2-氯汞-4-硝基苯酚对人肌肌酸激酶巯基的修饰

本文用含汞试剂MNP修饰人肌肌酸激酶,结果表明,人肌肌酸激酶有6个可反应巯基。MNP首先修饰了一对与活力无关的非必需巯基,增大MNP摩尔比,则进一步修饰另外四个与活性有关的巯基。修饰酶的差吸收光谱、荧光光谱表明这三对巯基的微环境各不相同。其中第二对巯基很可能是位于活性部位的必需巯基,而第三对巯基则是由于第二对巯基,也就是必需巯基,被修饰后,微区构象发生改变而暴露出来的。比较MNP修饰人肌肌酸激酶、鸡胸脯肌肌酸激酶、兔肌肌酸激酶的结果,探讨了MNP对肌酸激酶的修饰以及人肌肌酸激酶可反应巯基的化学微环境。     

The tyrosyl residues in creatine kinase. Modification by iodine.

The effect of the iodination of tyrosyl residues in creatine kinase from rabbit muscle has been investigated at alkaline pH after reversible masking of the reactive thiol groups. The conversion of 4-5 tyrosyl residues to monoiodotyrosines as measured by spectrotitration and by radioactive iodine labelling resulted in almost total loss of enzymic activity. The modified enzyme was unable to bind its nucleotide substrates but no significant conformational change was revealed by optical rotatory dispersion or Stokes radius measurements. However, change in the reactivity of some non-essential thiol groups, presumably those located near the active thiol groups, was observed.     

Modification of creatine kinase by S-nitrosothiols: S-nitrosation vs. S-thiolation

Creatine kinase is reversibly inhibited by incubation with S-nitrosothiols. Loss of enzyme activity is associated with the depletion of 5,5'-dithiobis (2-nitrobenzoic acid)-accessible thiol groups, and is not due to nitric oxide release from RSNO. Full enzymatic activity and protein thiol content are restored by incubation of the S-nitrosothiol-modified protein with glutathione. S-nitroso-N-acetylpenicillamine, which contains a more sterically hindered S-nitroso group than S-nitrosoglutathione, predominantly modifies the protein thiol to an S-nitrosothiol via a transnitrosation reaction. In contrast, S-nitrosoglutathione modifies creatine kinase predominantly by S-thiolation. Both S-nitroso-N-acetylpenicillamine and S-nitrosoglutathione modify bovine serum albumin to an S-nitroso derivative. This indicates that S-thiolation and S-nitrosation are both relevant reactions for S-nitrosothiols, and the relative importance of these reactions in biological systems depends on both the environment of the protein thiol and on the chemical nature of the S-nitrosothiol.     

The role of thiol groups in the structure and mechanism of action of arginine kinase

1. A detailed study of the reaction of iodoacetamide with arginine kinase has been carried out. 2. The enzyme contains five reactive thiol groups per 37000g. of protein, all of which can be alkylated. 3. Below pH8.5 loss of activity is substantially independent of pH and can be correlated with the alkylation of a single pH-independent thiol. 4. One catalytic site per enzyme molecule is inferred. 5. The progress curves of the alkylation reaction are polyphasic and reveal a pH-and time-dependent sequential release of thiols which is dependent upon the alkylation of the first pH-independent thiol. This is supported by electrophoretic investigations. 6. Comparison of alkylation rate and rate of loss of activity suggests that two thiol groups are not essential for catalytic activity. Variability in enzyme preparations with respect to alkylation rate appears to be associated with these two groups. 7. A complex protection pattern is revealed by the effects of various substrate combinations on rates of alkylation and of loss of activity. It is inferred that two thiol groups participate in conformational changes and nucleotide interactions. 8. Comparison with creatine kinase suggests a fundamentally similar catalytic mechanism, although for arginine kinase certain additional restrictions are necessary because of the protection observed with nucleotide substrates.     

An essential tryptophan residue for rabbit muscle creatine kinase

The tryptophan residues in rabbit muscle creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) have been modified by dimethyl(2-hydroxy-5-nitrobenzyl) sulfonium bromide after reversible protection of the reactive SH groups. The modification of two tryptophan residues as measured by spectrophotometric titration leads to complete loss of enzymatic activity. Control experiments show that reversible protection of the reactive SH groups as S-sulfonates followed by reduction results in nearly quantitative recovery of enzyme activity. The presence of a 410 nm absorption maximum and the decrease in fluorescence of the modified enzyme indicate the modification of tryptophan residues. At the same time, SH determinations after reduction of the modified enzyme show that the reagent has not affected the protected SH groups. Quantitative treatment of the data (Tsou, C.-L. (1962) Sci. Sin. 11, 1535 1558) shows that among the tryptophan residues modified, one is essential for its catalytic activity. The presence of substrates partially protects the modification of tryptophan residues as well as the inactivation, suggesting that the essential tryptophan residue is situated at the active site of this enzyme.     

The presence of reactive SH groups in the enzymatically active dicyano derivative of creatine kinase

It has been shown that the active dicyano derivative of creatine kinase (ATP:creatine N-phosphotransferase) obtained by cyanolysis of the 5,5'-dithiobis(2-nitrobenzoic acid)-modified and inactivated enzyme contains, as does the native enzyme, two reactive SH groups. Modification of these two SH groups leads to complete inactivation of the dicyano enzyme. Reaction with 4-iodoacetamido-1-naphthol introduces fluorescent labels at these reactive SH groups of the native and the dicyano enzymes. Following tryptic digestion, the respective fluorescent-labelled peptides have been separated by HPLC and the amino acid composition analysis of these peptides has shown that they are consistent with the sequence of the peptide segment containing the active-site SH of Cys-282 of creatine kinase for both the native and the dicyano enzymes, showing that the active SH groups are free in the dicyano enzyme. Upon mild denaturation in 3 M urea, it can be shown that two of the SH groups partially buried in the native enzyme have been cyanylated in the dicyano enzyme. The two reactive SH groups are therefore essential for the activity of creatine kinase and the two cyanylated SH groups are internal groups which probably contributes partially to the stabilization of an active conformation of the enzyme molecule.     

ATP binding site of mitochondrial creatine kinase. Affinity labelling of Asp-335 with C1RATP

The ATP binding site of mitochondrial creatine kinase from chicken heart has been studied by modifying the purified enzyme with a 14C-labelled ATP analogue, C1RATP, in which the reactive label was covalently bound to the gamma-phosphate group of ATP. The modified enzyme was digested by pepsin, and a single radioactive nonapeptide was isolated by HPLC. Amino acid analysis and direct sequence determination revealed that the isolated peptide corresponds to amino acids 335-343 within the C-terminal region of Mi-CK, this peptide being highly preserved throughout evolution. Asp-335 is very likely the site of modification by C1RATP. The specificity of the ATP analogue for the active site of creatine kinase was demonstrated by the inhibition of the enzymatic activity of Mi-CK by C1RATP and by the prevention of this inhibition bij ADP.     

The reactivity of thiol groups and the subunit structure of aldolase

1. Seven unique carboxymethylcysteine-containing peptides have been isolated from tryptic digests of rabbit muscle aldolase carboxymethylated with iodo[2-(14)C]acetic acid in 8m-urea. These peptides have been characterized by amino acid and end-group analysis and their location within the cyanogen bromide cleavage fragments of the enzyme has been determined. 2. Reaction of native aldolase with 5,5'-dithiobis-(2-nitrobenzoic acid), iodoacetamide and N-ethylmaleimide showed that a total of three cysteine residues per subunit of mol.wt. 40000 were reactive towards these reagents, and that the modification of these residues was accompanied by loss in enzymic activity. Chemical analysis of the modified enzymes demonstrated that the same three thiol groups are involved in the reaction with all these reagents but that the observed reactivity of a given thiol group varies with the reagent used. 3. One reactive thiol group per subunit could be protected when the modification of the enzyme was carried out in the presence of substrate, fructose 1,6-diphosphate, under which conditions enzymic activity was retained. This thiol group has been identified chemically and is possibly at or near the active site. Limiting the exposure of the native enzyme to iodoacetamide also served to restrict alkylation to two thiol groups and left the enzymic activity unimpaired. The thiol group left unmodified is the same as that protected by substrate during more rigorous alkylation, although it is now more reactive towards 5,5'-dithiobis-(2-nitrobenzoic acid) than in the native enzyme. 4. Conversely, prolonged incubation of the enzyme with fructose 1,6-diphosphate, which was subsequently removed by dialysis, caused an irreversible fall in enzymic activity and in thiol group reactivity measured with 5,5'-dithiobis-(2-nitrobenzoic acid). 5. It is concluded that the aldolase tetramer contains at least 28 cysteine residues. Each subunit appears to be identical with respect to number, location and reactivity of thiol groups.     

The amino acid sequence of the peptide containing the thiol group of creatine kinase from normal and dystrophic chicken breast muscle. Comparison of some of the immunological properties of the antibodies developed in rabbits against these enzymes

The major (14)C-labelled peptides from creatine kinase from normal and dystrophic chicken muscle obtained by carboxymethylating the reactive thiol groups with iodo[2-(14)C]acetic acid and digestion with trypsin were purified by ion-exchange chromatography on Dowex-50 (X2) and by paper electrophoresis. The chromatographic characteristics of the (14)C-labelled peptides, their electrophoretic mobilities at pH6.5, and their amino acid compositions were identical for the two enzymes. The sequence of amino acids around the essential thiol groups of creatine kinase from normal and dystrophic chicken muscle was shown to be Ile-Leu-Thr-CmCys-Pro-Ser-Asn-Leu-Gly-Thr-Gly-Leu-Arg (CmCys, carboxymethylcysteine). This sequence is almost identical with that for the creatine kinases in human and ox muscle and bovine brain and is very similar to that of arginine kinase from lobster muscle. Antibodies to the enzymes were raised in rabbits and their reaction with the creatine kinase from normal and dystrophic muscles in interfacial, immunodiffusion and immunoelectrophoretic experiments was studied. The cross-reaction between normal muscle creatine kinase and antisera against the dystrophic muscle enzyme (or vice versa) observed by immunodiffusion and by immunoelectrophoretic experiments further suggests that the enzymes from normal and dystrophic chicken muscle are similar in structure. The results of the present study, the identical amino acid sequence of the peptides containing the reactive thiol group from both the normal and dystrophic chicken muscle enzymes and the immunological similarities of the two enzymes are in accord with the similarity of the two enzymes observed by Roy et al. (1970).     

Immobilized bacterial luciferase for microscale analysis of creatine kinase activity

The commercially available bacterial luciferase: oxidoreductase system obtained from Vibrio fischerii has been immobilized in a bovine serum albumin (BSA) gel. The gel was cut in the shape of a disc and held to the bottom of a reaction cell, gel upwards. The immobilized enzyme gels are stable, reusable and easily cleaned of spent reagents. NADH and NADPH have been assayed down to nanomolar concentrations, although with an error of ± 15%. The system has been coupled to an NADPH-producing commercial assay for creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) activity. The kinetic assay gives a linear reaction rate vs. creatine kinase activity plot in the clinically important range.     

DTNB对人肌肌酸激酶不可逆抑制作用的研究

本文研究了DTNB对人肌肌酸激酶的不可逆抑制作用.研究结果表明,与兔肌肌酸激酶不同,人肌肌酸激酶分子中有四个可反应SH.用邹承鲁作图法定量处理结果表明,在这四个可反应的SH中,有一个快反应SH,两个慢反应SH且这两个慢反应SH是酶的必需基团,此外还有一个反应很慢的SH.用邹承鲁提出的在抑制剂存在条件下,酶活性不可逆改变动力学方法,测定了一系列动力学常数,并对DTNB的作用机制进行了探讨.     

Purification and properties of adenosine triphosphate–creatine phosphotransferase from muscle of the dogfish Scylliorhinus canicula

1. Creatine kinase occurs in high concentration in the soluble proteins of dogfish muscle. A fourfold purification gives essentially pure enzyme but with a low specific activity. This appears to be a property of the native enzyme and not a result of the isolation procedures used. 2. The amino acid composition is similar to that of other phosphagen kinases, but the enzyme differs from mammalian creatine kinases in having four thiol groups readily reactive towards 5,5′-dithiobis-(2-nitrobenzoic acid). Titration of two thiol groups is accompanied by almost complete loss of activity. The remaining two thiol groups react at different rates, suggesting that modifying the third thiol group affects the reactivity of the fourth thiol group. 3. The enzyme is markedly protected against inactivation by iodoacetamide by MgATP or MgADP. Addition of creatine to MgADP decreases protection, but the further addition of Cl⁻ restores protection to the original value. The quaternary MgADP–creatine–enzyme–nitrate complex protects very strongly as is found for the rabbit enzyme. The involvement of the conformational state of the enzyme in such effects is discussed. 4. Creatine kinase from both dogfish and rabbit is equally sensitive to urea denaturation. Urea protects the dogfish enzyme by about 9% against inhibition by iodoacetamide. 5. The formation of a hybrid between the dogfish and rabbit enzymes in vitro has been demonstrated. 6. At high substrate concentrations the dogfish enzyme shows apparent ordered kinetics. The effect of temperature on V_max. and the Michaelis constants for MgATP and creatine were determined. These and changes in the apparent activation energy suggest that limited adaptation has occurred commensurate with physiological need.     

o-Quinones formed in plant extracts. Their reaction with bovine serum albumin

1. The reactions between chlorogenoquinone, the o-quinone formed during the oxidation of chlorogenic acid, and bovine serum albumin depend on the ratio of reactants. 2. When the serum albumin is in excess, oxygen is not absorbed and the products are colourless. This reaction probably involves the thiol group of bovine serum albumin; it does not occur with bovine serum albumin which has been treated with p-chloromercuribenzoate, iodoacetamide or Ellman's reagent. 3. When bovine serum albumin reacts with excess of chlorogenoquinone, oxygen is absorbed and the products are red. The red colour is probably formed by reaction of the lysine in-amino groups of bovine serum albumin, as it is prevented by treating the protein with formaldehyde, succinic anhydride or O-methylisourea. 4. Bovine serum albumin modified by a 1.5-fold (BSA-Q) and a fivefold (BSA-Q2) excess of chlorogenoquinone were separated by chromatography on DEAE-Sephadex A-50, and some of their properties observed. 5. Reaction of BSA-Q2 with fluorodinitrobenzene suggests that the terminal alpha-amino group, as well as lysine in-amino groups, are combined with chlorogenoquinone.     

Photonic immobilization of BSA for nanobiomedical applications: creation of high density microarrays and superparamagnetic bioconjugates

Light assisted molecular immobilization has been used for the first time to engineer covalent bioconjugates of superparamagnetic nanoparticles and proteins. The technology involves disulfide bridge disruption upon UV excitation of nearby aromatic residues. The close spatial proximity of aromatic residues and disulfide bridges is a conserved structural feature in proteins. The created thiol groups bind thiol reactive surfaces leading to oriented covalent protein immobilization. We have immobilized a model carrier protein, bovine serum albumin, onto Fe(3)O(4)@Au core-shell nanoparticles as well as arrayed it onto optically flat thiol reactive surfaces. This new immobilization technology allows for ultra high dense packing of different bio-molecules on a surface, allowing the creation of multi-potent functionalized active new biosensor materials, biomarkers identification and the development of nanoparticles based novel drug delivery system.     

Kinetics of inactivation of creatine kinase during modification of its thiol groups

Kinetics of inactivation and modification of the reactive thiol groups of creatine kinase by 5,5'-dithiobis(2-nitrobenzoic acid) or iodoacetamide have been compared, the former by following the substrate reaction in presence of the inactivator [Wang, Z.-X., & Tsou, C.-L. (1987) J. Theor. Biol. 127, 253]. The microscopic constants for the reaction of the inactivators with the free enzyme and with the enzyme-substrate complexes were determined. From the results obtained it appears that with respect to ATP both inactivators are noncompetitive whereas for creatine iodoacetamide is competitive but DTNB is not. The formation of the ternary complex protects against the inactivation by both DTNB and iodoacetamide. The inactivation kinetics is monophasic with both inactivators, but under similar conditions, the modification reactions in the presence of the transition-state analogue of creatine-ADP-Mg2+-nitrate show biphasic kinetics as also reported by Price and Hunter [Price, N.C., & Hunter, M.G. (1976) Biochim. Biophys. Acta 445, 364]. If the reactive ternary complex and the enzyme complexed with the transition-state analogue react in the same way with these reagents, the modification of one fast-reacting thiol group for each enzyme molecule leads to complete inactivation, indicating that the enzyme has to be in the dimeric state to be active.     

Brain adenosine 5′-triphosphate–creatine phosphotransferase. Purification, thiol group reactivity and the amino acid sequence around the reactive thiol groups

1. The purification of creatine kinase (ATP-creatine phosphotransferase, EC 2.7.3.2) from ox brain by a method that is quicker, simpler and gives much higher yields than other published procedures is described. 2. Stoicheiometric inhibition studies with iodoacetate showed that the enzyme, like that from muscle, has two reactive thiol groups that are essential for enzyme activity. 3. The amino acid sequence around the essential thiol groups was determined and found to be virtually identical with that in creatine kinases from rabbit and ox muscle, and very similar to that found in arginine kinase; the evolutionary significance of this is discussed. 4. The identification of DNS-amino acids on thin layers of silica gel was found to have, in many cases, distinct advantages over that on polyamide layers.     

Inhibition of kinase activity of the adrenal pyruvate dehydrogenase complex by the mitochondrial component

A component inhibiting the phosphorylation-linked inactivation of the adrenal pyruvate dehydrogenase complex in the presence of ATP was revealed during purification of the complex from bovine adrenal mitochondria. The degree of the kinase activity inhibition is greater at lower concentrations of ATP. It was assumed that the mitochondrial component screens the kinase active site or the phosphorylation sites of pyruvate dehydrogenase, thus limiting the ATP access to them. Proteins and lipids are incorporated into the component at a ratio 2:1, which is suggestive of its lipoprotein nature. The effect of the mitochondrial component on the kinase activity of the pyruvate dehydrogenase complex is somewhat specific and is unaffected by bovine serum albumin or blood serum lipoproteins.     

The recBC enzyme of Escherichia coli K12: premature cessation of catalytic activities in vitro and reactivation by potassium ions.

It is shown that in vitro the degradation of native and single-stranded DNA as well as the hydrolysis of ATP by purified recBC enzyme ceases 2-3 min after the start of the reaction. The presence of potassium ions (60-100 mM), bovine serum albumin (1 mg/ml) or protein from cell-free Escherichia coli extract (10 microgram/ml) prevents the cessation of the activity. Once the cessation has occurred, the activity of the enzyme can be completely restored by the addition of potassium ions, but not by bovine serum albumin. Sedimentation studies revealed that, in contrast to the active recBC enzyme, the 'silent' enzyme is no longer associated with substrate DNA of high molecular weight. On the basis of these results and other observations it is hypothesized that during the degradation of DNA in the absence of potassium ions or bovine serum albumin the recBC enzyme is subject to an alteration of its molecular conformation which results in an inactive form.     

Properties and reaction with iodoacetamide of adenosine 5′-triphosphate–creatinine phosphotransferase from human skeletal muscle. Further evidence about the role of the essential thiol group in relation to the mechanism of action

1. The purification of creatine kinase from human and monkey skeletal muscle by horizontal electrophoresis on Sephadex blocks is described. 2. The purified enzymes are shown to have similar chemical and kinetic properties to the rabbit muscle enzyme and a common mechanism is inferred. 3. Iodoacetamide has a similar apparent second-order inhibition constant with the human and rabbit enzymes, but the inhibition does not go to completion with the former. This is even more marked with the monkey enzyme, which has more reactive thiol groups, but inhibition is only about 50%. 4. Single substrates have little effect on the inhibition by iodoacetamide, but with the primate enzymes, in contrast with the rabbit enzyme, high concentrations of ADP-Mg(2+) plus creatine convert the essential thiol group from being pH-independent into one with a normal ionization. Low concentrations of ADP-Mg(2+) plus creatine first enhance the rate of inactivation, but cause protection as the reaction proceeds. These results are interpreted to indicate an activation of the thiol group on the subunit to which the substrates bind and a co-operatively induced decrease in the activity of the thiol group on the other subunit which lacks substrates. 5. The effects of a substrate equilibrium mixture on the rate of inhibition are essentially those of ADP-Mg(2+) plus creatine. 6. Since no substrate combination affords significant protection to the thiol group associated with the catalytic site to which the substrates are bound, it is concluded that any mechanism involving the thiol group in a direct participation in the transition-state complex of the catalytic reaction must be abandoned unless the transition state is only a small part of the time taken for one catalytic cycle.     

Immobilized bovine lactose synthase. A method of topographical analysis of the active site.

Bovine galactosyltransferase (UDPgalactose: D-glucose 4beta-galactosyltransferase, EC 2.4.1.22) was covalently coupled to Sepharose 4B by reaction at pH 5.0 with the activated mixed disulfide Sepharose-glutathione-2(5-nitropyridyl)-disulfide. The Sepharose-protein conjugate was presumably coupled via the unique highly reactive cysteine of those thiols on the bovine enzyme. The gel-bound N-acetyllactosamine and lactose synthase activity of about 0.4% was consistent with the affects of diffusion and the 90% activity reduction noted upon thiol modification of the dissolved enzyme. The residual lactose biosynthetic activity of the bound enzyme appeared possible only if the reactive thiol were physically distinct from the active site since the bulky Sepharose-glutathione group must not obscure the alpha-lactalbumin binding region.