Sybil Seitzinger Albert Bleeker Nancy B. Dise A. M. Roxana Petrescu Allison M. Leach Wim de Vries 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》2013,368(1621)
The demand for more food is increasing fertilizer and land use, and the demand for more energy is increasing fossil fuel combustion, leading to enhanced losses of reactive nitrogen (Nr) to the environment. Many thresholds for human and ecosystem health have been exceeded owing to Nr pollution, including those for drinking water (nitrates), air quality (smog, particulate matter, ground-level ozone), freshwater eutrophication, biodiversity loss, stratospheric ozone depletion, climate change and coastal ecosystems (dead zones). Each of these environmental effects can be magnified by the ‘nitrogen cascade’: a single atom of Nr can trigger a cascade of negative environmental impacts in sequence. Here, we provide an overview of the impact of Nr on the environment and human health, including an assessment of the magnitude of different environmental problems, and the relative importance of Nr as a contributor to each problem. In some cases, Nr loss to the environment is the key driver of effects (e.g. terrestrial and coastal eutrophication, nitrous oxide emissions), whereas in some other situations nitrogen represents a key contributor exacerbating a wider problem (e.g. freshwater pollution, biodiversity loss). In this way, the central role of nitrogen can remain hidden, even though it actually underpins many trans-boundary pollution problems. 相似文献
15.
16.
Towards a structure-function analysis of bovine lactoferricin and related tryptophan- and arginine-containing peptides. 总被引:14,自引:0,他引:14
Hans J Vogel David J Schibli Weiguo Jing Elke M Lohmeier-Vogel Raquel F Epand Richard M Epand 《Biochimie et biologie cellulaire》2002,80(1):49-63
The iron-binding protein lactoferrin is a multifunctional protein that has antibacterial, antifungal, antiviral, antitumour, anti-inflammatory, and immunoregulatory properties. All of these additional properties appear to be related to its highly basic N-terminal region. This part of the protein can be released in the stomach by pepsin cleavage at acid pH. The 25-residue antimicrobial peptide that is released is called lactoferricin. In this work, we review our knowledge about the structure of the peptide and attempt to relate this to its many functions. Microcalorimetry and fluorescence spectroscopy data regarding the interaction of the peptide with model membranes show that binding to net negatively charged bacterial and cancer cell membranes is preferred over neutral eukaryotic membranes. Binding of the peptide destabilizes the regular membrane bilayer structure. Residues that are of particular importance for the activity of lactoferricin are tryptophan and arginine. These two amino acids are also prevalent in "penetratins", which are regions of proteins or synthetic peptides that can spontaneously cross membranes and in short hexapeptide antimicrobial peptides derived through combinatorial chemistry. While the antimicrobial, antifungal, antitumour, and antiviral properties of lactoferricin can be related to the Trp/Arg-rich portion of the peptide, we suggest that the anti-inflammatory and immunomodulating properties are more related to a positively charged region of the molecule, which, like the alpha- and beta-defensins, may act as a chemokine. Few small peptides are involved in as wide a range of host defense functions as bovine and human lactoferricin. 相似文献
17.
18.
19.
The thermostability and thermodinamics of formation of the enzyme-substrate complex of two oxidation products of chicken egg lysozyme with the tryptophane-62 residue modified to N'-formylkinurenine (with 2.5% activity) and kinurenine (with 27.5% activity) have been studied. In thermostability and pH effect on the substrate binding the lysozyme oxidation products do not differ from native lysozyme. The data obtained and thermodynamical characteristics of the enzyme-substrate complex formation suggest that the chemical nature of the 62 residue does not significantly affect the conformational properties of lysozyme, however, having a strongly pronounced effect on the binding of substrate and hence the total enzyme activity. 相似文献
20.
Consequences of a subtle sialic acid modification on the murine polyomavirus receptor. 总被引:2,自引:1,他引:1
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
M Herrmann C W von der Lieth P Stehling W Reutter M Pawlita 《Journal of virology》1997,71(8):5922-5931
Polyomaviruses are small, nonenveloped DNA tumor viruses with restricted host ranges. Virus binding to cell surface receptors is one determinant of viral tropism. Although murine polyomavirus is among the best characterized viruses, little is known about the sialic acid-containing receptor and its interaction with viral particles. By using nonradioactive virus binding assays as recently described for the B-lymphotropic papovavirus, murine polyomavirus particles were found to bind in a saturable and noncooperative manner to 25,000 receptors per 3T6 mouse fibroblast. The virus-receptor interaction at 4 degrees C was of high affinity (Kd = 1.8 x 10(-11) M), very fast (k1 = 1.7 x 10(7) M(-1) s(-1)), and stable (half-life = 38 min). Elongation of the N-acyl side chain of sialic acid by biosynthetic modulation with synthetic precursor analogs has been shown for other polyomaviruses to influence both sialic acid-dependent binding and infection (O. T. Keppler, P. Stehling, M. Herrmann, H. Kayser, D. Grunow, W. Reutter, and M. Pawlita, J. Biol. Chem. 270:1308-1314, 1995). In 3T6 cells in which about one-third of the sialic acids were modified, infection and binding of polyomavirus particles were significantly reduced. The number of receptors per cell was decreased to 18,000, with the remaining receptors displaying the same affinity as in untreated cells. Molecular modeling studies based on the three-dimensional structure of a mouse polyomavirus-sialyllactose complex recently solved by T. Stehle and coworkers (T. Stehle, Y. W. Yan, T. L. Benjamin, and S. C. Harrison, Nature 369:160-163, 1994) were performed. They suggest that the elongation of the N-acyl side chain by a single methylene group leads to steric hinderence, with the peptide backbone of a loop walling the tip of the shallow sialic acid binding groove. This collision appears to be incompatible with functional binding. The data are taken as a basis to discuss possible features of the organization and topology of the cellular receptor for mouse polyomavirus. 相似文献