The reactivity of the thiol groups of calf thymus deoxyribonucleohistone

The reactivities of the two cysteine thiol groups of calf thymus F3 histone were investigated using 5,5'-dithiobis-[2- nitrobenzoic acid], (DTNB). In isolated histone, both thiol groups were available for reaction. However, analysis of reaction profiles of native deoxyribonucleohistone, (DNH), in various solvent conditions, together with gel electrophoresis studies of DNH modified with DTNB, showed that only one of the thiol groups is normally modified by the reagent. If NaCl is present (above 1.OM) the other thiol group can also be modified. The reactivities of both groups were largely independent of the degree of DNH supercoiling and of the binding of F3 to the DNA.     

The reactivity and function of thiol groups in trout actin

1. Considerable differences were found between the rates and degrees of modification of native trout actin with iodo[2-(14)C]acetate and iodo[1-(14)C]acetamide. 2. With iodoacetate, G- and F-actin were both labelled in the N-terminal peptide only. This modification had little effect on the ability of the actin to polymerize. 3. Iodoacetamide labelled three cysteine residues in both G- and F-actin. The modified cysteine residues were identified from the position of the corresponding tryptic peptides on peptide ;maps'. 4. The modification had little effect on the ability of G-actin to polymerize, to bind ATP or to bind Ca(2+), or on the ability of F-actin to depolymerize. 5. It is concluded that the three cysteine residues present on the ;surface' of the native trout actin molecule have no direct role in the polymerization processes, the binding of ATP, or the binding of Ca(2+).     

A study of the subunit structure and the thiol reactivity of bovine liver uridine diphosphate glucose dehydrogenase

1. The amino acid analysis of UDP-glucose dehydrogenase is reported. 2. N-Terminal-group analysis indicates only one type of N-terminal amino acid, methionine, to be present. 3. Peptide ;mapping' in conjunction with the amino acid analysis indicates that the subunits of the enzyme are similar if not identical. 4. The various kinetic classes of thiol group were investigated by reaction with 5,5'-dithiobis-(2-nitrobenzoate). 5. NAD(+), UDP-glucose and UDP-xylose protect the two rapidly reacting thiol groups of the hexameric enzyme. 6. Inactivation of the enzyme with 5,5'-dithiobis-(2-nitrobenzoate) indicates the involvement of six thiol groups in the maintenance of enzymic activity. 7. The pH-dependence of UDP-xylose inhibition of the enzyme was investigated. 8. The group involved in the binding of UDP-xylose to the protein has a heat of ionization of about 33kJ/mol and a pK of 8.4-8.6. 9. It is suggested that UDP-xylose has a cooperative homotropic effect on the enzyme.     

Re-evaluation of the role of thiol groups in rabbit muscle aldolase A

Exposed thiol groups of rabbit muscle aldolase A were modified by 5,5'-dithiobis(2-nitrobenzoic) acid with concomittant loss of enzyme activity. When 5-thio-2-nitrobenzoate residues bound to enzyme SH groups were replaced by small and uncharged cyanide residues the enzyme activity was restored by more than 50%. The removal of a bulky C-terminal tyrosine residue from the active site of aldolase A resulted in enzyme which was inhibited by 5,5'-dithiobis(2-nitrobenzoic) acid only by 50% and its activity was nearly unchanged after modification of its thiol groups with cyanide. The results obtained show directly that rabbit muscle aldolase A does not possess functional cysteine residues and that the inactivation of the enzyme caused by sulfhydryl group modification reported previously can be attributed most likely to steric hindrance of a catalytic site by modifying agents.     

Nucleotide in monomeric actin regulates the reactivity of the thiol groups

A new thiol reagent, 2,4-dinitrophenyl glutathionyl disulfide, allowed the characterization of four thiol groups in monomeric actin by stoichiometric reaction. The number of thiol groups exposed to the reagent was found to depend on the nucleotide bound. In the absence of ATP, G-actin exposed four thiol groups ( G4s ). On the addition of ATP (1 equiv), three of them were shielded. The resulting actin with one thiol group exposed ( G1s ) is the form of monomeric actin normally produced by depolymerization of F-actin in buffers containing ATP. G1s is stable over hours, while G4s , i.e., monomeric actin in ATP-free solution, is not. This must be concluded from the fact that the shielding effect of thiol groups induced by addition of ATP was lost within ca. 30 min probably due to denaturation of G4s to G4s *. Therefore, denaturation of monomeric actin must be understood in terms of loss of thiol shielding, rather than by oxidation of the thiol groups. Addition of equimolar amounts of Ca2+ significantly retarded the denaturation process. ADP (50 equiv) shielded only ca. two of the four thiol groups but, similar to ATP, protected actin from denaturation. Three ATP analogues (10 equiv) were tested but had no shielding effect. In the presence of these analogues actin ( G4s ) rapidly denatured (to G4s *) as in the absence of added nucleotides. It was shown that the thiol-shielding activity and the protective capacity of a nucleotide are interrelated with its binding capability to monomeric actin. G1s was found to be polymerizable as was G approximately 2s on the addition of ATP. No polymerization could be detected for G4s or G4s *.(ABSTRACT TRUNCATED AT 250 WORDS)     

Possible role of thiol groups in the abnormal kinetics of aldolase in hereditary fructose intolerance

In Hereditary Fructose Intolerance, the apparent Km of liver aldolase for Fructose-1-Phosphate was shown to be very high. The addition of β mercaptoethanol normalizes this low affinity. It seems that the primary defect of this genetic disease involves directly or indirectly the thiol groups of aldolase B.     

Topography of Escherichia coli ribosomal proteins. The order of reactivity of thiol groups

1. 30S and 50S ribosomal subunits of Escherichia coli were treated with N-[2,3-(14)C]-ethylmaleimide and iodo[(14)C]acetamide. 2. The proteins in the native subunits which reacted with the reagents were S1,double dagger S2, S12, S13, S18, S21, L2, L5, L6, L10, L11, L15, L17, L20, L26+28 and L27. 3. Several proteins, such as S1, S12, S14, S18, L2, L6, L10, L11 and either L26 or 28, had thiol groups in an oxidized form and reacted to a greater extent after reduction with beta-mercaptoethanol or dithiothreitol. 4. The total number of thiol groups in 30S and 50S subunits was determined as 16-17 and 26-27 respectively. The total number of thiol groups in each ribosomal protein was also determined. 5. The reaction of 30S and 50S subunits with iodoacetamide under several different conditions established the order of reactivity of thiol groups.     

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.     

Probe of beta-galactosidase structure with iodoacetate. Differential reactivity of thiol groups in wild-type and mutant forms of beta-galactosidase.

Carboxymethylation with 14 C-labeled iodoacetate of cysteine residues in wild-type beta-galactosidase from Escherichia coli and in a defective beta-galactosidase from deletion mutant strain M15 was investigated in order to determine accessible positions in the tetrameric wild-type form and the dimeric mutant M15 protein. The extent of carboxymethylation, the effects on biological activity, antibody activation, physical stability, and the labeling of particular residues were studied. The results distinguish three groups of spatial relationships for cysteine residues in the protein, define possible regions for subunit interactions, and confirm that no cysteine residue is specifically involved in catalysis. Residue 1019 and to a lesser extent 498 are accessible in the tetrameric protein and probably represent exposed areas. In the M15 protein, these two, and three additional residues, at 76,387 and 600, were found to react significantly with reagent. One or more of the latter are suggested to be in the dimer-dimer interface. Complementation and activation by antibody are inhibited by carboxymethylation of M15 protein.     

The thiol groups of the Folch-Pi protein from bovine white matter. Exposure, reactivity and significance.

The number and the reactivity of accessible thiol groups of the Folch-Pi apoprotein and proteolipid (50% of myelin proteins) were studied, by using a specific thiol-disulphide interchange reaction, in connection with the known solubility of this protein in organic and aqueous solvents. The high reactivity of 2,2'-dipyridyl disulphide towards thiol groups leads to the titration of 4.8 mol of SH groups/mol of protein (Mr 30000) in alkaline and acidic chloroform/methanol (2:1, v/v). Unlike previous findings, this value was consistently found from batch to batch and remained stable with time. In the proteolipid 1 mol of SH groups/mol was not accessible as compared with the apoprotein. In aqueous solvents, a similar number of 4.4 mol of SH groups/mol was also found. For the first time, kinetic studies carried out in chloroform/methanol discriminated between two classes of thiol groups. The reaction of 2 mol of SH groups/mol was characterized by apparent second-order rate constants whose values were 5-10-fold higher than those of the other class. Kinetic studies and cyanylation experiments in aqueous solvents also indicated the high reactivity of these thiol groups with Ellman's reagent. Together with kinetic results, studies on the stoichiometry of the interchange reaction of equimolar solutions of protein and disulphide indicate that these highly reactive thiol groups are near to each other in the amino acid sequence. The location of the thiol groups at the boundary between hydrophilic and hydrophobic domains of the Folch-Pi protein is suggested in connection with their possible structural and biological significance.