首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 414 毫秒
1.
Azo coupling reactions of N-α-acetylhistidine, N-α-acetyltyrosine, and N-α-acetyllysine with p-methylbenzenediazonium ion were investigated as model reactions to obtain information on the relative reactivity of the histidine, tyrosine, and lysine moieties of protein, separated from structural effects. The azo coupling yields of the amino acids increased as the pH of the reaction medium was increased, indicating that the ractive species are the imidazole anion of histidine, the phenolate anion of tyrosine, and the neutral ε-amino group of lysine. It was calculated, based on percentage yields of the azo products, that the imidazole anion is more reactive than the phenolate anion and the ε-amino group, respectively.  相似文献   

2.
The rates of the trinitrophenylation of the amino groups of ribonuclease A (RNAse) with the specific reagent trinitrobenzene sulfonic acid have been studied at 27°C, between pH 7.0 and 9.9. From the variation of the velocity constants with pH it has been shown that the reaction is biphasic in the sense that for each amino group two pKs have been found: one (pK = 7.3–7.52) in the range of pH between 7.0 and 8.3 and the other (pK = 9.28–9.69) in the pH range 8.5–9.9. It is pointed out that when the experimental conditions approached one another, there was agreement between the pK values obtained from titrimetric and kinetic studies. Evidence is presented from the literature concerning the validity of the pK value near 7.5 for the ε-amino groups in RNAse. The studies were repeated with performic acid oxidized RNAse and the 10 ε-amino groups were found to be monophasic with pK values between 8.01 and 8.10. The α-amino group of the N-terminal lysine was biphasic with a pK of 7.26 (pH range 7–8) and 8.13 (pH range 8.2–9.5).  相似文献   

3.
《Phytochemistry》1987,26(5):1299-1300
The effect ofpH on Km and Vmax values of coconut α-galactosidase indicates the involvement of two ionizing groups with pKa values of 3.5 and 6.5 in catalysis. Chemical modification has indicated the presence of two carboxyl groups, a tryptophan and a tyrosine, at or near the active site of α-galactosidase. Based on these facts a new mechanism of action for α-galactosidase is proposed in which the ionizing group with a pKa of 3.5 is a carboxyl group involved in stabilizing a carbonium ion intermediate and the ionizing group with a pKa of 6.5 is a carboxyl group perturbed due to the presence of a hydrophobic residues in its vicinity which donates a H+ ion in catalysis.  相似文献   

4.
A mixture of acids with known pK′ and in a known concentration was dissolved to simulate the ionic character of hemoglobin. The titration curves of the mixture were obtained in water and 3.6 M KCl at 20°C. These curves were subjected to analysis by a reiterative curve-fitting procedure to determine if one could evaluate both the group ionization constants and the numbers of groups. This approach was successful in that the calculated parameters were within the experimental error encountered in obtaining these constants on each of the model compounds. However, analysis for the electrostatic interaction parameter w indicated that there was a possible effect on the ionization of formic acid, pyridine, imidazole, and the α-amino group of glycylglycine in water and on imidazole and the α-amino group of glycylglycine in 3.6 M KCl.  相似文献   

5.
Guanidinated, carbamylated, and acetylated glucagon largely retains the ability to stimulate the formation of cyclic AMP in rat liver homogenates, while with N-ethoxyformyl-acetylated glucagon this ability is completely lost. This latter derivative can be reconverted to a biologically active peptide by treatment with hydroxylamine. These results indicate that the imidazole group of the amino terminal histidine, but not the α or ? amino groups of glucagon, is essential for activity. Histidine amide does not stimulate the activity of adenyl cyclase even at 0.2 m concentration. The titration behavior of glucagon shows a normal pK for the amino terminal histidine.  相似文献   

6.
The pKa values for the proton dissociation of carboxyl, imidazolium, and ammonium groups for histidine and ten of its derivatives were determined electrometrically at seven temperatures in the range 10–40°C. The ΔH and ΔS values were estimated from the temperature dependence of the dissociation constants of histidine and its derivatives. These results and the pKa values compared in terms of inductive effect suggest an ion-dipole interaction between the protonated amino group and the unprotonated imidazole ring. The charge and the solvation effects of the neighboring groups are the main factors that determine the imidazole group pKa in histidine and its studied derivatives. The Nτ-H tautomer is favored over the Nπ-H by 1.6 kcal/mol, indicating that the inductive substituent effect at position 4 of the imidazole ring is the major component in determining this tautomeric preference.  相似文献   

7.
《Insect Biochemistry》1990,20(6):645-652
Post-emergence levels of 3,4-dihydroxyphenylacetic acid (DOPAC) and ketocatechol were determined in cuticle from adult Tenebrio molitor. Possible pathways for biosynthesis of DOPAC were studied by comparing the incorporation of injected [U-14C]tyrosine, [7-14C]dopamine, [7-14C]DOPA, [7-14C]tyramine, [U-14C]p-hydroxyphenylpyruvic acid (p-HPPA) and [ring-3H]p-hydroxyphenylacetic acid (p-HPAA) into cuticular DOPAC during its period of maximal increase 1–3 days after adult emergence. Increased incorporation of [U-14C]tyrosine between days 0 and 3 suggests rapid de novo biosynthesis of DOPAC from this primary precursor. Of the putative intermediates tested, only p-HPPA had a pattern of incorporation similar to that seen with tyrosine. Since p-HPAA was poorly incorporated into both cuticle and DOPAC, a tentative pathway tyrosine → p-HPPA → 3,4-dihydroxyphenylpyruvic acid → DOPAC is proposed.  相似文献   

8.
α-Amylases have been found to convert starch and glycogen, in part, to products other than hemiacetal-bearing entities (maltose, maltodextrins, etc.)—hitherto, the only products obtained from natural α-glucans by α-amylolysis. Glycosides of maltosaccharides were synthesized by purified α-amylases acting on starch or bacterial glycogen in the presence of p-nitrophenyl α- or β-d-glucoside. From a digest with crystallized B. subtilis var. amyloliquefaciens α-amylase, containing 4 mg/ml of [14C]glycogen and 40 mmp-NP β-d-glucoside, three pairs of correspondingly labeled glycosides and sugars were recovered: p-NP α-d-[14C]glucopyranosyl (1 → 4) β-d-glucopyranoside, and [14C]glucose; p-NP α-[14C]maltosyl (1 → 4) β-d-glucopyranoside, and [14C]maltose; p-NP α-[14C]maltotriosyl (1 → 4) β-d-glucopyranoside, and [14C]maltotriose. The three glycosides accounted for 11.4% of the [14C]glycogen donor substrate; the three comparable sugars, for 30.4%; higher maltodextrins, for 58.2%. Calculations based on the molar yields of all reaction products show that [14C]glycosyl moieties were transferred from donor to p-NP β-d-glucoside with a frequency of 0.234 relative to all transfers to water. This is a very high value considering the minute molar ratio (0.0007) of β-d-glucoside-to-water concentration. Less striking but similar findings were obtained with cryst. hog pancreatic and Aspergillus oryzae α-amylases. The results extend earlier findings (Hehre et al., Advan. Chem. Ser. (1973) 117, 309) in showing that α-amylases have a substantial capacity to utilize the C4-carbinols of certain d-glucosyl compounds as acceptor sites.  相似文献   

9.
We describe herein the practical post-modification synthesis of oligodeoxynucleotides (ODNs) containing 4,7-diaminoimidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidine nucleoside (ImNN). Since the ImNN nucleoside unit possessing tribenzoyl groups on its exocyclic amino groups as the protecting group was quite unstable under acidic conditions, cleavage of its glycosidic linkage in ODN has been suggested throughout the conditions of solid-phase synthesis. As an alternative approach, we investigated a post-modification synthesis of the desired ODNs containing the ImNN unit. Starting with protected 4-amino-7-chloro-1-(2-deoxy-β-d-ribofuranosyl)imidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidine derivative 1, conversion into the corresponding phosphoramidite unit was examined. The p-bromobenzoyl group (p-BrBz) was the best protecting group of 4-amino group of 1 to give the phosphoramidite unit 9 for the post-modification synthesis. After carrying out the ODN synthesis linked to the controlled pore glass (CPG) support, the support was treated with ammonium hydroxide at 55 °C to remove the protecting groups, detach the ODN form the CPG support, and convert the 7-chloro group into a desired amino group. As a result, the desired ODNs containing ImNN were obtained in good yield.  相似文献   

10.
Carboxy-p-fluorosulfonyl[14C]benzoyl-5′-adenosine has been synthesized with the radiolabel ultimately derived from carboxy-p-amino[14C]benzoic acid by a synthetic route employing four reaction steps. Starting with 1 mmol of p-amino[14C]benzoic acid, p-fluorosulfonyl[14C]benzoyl-5′-adenosine is obtained with an overall yield of 25–30%.  相似文献   

11.
α-Bungarotoxin (α-Bgt), an α-neurotoxin, has been 14C-methylated by treatment with [14C]formaldehyde following NaCNBH3 reduction. The methylation rate is fast (about 84% methylation in 15 min), with 12 methyl groups incorporated per mole of α-Bgt or a mean of 1.7 methyl groups per available amine residue. The specific activity of α-[14C]Bgt is 768 mCi/mmol. Unlike most of the reported chemical modifications of α-neurotoxins, involving a high decrease of the toxin activity after modification, α-[14C]Bgt retains 100% of its unmodified ability to bind to both isolated acetylcholine receptor (AcChR) and AcChR-enriched membrane fragments prepared from Torpedo californica. This lysyl residue modification does not perturb the toxin binding activity, probably, because the net positive charges of the ?-amino groups and amino-terminal residue remain unaltered. 14C-Methylated α-Bgt appears better suited than 125I-α-Bgt for use in AcChR binding studies because of the longer half-life of the isotope, and the apparent high uniformity of labeling of the toxin preparations.  相似文献   

12.
Abstract— A 100,000 g supernatant fraction from rat brain that was passed through a column of Sephadex G-25-40 was able, after addition of some factors, to incorporate [I4C]arginine (apparent Km= 5 μM) and [14C]tyrosine (apparent Km= 20 μM) into its own proteins. The factors required for the incorporation of [14C]arginine were: ATP (optimal concentration = 0-25-2 μM) and Mg2+ (optimal concentration 5 mM). For the incorporation of [I4C]tyrosine the required factors were: ATP (apparent Km= 0-75 μM), Mg2+ (optimalconcentration 8-16 mM) and K+ (apparent Km= 16 mM). Addition of 19 amino acids did not enhance these incorporations. Optimal pHs were: for [14C]arginine and [14C]tyrosine, respectively, 7-4 and 7-0 in phosphate buffer and 7–9 and 7-3-8-1 in tris-HCl buffer. Pancreatic ribonuclease abolished the incorporation of [14C]arginine but had practically no effect in the incorporation of [14C]tyrosine. Furthermore, [14C]arginyl-tRNA was a more effective donor of arginyl groups than [14C]arginine, whereas [14C]tyrosyl-tRNA was considerably less effective than [14C]tyrosine. The incorporations of [14C]arginine and [14C]tyrosine into brain proteins were from 25- to 2000-fold higher than for any other amino acid tested (12 in total). In brain [14C]arginine incorporation was higher than in liver and thyroid but somewhat lower than in kidney. In comparison to brain, the incorporation of [14C]tyrosine was negligible in liver, thyroid or kidney. Kinetic studies showed that the macromolecular factor in the brain preparation was complex. The protein nature of the products was inferred from their insolubilities in hot TCA and from the action of pronase that rendered them soluble. [14C]Arginine was bound so that its a-amino group remained free. Maximal incorporation of [14C]tyrosine in brain of 30-day-old rats was about one-third of that in the 5-day-old rat. The changes with postnatal age in the incorporation of [14C]arginine were not statistically significant.  相似文献   

13.
Angiosperms possess a retaining trans-α-xylosidase activity that catalyses the inter-molecular transfer of xylose residues between xyloglucan structures. To identify the linkage of the newly transferred α-xylose residue, we used [Xyl-3H]XXXG (xyloglucan heptasaccharide) as donor substrate and reductively-aminated xyloglucan oligosaccharides (XGO–NH2) as acceptor. Asparagus officinalis enzyme extracts generated cationic radioactive products ([3H]Xyl·XGO–NH2) that were Driselase-digestible to a neutral trisaccharide containing an α-[3H]xylose residue. After borohydride reduction, the trimer exhibited high molybdate-affinity, indicating xylobiosyl-(1→6)-glucitol rather than a di-xylosylated glucitol. Thus the trans-α-xylosidase had grafted an additional α-[3H]xylose residue onto the xylose of an isoprimeverose unit. The trisaccharide was rapidly acetolysed to an α-[3H]xylobiose, confirming the presence of an acetolysis-labile (1→6)-bond. The α-[3H]xylobiitol formed by reduction of this α-[3H]xylobiose had low molybdate-affinity, indicating a (1→2) or (1→4) linkage. In NaOH, the α-[3H]xylobiose underwent alkaline peeling at the moderate rate characteristic of a (1→4)-disaccharide. Finally, we synthesised eight non-radioactive xylobioses [α and β; (1↔1), (1→2), (1→3) and (1→4)] and found that the [3H]xylobiose co-chromatographed only with (1→4)-α-xylobiose. We conclude that Asparagus trans-α-xylosidase activity generates a novel xyloglucan building block, α-d-Xylp-(1→4)-α-d-Xylp-(1→6)-d-Glc (abbreviation: ‘V’). Modifying xyloglucan structures in this way may alter oligosaccharin activities, or change their suitability as acceptor substrates for xyloglucan endotransglucosylase (XET) activity.  相似文献   

14.
A system for radioactive labeling of compounds of biological interest that, due to their low electronic density, cannot be labeled by the standard iodination techniques is described. Using p-nitroanisole as a model, we have prepared 2-[125I]iodo-4-nitroanisole by treatment with thallium trifluoroacetate, with later displacement of the thallium by iodide according to A. McKillop et al. (J. Amer. Chem. Soc.93, 4841–4844 (1970)). The labeled iodonitroanisole has been used as a photoactive reagent to label a protein (bovine serum albumin), showing that under the irradiation conditions used, the label is incorporated into the polypeptide mainly through modification of ?-amino groups of the lysine residues.  相似文献   

15.
The potentiometric acid-base titration curve of fully protonated lysozyme at ionic strengths of 0.10 and 1.0 m has been performed. The stoichiometry and the pKa values of each titratable group have been determined through the linearization of titration curves. Two types of carboxylic groups with pKa values of 3.76 and 5.02, the imidazole group with pKa 7.37 and the amine group with pKa 9.63, have been identified at an ionic strength of 0.10 m at 25.0°C. The number of titratable groups found per mole of protein has been 5.12 and 5.60 for the two types of carboxylic groups, 1.13 for the imidazole group, and 3.19 for the amino groups. The endpoint of the titration of the protein obtained by this method accords quite well with the endpoint obtained by the use of Gran function applied to the excess of strong base.  相似文献   

16.
Adult rat brain capillaries were isolated by a simplified procedure and showed an enrichment of the marker enzyme, γ-glutamyltranspeptidase. The uptake of [35S]cystine at 37°C by this preparation can be divided into two components, a sodium- and energy-dependent transport process for the free amino acid pool, with an apparent Km of 36 μm , and a binding process, with an apparent Km of 1.13 mm . Chemical analysis of the amino acid pool indicates that cystine is the major form of intracapillary 35S. Cystine transport was not inhibited by lysine, but glycine, α-methylaminoisobutyric acid and β-2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid were inhibitory to a small extent.  相似文献   

17.
The absorption of lysine, arginine, phenylalanine and methionine by Taenia crassiceps larvae is linear with respect to time for at least 2 min. Arginine uptake occurs by a mediated system and diffusion, and arginine, lysine and ornithine (in order of decreasing affinity) are completely competitive inhibitors of arginine uptake. The basic amino acid transport system has a higher affinity for l-amino acids than d-amino acids, and blocking the α-amino group of an amino acid destroys its inhibitory action. Phenylalanine uptake by T. crassiceps larvae is inhibited in a completely competitive fashion by serine, leucine, alanine, methionine, histidine, phenylalanine, tyrosine and tryptophan (in order of increasing affinity). Methionine apparently binds non-productively to the phenylalanine (aromatic amino acid-preferring) transport system. l-methionine uptake by larvae is inhibited more by d-alanine and d-valine than by their respective l-isomers, while d- and l-methionine inhibit l-methionine uptake equally well. The presence of an unsubstituted α-amino group is essential for an inhibitor to have a high affinity for the methionine transport system. Uptake of arginine, phenylalanine and methionine is Na+-insensitive, and both phenylalanine and methionine are accumulated by larvae against a concentration difference in the presence or absence of Na+. Arginine accumulation is precluded by its rapid metabolism to proline, ornithine and an unidentified compound.  相似文献   

18.
Oxidized cytochrome c is known to undergo a restricted conformational refolding of its haem area at around pH 9. Methionine 80, the sixth ligand of the ferric haem iron in the biologically active neutral conformational state, is replaced by a new strong-field ligand in the biologically inactive alkaline state of the molecule. It had been proposed that a lysine residue, possibly lysine 79. is the new haem ligand.We have tested this proposition by a more direct approach than hitherto employed, namely by measuring the relative chemical reactivity of lysines in the oxidized eytochrome c and in fragment 66–80 cut out of the native molecule. The relative rates of acetylation of lysine 79, measured between pH 7 and pH 11, are virtually identical in the intact molecule and in the haem-free fragment 66–80. Similarly, the rates are also the same for the amidination reaction with isethionylacetimidate. When the relative rates of acetylation and amidination of lysines 72 + 73 were compared there was again no significant difference between the intact molecule and fragment 66–80. These results contradict the involvement of any of the three lysines in the alkaline isomerization, as a haem-bound ?-amino group would be much less reactive than its freely accessible counterpart in fragment 66–80.To corroborate the above finding, the pK value and absolute rate constant of acetylation of lysine 79 were determined and compared with the respective values for lysines 39 and 60. The latter two residues are on the side opposite to the haem pocket and hence unable to bind to the haem iron.The three pK values and rate constants k obey the Brønsted relationship: log κ = α + βpK with β = 0.48, a value characteristic of the acetylation of freely accessible primary amino groups.Taken together, these results oppose an ?-amino: haem iron co-ordination in the alkaline state of oxidized eytochrome c.  相似文献   

19.
Activated folate formed by reaction of folic acid and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide irreversibly inhibits the folate transport system of Lactobacillus casei. Complete inhibition of both folate binding to the carrier protein and folate transport was achieved by pretreatment of the cells at low temperature (4 °C) and at neutral pH with 200 nm activated folate. Fifty percent inhibition of binding and transport occurred at 35 and 40 nm activated folate, respectively. Specificity was demonstrated by the fact that excess nonactivated folate added during the pretreatment step afforded complete protection of the binding protein against inhibition, and that activated folate had no effect on the binding or transport of thiamine. Rapid measurements at 4 °C were employed to show that, prior to the appearance of irreversible inhibition, activated folate (Ki = 15 nM) interacted reversibly with the binding site for folate (Kd = 0.8 nM). Cells treated with activated [3H]folate incorporated 1 mol of folate per mole of binding protein. Purification of the labeled protein followed by digestion with Pronase led to the isolation of a compound identified as ?-N-folyl lysine. The ?-amino group of a lysyl residue thus appears to be the nucleophilic group at the binding site that reacts with activated folate.  相似文献   

20.
Various modified substrates of lysine monooxygenase were examined to determine whether they were oxygenated or oxidized. Among various methyllysines tested, N?- and δ-methyllysine underwent predominantly an oxygenative decarboxylation, producing the corresponding acid amides, while γ-methyllysine underwent predominantly an oxidative deamination with an α-keto acid as the reaction product. β-Methyllysine was inactive as substrate. All four methyllysines decreased the cooperativity of the enzyme with the normal substrate, lysine. Furthermore, lysine oxygenation was competitively inhibited by all of them except for β-methyllysine, which was much less inhibitory than the other methyllysines. Other analogs with a chloro or hydroxyl group at either the δ or the γ position were both oxygenated and oxidized. Analogs with a modified carboxyl or α-amino group were inactive as substrates.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号