Specific SH Groups Research Articles

Photoactivation of Phthalocyanine-loaded Low Density Lipoproteins Induces a Local Oxidative Stress that Propagates to Human Erythrocytes: Protection by Caffeic Acid

Toxic effects imposed to human erythrocytes by low density lipoproteins carrying phthalocyanines used in photodynamic therapy (PDT) of tumors are described. This study was aimed at evaluating cytotoxic effects induced by reactive species produced locally in photosensitizer-loaded lipoproteins and further transferred to the cells. The experimental set up designed to examine these interactions starts with the loading of human plasma with the photosensitizer, the subsequent rapid purification and dialysis of the LDL fraction and incubation with human erythrocytes. This experimental model was assessed by following leakage of endogenous K + from cells, electrochemical detection of oxygen, spectroscopic determination of conjugated dienes, phthalocyanine, SH groups and hemoglobin, analysis of fatty acids by gas chromatography and identification of f -tocopherol by HPLC. Photosensitizer-loaded lipoproteins become more susceptible to oxidation, exhibiting shorter lag phases of lipid oxidation, higher rates of oxidation and increased loss of endogenous f -tocopherol when challenged with peroxyl radicals and copper, as compared with native lipoproteins from the same plasma sample. Incubation of photosensitized lipoproteins with erythrocytes under light (>560 nm) results in a sigmoidal efflux of K + followed by hemolysis. The phenolic antioxidant caffeic acid inhibits lipoprotein oxidation induced by peroxyl radicals, either in native or photosensitizer-loaded fractions, delays hemolysis of erythrocytes and partially prevents membrane loss of SH groups in ghosts, but not the efflux of K + . Mechanistically, a chain lipid peroxidation reaction does not participate in the toxic effects to cells but a specific pool of membrane SH groups sensitive to caffeic acid is likely to be involved. This study suggests that an oxidative stress occurring locally in phthalocyanine-loaded low density lipoproteins may further induce cytotoxic effects by targeting specific SH groups at the cell membrane level. The physiological relevance of these findings and the beneficial use of antioxidants are discussed in the context of PDT.

Effects of lead and selected pyrethroids/piperonyl butoxide alone and in combination on Na, K‐ATPase

In vitro effects of Pb2+, the pyrethroid insecticides cypermethrin, fenvalerate and the syner‐gist piperonyl butoxide on sodium‐potassium‐activated adenosine triphosphatase (Na,K‐ATPase) from dog kidney were determined. Pb2+ with an estimated IC50 value of 5.2 μM was found to be a potent inhibitor of Na,K‐ATPase activity, whereas Na,K‐ATPase was less sensitive to the pyrethroids tested and piperonyl butoxide. Investigation with circular dichroism (CD) spec‐troscopy showed that inhibition occurs through conformational changes of the α‐subunit of the enzyme. The kinetic characteristics of inhibition of Na,K‐ATPase with varying substrate (ATP) concentrations as well as with varying Na+ concentrations exhibited a competitive type of inhibition with Pb2+ in the μM range. With Pb2+ alone in the enzyme assay no conformational changes of the protein could be observed which confirmed the assumption that Pb2+ can bind to the Na+ binding site of the α‐subunit. Uncompetitive type of inhibition occurred with varied K+ concentrations demonstrating that this cation binding site is not affected directly by Pb2+. Complete reversal of Pb2+ by DTT confirms that a possible target for interaction of this heavy metal ion with Na, K‐ATPase are specific SH groups. Synergistic effects could only be determined with higher Pb2+ concentrations of 3, 5 and 7 μM plus piperonyl butoxide while all other combinations with this heavy metal plus organic substances where of the additive type. With CD spectroscopy also only additive effects were observed. These results demonstrate that higher concentrations of piperonyl butoxide favor the binding of Pb2+ to the Na+ binding site by conformational changes of the protein.

Binding of the activating ion Co(II) to myo-inositol monophosphatase monitored by fluorescence and phosphorescence spectroscopy.

Two extrinsic probes, pyrene-maleimide and eosin-maleimide, were used to label specific SH groups of the enzyme myo-inositol monophosphatase. The fluorescence of pyrene-monophosphatase is enhanced upon addition of the activating metal ions Co(II) and Mg(II). Co(II) ions bind with a dissociation constant of 4 microM, whereas the apparent activation constant Ka is 0.4 mM. Energy transfer measurements demonstrated that the pyrene chromophore, covalently linked to Cys-218, is within 9 A of the metal ion Tb(III) coordinated to the metal-binding site. The phosphorescence emitted by eosin covalently linked to the protein is quenched by the addition of the activating cations Co(II) and Mg(II). Phosphorescence titrations conducted under anaerobic conditions were used to determine a dissociation constant of approximately 3 microM for the binding of Co(II) ions. The results are consistent with the hypothesis that two activating ions per monomeric subunit participate in the catalytic mechanism. The affinity of the tightly bound ion is at least 100-fold greater than the affinity of the weakly bound ion.

19] Chemical derivatization of Ca2+-pump protein from skeletal muscle with N-substituted maleimides and 5-(2-iodoacetamidoethyl)aminonaphthalene 1-sulfonate

This chapter focuses on the chemical derivatizaton of Ca 2+ -pump protein from skeletal muscle with N-substituted Maleimides and 5-(2-Iodoacetamidoethyl)aminonaphthalene 1-Sulfonate. It has been difficult to achieve selective modification of a particular SH group, which is well-defined in terms of either its location or functional role, as a large number of SH groups are present in Ca 2+ ,Mg 2+ -ATPase of the sarcoplasmic reticulum (12–17 reactive SH groups in an ATPase polypeptide). In spite of the fact that the ATPase molecule has long been known to contain SH groups essential for the functioning of the transport system, and that their number has been suggested to be rather small, they have not yet been identified. SH groups were also utilized occasionally, as a target for covalent attachment of various probes to monitor conformational changes of transport ATPase during its catalytic cycle. The chapter describes simple procedures for selective derivatization of a limited number of specific SH groups with N-ethylmaleimide (MalNEt), and related fluorescent and paramagnetic probes, as well as a procedure for labeling with 5-(2-iodoacetamidoethyl)aminonaphthalene 1-sulfonate (IAEDANS), a fluorescent derivative of iodoacetamide.

Coincidence of H+ binding and Ca2+ dissociation in the sarcoplasmic reticulum Ca-ATPase during ATP hydrolysis.

H+ and Ca2+ concentration changes in the reaction medium following MgATP addition at pH 6.0 were determined with the partially purified Ca-ATPase from sarcoplasmic reticulum vesicles in the presence of 25-50 microM CaCl2 and 5 mM MgCl2 at 4 degrees C. Previously, we showed a sequential occurrence of H+ binding and H+ dissociation in the Ca-ATPase during ATP hydrolysis and further suggested that the H+ binding takes place inside the vesicles (Yamaguchi, M., and Kanazawa, T. (1984) J. Biol. Chem. 259, 9526-9531). The present results demonstrate that the H+ binding occurred coincidently with Ca2+ dissociation from the enzyme upon conversion of the phosphoenzyme (EP) intermediate from the ADP-sensitive form to the ADP-insensitive form in the catalytic cycle of ATP hydrolysis. As KCl decreased in the medium, the extent of the H+ binding increased almost proportionately with the extent of either the Ca2+ dissociation or the accumulation of ADP-insensitive EP. Both the H+ binding and the Ca2+ dissociation were prevented by a modification of the specific SH group of the enzyme essential for the conversion of ADP-sensitive EP to ADP-insensitive EP. In the late stage of the reaction, H+ dissociation from the enzyme occurred coincidently with Ca2+ binding to the dephosphoenzyme which was formed by EP decomposition. These results are consistent with the possibility that the H+ ejection during the Ca2+ uptake with the intact vesicles previously shown by several investigators takes place through a Ca2+/H+ exchange directly mediated by the membrane-bound Ca-ATPase.

Protonation of the sarcoplasmic reticulum Ca-ATPase during ATP hydrolysis.

pH changes of the reaction medium (pH 6.35) following addition of MgATP were determined at 4 degrees C with sarcoplasmic reticulum vesicles in the presence of 0.1 mM CaCl2 and 5 mM MgCl2. With intact vesicles, a pronounced acidification following a slight alkalinization was induced by MgATP, indicating H+ ejection from vesicles. This agrees with previous observations by several investigators that suggested H+ counter-transport coupled to Ca2+ uptake. When vesicles were made leaky, a markedly enhanced alkalinization occurred after addition of MgATP, and then the pH returned to the level before the MgATP addition. The decay of this alkalinization coincided with the disappearance of phosphoenzyme (EP) which was formed from MgATP and the Ca-ATPase of sarcoplasmic reticulum. The CaCl2 concentration dependence of the alkalinization agreed well with that of EP formation. Furthermore, the alkalinization was prevented by modification of a specific SH group of the enzyme essential for EP decomposition. These findings give evidence that the alkalinization with leaky vesicles represents protonation of the enzyme during ATP hydrolysis. The enhanced alkalinization with leaky vesicles suggests protonation occurring inside vesicles. When CaATP in place of MgATP was added in the absence of MgCl2, neither the H+ ejection from intact vesicles nor the enhanced alkalinization with leaky vesicles occurred. Under these conditions, CaATP did not efficiently support active Ca2+ uptake. These results are compatible with the view that the observed protonation could represent an initial event occurring inside vesicles in the H+ countertransport coupled to Ca2+ uptake.

Studies on Conformational Transitions of Ca2+,Mg2+-Adenosine Triphosphatase of Sarcoplasmic Reticulum. II. Conformational Characteristics of Stabilized Reaction Intermediates as Revealed by Fluorescent and Paramagnetic Probes1

Various reaction intermediates of sarcoplasmic reticulum Ca2+,Mg2+-ATPase were stabilized and accumulated by modifying a specific SH group or by using nucleotide analogs. Conformational changes of the Ca2+,Mg2+-ATPase during the catalytic cycle were studied in the stabilized intermediates by the use of fluorescent and spin probes, which were introduced at specific SH groups of ATPase, namely one highly reactive but functionally nonessential (SHN) and one essential for the decomposition of the E-P intermediate (SHD) [Kawakita, M., et al. (1980) J. Biochem. 87, 609-617]. The fluorescence intensity of N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide attached to SHD decreased by 2.5% upon addition of 10 microM AMP-P(NH)P provided that Ca2+ was also present. The AMP-P(NH)P-induced fluorescence change could also be detected by using other fluorescent probes such as N-[p-(2-benzimidazolyl)phenyl]maleimide and N-(1-anilinonaphthyl-4)maleimide. Moreover, labeling at SHN gave similar results. When SHN was labeled with N-[p-(2-benzimidazolyl)phenyl]maleimide, the fluorescence intensity also decreased by 2.5% upon addition of ATP only in the presence of Ca2+, where E-P formation took place. A conformational difference between ECa1-P X ADP and ECa1-P was suggested from saturation transfer ESR measurement of spin-labeled ATPase by using ADP beta S as an ADP analog to cause accumulation of ECa1-P X ADP beta S complex. Possible structural similarities among some of the intermediates are discussed based on these findings.

Some functional and structural properties of Bufus paracnemis and Pipa pipae hemoglobins

1. 1. Hemoglobin from terrestrial and an aquatic amphibia Bufo paracnemis and Pipa pipae respectively both living in the same region (Belém, Pará) were compared. 2. 2. The number of hemolysate components were determined by starch-gel electrophoresis and CMC-chromatography. P. pipae hemoglobin presented 4 components and B. puracnemis 2, all of anodic mobility. 3. 3. Functional properties of the hemoglobins (oxygen affinity. Bohr effect, carbon monoxide equilibrium) were determined and compared. 4. 4. The differences found in the functional properties were correlated with the different habitat: aquatic or terrestrial for the amphibia. 5. 5. The study of the oxygen functional properties of the hemoglobin showed in the stripped proteins of P. pipae in the presence of ATP. an oxygen affinity ( P 50 = 7.24 mmHg) that of B. paracnemis at the same pH ( P 50 = 15.84 mmHg). At high pH the P 50 values are different being 15.84 mmHg for the terrestrial frog and 5 mmHg for P. pipae haemoglobin both at pH 8. In addition Bohr effect was noted only in P. pipae hemolysate in the presence of ATP. 6. 6. The CO-equilibrium affinity constant in the presence of ATP are similar in both frogs, of about, log C 50 = −6.9. The ratio P CO/ P O 2 for B-paracnemis hemoglobin was about 300 whereas for that of P. pipae was about 100 only. 7. 7. The kinetic study of reactive sulfhydryl groups in both frog hemoglobin with 4-PDS (specific SH group reagent was used and shown high pseudo-first order constant for B. paracnemis hemoglobin) ( k' = 0.46/min) either in presence or not of ATP, compared for that of P. pipae where values were k' = 0.003/min in the stripped protein and k' = 0.014/min in the presence of ATP. 8. 8. Denaturation kinetic studies of the hemoglobin with sodium benzoate was performed and the results were compared with that of Rana catesheiana. i.e. the pseudo-first order of the hemoglobin denaturation reaction are low for the aquatic P. pipae adult although for the B. paracnemis high molecular resistant was also noted. For P. pipae hemoglobin, nevertheless with ATP such resistance becomes higher. This does not occur with B. paracnemis hemoglobins.

13] Cleavage at cysteine after cyanylation

Publisher Summary The SH groups of denatured proteins can be converted quantitatively to SCN groups with 2-nitro-5-thioeyanobenzoate (NTCB) 1,2, or with other reagents. NTCB is readily synthesized and can be labeled with [ 14 C] cyanide. Exposure to pH 8-9 results in cleavage at the amino group of the modified cysteine residue in excellent yield. Because disulfides do not react with NTCB, selective cleavage at cysteine can be obtained in the presence of cystine on the basis of this reaction. Specific SH groups of undenatured papain, aspartate transcarbamylase, isocitrate dehydrogenase, aspartate aminotransferase, NAD-specific glutamate dehydrogenase, glyceraldehyde-3-P dehydrogenase, and myosin can be modified with NTCB, or with 5,5'-dithiobis plus cyanide. Specific cleavage at cysteine residues specifically modified in native proteins provides a convenient method for locating residues. The use of disulfides formed from the 6-thio analog of ATP followed by cyanide, or direct cyanylation of protein SH groups with a 6-thiocyano analog of ATP has led to the incorporation of label and specific clearage in several cases.

Structure and function of the two heads of the myosin molecule. III. Cooperativity of the two heads of the myosin molecule, shown by the effect of modification of head A with rho-chloromercuribenzoate on the interaction of head B with F-actin.

Subfragment-1 of HMM was prepared by tryptic [EC 3.4.21.4] digestion of HMM, which had been modified with 1 mole of CMB per mole of HMM at a specific SH group, SHr. S-1(T) obtained from CMB-HMM retained almost all the CMB, and the amount of bound CMB was about 0.8-0.9 mole per 2 moles of S-1(T). S-2 of CMB-HMM contained no bound CMB. The ATPase [EC 3.6.1.3] activity of HMM increased gradually with increase in the concentration of FA, and the acto-HMM ATPase was inhibited by excess substrate or removal of Ca2+ ions in the presence of RP. The ATPase activity of CMB-HMM increased to a maximum level on adding a small amount of FA, and the acto-CMB-HMM ATPase showed neither substrate inhibition nor Ca2+ sensitivity in the presence of RP. On the other hand, the dependence on the concentration of FA of the ATPase activity of acto-S-1(T) was unaffected by modification of S-1 with CMB. The Ca2+ sensitivity of the ATPase activity of acto-S-1(T) in the presence of RP was also unaffected by the modification. Acto-S-1(T) dissociated almost completely, while acto-CMB-S-1(T) was only 50% dissociated on adding ATP. More than 80% of the bound CMB was contained in S-1(T) undissociated from FA. Furthermore, superprecipitation of actomyosin induced by ATP was completely inhibited by adding about 2 moles of CMB-S-1(T) per mole of actin monomer. On the other hand, about 90% of the burst size of Pi liberation was retained in S-1(T) dissociated from FA. It was concluded that the two heads of the myosin molecule are different: one shows the initial burst of Pi liberation, and does not contain the SHr group which binds CMB (head B), and the other does not show the initial burst and contains the SHr group (head A). It was also concluded that modification of head A of HMM or myosin with CMB increases its binding strength to FA, and consequently the substrate inhibition and Ca2+ sensitivity of acto-HMM or actomyosin ATPase at head B are lost on modification of head A with CMB. CMB-S-1(CT) was prepared by chymotryptic [EC 3.4.21.1] digestion of CMB-myosin, and separated into two fractions by ultracentrifugation of acto-CMB-S-1(CT) in the presence of ATP. Three components of CMB-S-1(CT) with molecular weights of 9, 2.4, and 1.2 X 10(4) were separated by SDS-polyacrylamide gel electrophoresis. The ratios of the peak areas of the three components in electrophoretograms were the same in CMB-S-1(CT) and in the two fractions (1 : 0.18 : 0.09), indicating that heads A and B have the same subunit structure.

Interaction of SH 1 -blocked HMM with actin and ATP.

NUMEROUS studies have been performed on the effect of blocking the SH groups of myosin with sulphydryl reagents1–8. In particular, the effect of NEM has become quite clear; on blocking one specific SH group (referred to as SH1) per myosin subunit, the EDTA-ATPase of the myosin is completely inhibited whereas the Ca-ATPase is markedly activated9. However, the interaction of SH1-blocked myosin and actin has not been as thoroughly investigated. Sekine and Yanoguchi have reported that blocking SH1 with NEM does not affect the ATPase activity and superprecipitation of actomyosin10. On the other hand, the effect of native tropomyosin on the actomyosin ATPase has been reported to be abolished by NEM under certain conditions although differences were reported depending on whether myosin or actomyosin was exposed to the NEM11.

X-ray modification of the catalytic and allosteric functions of fructose-1,6-diphosphatase

1. 1. Native rabbit liver fructose-1,6-diphosphatase ( d-fructose-1,6-diphosphate 1-phosphohydrolase, EC 3.1.3.11) and enzyme stimulated by disulfide formation have been irradiated in solution with X-rays. 2. 2. The dose-response curve for the native enzyme showed an initial stimulation of catalytic activity, followed by subsequent exponential inactivation. The disulfide-modified enzyme was inactivated exponentially with no initial stimulation. The G values for inactivation of catalytic activity were 0.017 and 0.009 for the native and the disulfide-modified enzymes, respectively. 3. 3. The allosteric activity of the native enzyme, as measured by the inhibition by AMP, was much more radioresistant than the catalytic activity. In the modified enzyme, these two functions were of comparable radiosensitivity. 4. 4. The ability of the enzymes to be stimulated by mixed disulfide formation was about 10 times as sensitive to X-ray inactivation as was the catalytic function. The loss of sensitivity to stimulation by disulfides was due to destruction of 5–6 specific SH groups which were particularly radiosensitive ( G value, 1.2). 5. 5. The X-ray-induced reduction in enzyme activity was associated with destruction of enzyme SH groups of low radiosensitivity ( G value, 0.07. The loss of enzyme activity was partly reversed by addition of cysteamine after exposure. The results indicate that sulfhydryl groups play an important, but not exclusive role in the X-ray inactivation of fructose-1,6-diphosphatase. 6. 6. The presence of AMP during irradiation protected the enzyme almost completely against inactivation of the allosteric function. The results indicated that the X-ray inactivation of the allosteric function of fructose-1,6-diphosphatase is due to a specific modification of the binding sites for AMP.