The role of various amino acid residues in the functioning of NADP-specific isocitrate dehydrogenase from bovine adrenal cortex cytoplasm

The modification of SH-groups in the native isocitrate dehydrogenase accessible to 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) is accompanied by the enzyme inactivation. Isocitrate rather than NADP and MnCl2 protects two SH-groups of the enzyme from modification by DTNB and attendant inactivation. The isocitrate dehydrogenase inactivation by DTNB obeys pseudofirst-order reaction kinetics. The number of DTNB-titrated sulphydryl groups does not change after the isocitrate dehydrogenase denaturation by sodium dodecyl sulphate. In the presence of manganese ions isocitrate and to a lesser extent NADP protect isocitrate dehydrogenase from the inactivation induced by 2,3-butanedione, a specific modifier of arginine residues. It has also been shown that the methylene blue-sensitized photoinactivation of the enzyme associated with the photooxidation of histidine residues decreases in the presence of NADP. These data provide evidence for an essential role of the SH-groups, arginine residues and, probably, histidine in the functioning of NADP-dependent isocitrate dehydrogenase from adrenal cortex.     

Secondary structure and functional groups of the active center of glutamine synthetase of pea chloroplasts

The circular dichroism spectra of glutamine synthetase (EC 6.3.1.2) from pea chloroplasts were recorded. Based on these spectra the percentage of alpha-helix sites, beta-structures, beta-bends and disordered sites of the polypeptide chain was calculated and was found equal to 23, 57, 1 and 23%, respectively. Data from protein photooxidation in the presence of methylene blue and the type of pH-dependence of pKm suggest that glutamate binding takes place on the imidazole ring of the histidine molecule. The inhibition of native glutamine synthetase by p-chloromercurybenzoate and the presence of free SH-groups in the enzyme molecule (approximately two SH-groups per monomer) suggest that these groups are the functional groups of the enzyme active center.     

Retinal-sensitized photo-oxidation of rhodopsin

Kinetics of retinal photosensitized initiation of radicals of sulfhydryl groups of cysteine and rhodopsin is investigated by spin trapping. Photooxidation of both systems is the result of free radical mechanism. Photooxidation of SH-groups proceeds both with singlet oxygen participation and by direct interaction of photosensitizer with the substrate. The rate constants of these reactions are measured. The rate constant with oxygen participation (K0 = 1.1 X 10(9) M-1 S-1) is higher than the one without oxygen (K = 2.5 X 10(8) M-1 S-1) correspondingly. The lifetime of retinal triplet state in photoreceptor membrane is tau = 4 X 10(-6) s.     

Presence of SH-groups and histidine in the active site of Chlorella glutamine synthetase]

The presence of two cysteine residues per each six monomers comprising the oligomer of Chlorella glutamine synthetase (E.C.6.3.1.2) is demonstrated using homogenous enzyme preparation. p-Chloromercuribenzoate (p-CMB) is found to inhibit glutamine synthetase activity, the degree of inhibition depending on the inhibitor concentration. The following enzyme reactivation by dithiotreitol (10(-2) M) was observed only when the enzyme was inactivated with 10(-5) M p-CMB under 15 min. preincubation. Preincubation of the enzyme with 10(-4) M p-CMB for 45 min. did not result in its reactivation. Gel filtration of glutamine synthetase treated with 10(-4) M p-CMB has revealed the dissociation of the enzyme into inactive monomers. Incubation of glutamine synthetase with p-CMB at various pH values, incubation after pre-treatment with urea and experiments with HgCl2 indicate the presence of free and masked inside the globula SH-groups in the enzyme molecule. Competitive character of the enzyme inhibition with p-CMB with respect to ATP indicates that SH-groups of the active site participate in the ATP binding, probably, as Mg-ATP or Mn-ATP complexes. Data on the estimation of ionization constant of glutamate-binding group and experiments on the effect of histidine photooxidation on the enzyme activity indicate the presence of histidine residue in the enzyme active site, which participates in glutamate binding.     

The content and types of sulfhydryl groups in muscle creatine kinase detected by their accessibility to different reagents

Using three independent methods tte amperometric titration, Boyer's method and the method of Ellman it is shown that rabbit muscle creatine kinase contains 11-12 SH-groups, 3-6 of which are easily oxidized by atmospheric oxygen to form S-S-bonds. It is found that creatine kinase has four types of SH-groups distinguished by their accessibility to different SH-reagents. The first type related to the enzymatic activity is detected by the Ellman method (2 SH-groups), the first and the second ones taken together--by the Boyer method (4 SH-groups), the first, second and third ones--by the method of amperometric titration (6 SH-groups), all the 4 types together--when detecting SH-groups after protein denaturation--by any of the above methods (8-12 SH-groups).     

A kinetic study of the reactive capabilities of xanthine oxidase sulfhydryl groups with regard to n-chlormercuribenzoate]

Kinetic characteristics for reactivity of SH-groups of milk xanthine oxidase were obtained under different conditions. Two types of SH-groups with rate constant values, differing by a factor of about 50, were found in a phosphate buffer at pH 7.0. The slow stage of reaction is followed by protein precipitation. The number of fast- (12) and slowly-reacting (60) groups were calculated from the kinetic data. The blocking of the fast-reacting groups occurs without loss of the enzyme activity. The values of activation energy for the fast- and slowly-reacting groups are 15 and 48 kcal/mol respectively. The formation of the enzyme-substrate complex stabilizes the enzyme molecule; the number of fast-reacting SH-groups and the rate constant values for both types of groups remain unchanged, whereas the number of slowly-reacting SH-groups markedly decreases (37). The values of activation energy for both types of SH-groups show no changes in the presence of substrate. Conformations of the enzyme in different denaturating solvents were characterized by a number of SH-groups, reacting with p-chloromercurybenzoate. 54 groups are exposed in solutions of groups exposed in 7.0-8.5 M urea solutions is 35-38. In all solvents studied the protein molecule is probably not completely unfolded, since the number of exposed SH-groups is less than the full number of SH-groups determined by the amino acid analysis. Only 42 SH-groups reacted with 5,5'-dithiobis-(2-nitrobenzoic acid) under the same conditions.     

Modification of the Cl−-activated arginine aminopeptidases from rat liver and human erythrocytes: A comparative study

The amino acid residues important for the catalytic activity of the Cl^?-activated arginine aminopeptidases from human erythrocytes and rat liver were studied using enzyme modification. The general inhibition characteristics were similar with both enzymes. Inactivation with 5,5′-dithiobis-(2-nitrobenzoic acid) revealed one essential SH-group per active enzyme unit in both aminopeptidases. l-Arginyl-l-phenylalanine and N-l-arginyl-2-naphthylamide protected the enzymes against inactivation by DTNB, the former substrate being more effective. The rat liver enzyme was more sensitive to DTNB than the erythrocyte enzyme. Titration with DTNB revealed only fast reacting SH-groups in rat liver APB (mean 7.8). The erythrocyte enzyme, however, revealed SH-groups which reacted fast with low concentrations of DTNB, while high concentrations of DTNB or SDS treatment were needed to reveal all enzyme SH-groups (mean 8.0). The presence of at least one essential imidazole group in the erythrocyte enzyme was indicated by photooxidation in the presence of methylene blue, as previously found with the rat liver enzyme (5., 22.). The pH dependence curves of both enzymes also supported the presence of SH- and imidazole groups at or near the active site. Thus, the functional groups identified were the same for both enzymes. Neither enzyme had essential COOH or arginyl groups and they did not contain any zinc. The absence of Zn suggests that the reaction mechanism recently presented by other authors, based on the presence of Zn in the active center, does not apply to the Cl^?-activated arginine aminopeptidases. Accordingly, this enzyme group cannot be classified to metallopeptidases.     

Change in the molecular properties of sarcoplasmic reticulum Ca-ATPase during modification of the membranes with cholesterol

The thiol reagent NBD-chloride (4-chloro-7-nitro-benzo-2-oxo-1,3-diazole) was used to determine the amount and reactivity of SH-groups of Ca-ATPase of rat skeletal muscle sarcoplasmic reticulum during hypercholesterolemia. Modification of membranes with cholesterol brought about a decrease in the total amount and reactivity of SH-groups at the cost of reduction of rapid SH-groups and decrease of the modification constant of these SH-groups. The masking effect of high concentrations of ATP on the reactivity of SH-groups in hypercholesterolemia was noticed. It is inferred that the reduced efficacy of Ca-pump work found under the same experimental conditions before is a consequence of the modification of sarcoplasmic reticulum membranes with cholesterol and change in the molecular conformation of Ca-ATPase.     

Role of Sulfhydryl Groups in Functioning of Adenylyl Cyclase Signaling System

This review considers the literature data and author's own results on the role of SH-groups in functioning of the hormone-sensitive adenylyl cyclase system (ACS). It has been shown that the state of SH-groups affects crucially all main stages of the hormonal signal transudation: the ligand-binding properties of receptor and its coupling to G-proteins, interaction of G-proteins with adenylyl cyclase (AC) and its catalytic activity. It is noted that for the receptors, coupled to AC by a stimulating mode, the central aspect of the SH-dependent regulation of ACS is shifted to the receptor, while for the receptors coupled to AC by an inhibiting mode, it coincides with G-protein of the inhibiting type, which is sensitive to the SH-group state. Based on the performed comparative analysis of primary structures of signalling proteins—ACS components and of literature data, there are revealed the cysteine residues determining the functional activity of these proteins in the process of the hormonal signal transudation. The conclusion is made that the SH-group state (the ratio of free SH-groups and disulfide bonds) is the main factor determining the ACS reactivity to hormonal effects and selectivity of process of the signal transudation.     

Content of SH-groups in some fractions of rat hemoglobin

Eight fractions of rat hemoglobin obtained by electrophoresis in polyacrylamide gel were studied. Having used parachloromercuric benzoate (PChMB) and 5,5-dithio-bis (2-nitrobenzoic acid) it was established that fractions of 3 and 3a carboxyhemoglobin contain 6 available SH-groups and the rest one (1, 2, 2a, 4, 5, 6) about 4 SH-groups. In all fractions only 2 masked SH-groups are found. A problem is discussed on SH-groups localization in the polypeptide chains of certain hemoglobin fractions in rats.     

Non-equivalency of SH groups essential for the activity of mitochondrial creatine kinase

The properties of SH-groups of mitochondrial creatine kinase existing in solution as a hexamer with Mr of (240 +/- 12) X 10(3) Da, were investigated. The number and reactivity of SH-groups by specific modifiers--[5.5'-dithiobis-(2-nitrobenzoic acid), DTNB; 7-chloro-4-nitrobenzo-2-oxo-1.3-diazol, NBD-Cl; 2.2'-dithiopyridine, DTP] were determined. It was found that each subunit of the enzyme hexameric molecule contains two modified SH-groups, only one of which is protected against modification by Mg-ADP, Mg-ATP as well as during the formation of the transition state analog (TSA)--E-Mg X ADP-NO3-creatine--and is essential for the enzyme activity. These six essential SH-groups within the hexameric molecule of mitochondrial creatine kinase may be classified into two groups according to the rate of their interaction with DTNB, NBD-Cl and DTP. The rate constants of modification of three fast and three slow essential SH-groups differ 4-10 times. The kinetics of enzyme inactivation by iodoacetamide (IAA) is biphasic; each phase is characterized by a 50% loss of activity. The inactivation constants differ 30 times; both phases being protected by TSA; consequently, the inactivation is caused by the binding of IAA to the essential SH-groups. The unequal reactivity of essential SH-groups seems to be preexisting. Using a computer analysis, the dependence of the amount of residual activity on the number of modified SH-groups by NBD-Cl and DTNB was studied. The interaction of NBD-Cl and DTNB with the most reactive essential SH-groups in half of the subunits results in the inactivation of these subunits as well as in partial or complete inactivation of the other half of the non-modified subunits. The degree of inactivation of the latter 50% of subunits strongly depends on the nature of the modifier. The inactivating effect of the bound modifier is translated from one subunit to another in one direction. The experimental results point to asymmetrical association of mitochondrial creatine kinase subunits.     

Four free cysteine residues found in human IgG1 of healthy donors

Modifications with different thiol reagents demonstrated that 28 of 32 cysteine residues of human IgG1 are involved in the formation of disulfide bonds, and four cysteines remain free. So IgG1 is a protein possessing both free SH-groups and disulfide bonds. Only one of the four SH-groups is accessible for silver or mercury ions and hydrophobic reagents, whereas the remaining three SH-groups are masked and can be revealed only after deep denaturation of the protein. Detection of the masked cysteine residues was shown to depend on the kinetics of intramolecular changes occurring during denaturation of the protein and on the method of the assay of the SH-groups.     

Study on the role of SH-groups in the activity of muscle pyruvate dehydrogenase

The kinetics of inactivation of the pyruvate dehydrogenase component of the pigeon breast muscle pyruvate dehydrogenase complex in the presence of 5,5'-dithiobis (2-nitrobenzoate) is biphasic. The rate constants for the fast and slow phases of the inactivation reaction are close to those for modification of two classes of SH-groups differing in their reactivities towards the inhibitor. The reaction order with respect to the inhibitor concentration suggests that the two distinct SH-groups are essential for the enzyme activity. Modification of these SH-groups results in inhibition of the overall activity of the pyruvate dehydrogenase complex and of the 2-hydroxyethyl thiamine pyrophosphate - acceptor oxidoreductase activity of its decarboxylating component. Thiamine pyrophosphate exerts a protective effect on the enzyme only at the slow phase of the enzyme inactivation and SH-modification. As a result of interaction between the holoenzyme and pyruvate (or apoenzyme and 2-hydroxyethyl thiamine pyrophosphate) the rate of the enzyme inactivation is increased. This is associated with masking of non-essential SH-groups and with an increase of the accessibility of two essential SH-groups to the inhibitor. The data obtained suggest the interrelationship between the essential SH-groups and the 2-hydroxyethyl thiamine pyrophosphate-acceptor oxidoreductase activity of pyruvate dehydrogenase.     

Investigation of essential SH-groups of bacterial formate dehydrogenase]

Modification of two SH-groups in the molecule of formate dehydrogenase by dithiobisnitrobenzoate or to dacetamide results in the enzyme inactivation. Coenzymes, but not the substrate, protect the enzyme against the inactivation. NAD in the presence of potassium azide completely preserves the enzyme activity. Two SH-groups per enzyme molecule are protected from modification. The Km values for partially inactivated formate dehydrogenase remain constant for both substrates. The enzyme with modified SH-groups does not bind conezymes. The pH-dependence of the inactivation rate reveals the ionizable group with pK 9.6 (25 degrees C). The involvement of essential SH-groups in coenzyme binding is discussed.     

Study of reactivity of sulfhydryl groups of troponin]

The reactivities of SH-groups of troponin and its components were studied, using 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). At low concentrations of Ca2+ (pCa greater than 8) one rapidly- and two slowly-reacting SH-groups of troponin I and one SH-group of troponin C slowly reacting with NBD-chloride are titrated in the whole troponin complex. When Ca2+ concentration is increased up to pCa less than 5, only one slowly-reacting and one rapidly-reacting with NBD-chloride SH-groups of troponin I are titrated. The increase of Ca2+ concentration from pCa greater than 8 to pCa less than 5 results in a change of the environment polarity for the highly reactive SH-group of troponin I in the whole troponin complex. This phenomenon may suggest that the changes in troponin C structure during Ca2+ binding somehow induce changes in that of troponin I. The half-maximal change of troponin SH-groups reactivity is found at pCa 6.8. No cooperativity for Ca2+ binding by the troponin complex is observed using SH-groups titration by NBD-Cl.     

SH-groups of NAD kinase from the rabbit liver

Two SH-groups per enzyme subunit have been identified in the native preparation of rabbit liver NAD kinase, using DTNB. The titration curve is biphasic; one SH-group is modified at each step. There is a strict correlation between the loss of the enzyme activity and the rate of modification of fast and slow SH-groups. Substrates afford only a partial protection of NAD kinase against the DTNB-induced inactivation. The data obtained suggest that two SH-groups of NAD kinase are essential for the enzyme activity; however, these groups are not directly involved in the active center formation.     

Characteristics of sulfhydryl groups of histone H3 from the calf thymus using mercury-containing nitroxyl radicals

The interaction between total histone and deoxyribonucleoprotein (DNP) preparations from calf thymus with mercury-containing nitroxyl radicals in low ionic strength solutions, 2 M NaCl and urea was investigated. It was found that the label is rapidly incorporated into the SH-groups of histone H3 to produce characteristic EPR signals. Titration of SH-groups within DNP demonstrated that in low ionic strength solutions only one SH-group (presumably, the SH-group of the cysteine residue in position 110) is accessible to the reagents. After dissociation by 2 M NaCl, two SH-groups become titrable; however, the EPR spectra point to differences in the conformational state of these two groups. In 4 M urea, these differences are compensated for by structural disintegration. The spin labels may be used for the analysis of SH-groups under different conditions and at different functional states of nucleoproteins.     

Copper (II) Ions Affect <Emphasis Type="Italic">Escherichia coli</Emphasis> Membrane Vesicles’ SH-Groups and a Disulfide-Dithiol Interchange Between Membrane Proteins

The SH-groups in Escherichia coli membrane vesicles, prepared from cells grown in fermentation conditions on glucose at slightly alkaline pH, have a role in the F0F1-ATPase operation. The changes in the number of these groups by ATP are observed under certain conditions. In this study, copper ions (Cu2+) in concentration of 0.1 mM were shown to increase the number of SH-groups in 1.5- to 1.6-fold independent from K+ ions, and the suppression of the increased level of SH-groups by ATP was determined for Cu2+ in the presence of K+. Moreover, the increase in the number of SH-groups by Cu2+ was absent as well as the inhibition in ATP-dependent increasing SH-groups number by Cu2+ lacked when vesicles were treated with N-ethylmaleimide (NEM), specific thiol-reagent. Such an effect was not observed with zinc (Zn2+), cobalt (Co2+), or Cu+ ions. The increased level of SH-groups was observed in the hycE or hyfR mutants with defects in hydrogenases 3 or 4, whereas the ATP-dependent increase in the number of these groups was determined in hycE not in hyfR mutants. Both changes in SH-groups number disappeared in the atp or hyc mutants deleted for the F0F1-ATPase or hydrogenase 3 (no activity of hydrogenase 4 was detected in the hyc mutant used). A direct effect of Cu2+ but not Cu+ on the F0F1-ATPase is suggested to lead to conformational changes or damaging consequences, increasing accessible SH-groups number and disturbing disulfide-dithiol interchange within a protein-protein complex, where this ATPase works with K+ uptake system or hydrogenase 4 (Hyd-4); breaks in disulfides are not ruled out.     

Phosphate carrier of liver mitochondria: two equivalent SH-groups in the carrier unit

Phosphate carrier activity of mitochondria that had become swollen during the aerobic accumulation of calcium acetate was measured indirectly by monitoring the turbidity changes. The exchange between extramitochondrial phosphate and intramitochondrial acetate was inhibited by SH-group binding reagents. Part of the SH-groups of the carrier could be blocked by mersalyl without loss of activity whereas liberation of the same groups restored carrier activity when all the other SH-groups were irreversibly blocked. A symmetrical structure of the carrier is proposed with two equivalent SH-groups; each of them is able in itself to maintain carrier function.     

光系统Ⅱ反应中心D_1-D_2-Cyt b_(559)的光破坏作用研究

从高等植物叶绿体中分离得到的光系统Ⅱ(PSⅡ)反应中心D_1-D_2-Cytb(559)复合物很不稳定,极易受到光照的破坏。光照导致D_1-D_2-Cytb_(559)在红区(Qy带)的吸收光谱发生很大的变化,在最初光照45秒时间内,吸光度值升高,继续光照则吸光度值下降,而且680nm处的下降速度最大,吸收峰发生兰移,光照也导致荧光强度增大,发射峰兰移。所有这些结果表明,光破坏至少存在两个不同的过程,而且主要受到破坏的是原初电子供体P680。