Localization of tryptophan residues in Ca2+-ATPase from sarcoplasmic reticulum

By the methods of spectroscopy, fluorimetry and chemical modification of tryptophane residues with N-bromsuccinimide, the sarcoplasmic reticulum of rabbit sceletal muscle was shown to contain 18 +/- 1 tryptophane residues per Ca2+-ATPase molecule, 6 of which were, probably, inside the protein globule, in its hydrophobic region, and thus unavailable for modifier, while the rest 12 +/- 1 were easily transformed to the 6-oxyindole chromophore being the main source of the intrinsic fluorescence of the enzyme. The quantum yield for the rest four residues was equal to 0.015. Four tryptophane residues are located at the distance of less than 14 A from the ATP-binding site of the enzyme. The quantum yields of fluorescence for 8 of the tryptophane residues of Ca2+-ATPase were similar and equal to 0.03.     

Allosteric properties of CF1-ATPase form chloroplasts

The inhibiting effect of ADP and Mg2+ on CF1-ATPase from chloroplasts depending on their concentration, pH and the presence of stimulating agents of various origin was studied. It was shown that the low Mg-dependent activity of the soluble enzyme is due to non-competitive inhibition of the reaction by Mg2+ in the presence of ADP. The CF1-ATPase stimulators lower the inhibiting effect, thus allowing to detect the "true" Mg-dependent activity of the enzyme. The data obtained are indicative of the existence of Mg2+- and ADP-specific sitein the enzyme, which controls its catalytic activity. The properties and possible role of this site in photophosphorylation are discussed.     

粘质赛氏菌胞外蛋白酶的化学修饰

用九种化学修饰剂研究了粘质赛氏菌ＳｅｒｒａｔｉａＭａｒｃｅｓｃｅｎｓ４１００３（２）胞外蛋白酶分子中氨基酸侧链基团与酶催化活性的关系,结果表明组氨酸、丝氨酸、赖氨酸、精氨酸、谷氨酸及天冬氨酸等残基与酶活性无关;半胱氨酸残基与酶活性也无直接关系;而酪氨酸和色氨酸残基侧链的修饰引起酶活力大幅度下降,说明酪氨酸和色氨酸残基为酶活力必需．     

猪链球菌2型溶血素的化学修饰

用DTT、H2O2、DEPC、EDC、NAI、NBS、PCMB、2,3Diacetyl和SA 等9种化学修饰剂,处理猪链球菌2型江苏分离株提纯的溶血素,研究其分子中氨基酸侧链基团与其溶血活性的关系。结果表明,巯基、氨基、羧基和酪氨酸残基等与其溶血活性无关,而色氨酸、组氨酸和精氨酸的化学修饰引起溶血活性的大幅度下降,二硫键的化学修饰引起溶血活性的大幅度增强。显示色氨酸、组氨酸和精氨酸残基是该溶血素的活性必需基团,二硫键的断裂可引起其活性增强。     

嗜水气单胞菌胞外蛋白酶的化学修饰

 蛋白酶是嗜水气单胞菌 (Aeromonashydrophila)的重要致病因子 .为研究其结构与功能之间的关系 ,用DEPC、EDC、PMSF、N AI等 9种化学修饰剂处理嗜水气单胞菌J 1株胞外蛋白酶ECPase54,然后检测残余酶活力 ,借以研究酶分子中氨基酸侧链基团与酶活性中心的关系 .结果表明 ,羧基、丝氨酸、ε 氨基、胍基等残基与酶活性无关 ;半胱氨酸残基与酶活性也无直接关系 ;而色氨酸、组氨酸、酪氨酸残基侧链以及二硫键的化学修饰引起酶活性的大幅度的下降 ,说明色氨酸、组氨酸、酪氨酸残基以及二硫键是酶活力所必需的基团     

Reactivation of the chloroplast CF1-ATPase beta subunit by trace amounts of the CF1 alpha subunit suggests a chaperonin-like activity for CF1 alpha.

Incubation of tobacco and lettuce thylakoids with 2 M LiCl in the presence of MgATP removes the beta subunit from their CF1-ATPase (CF1 beta) together with varying amounts of the CF1 alpha subunit (CF1 alpha). These 2 M LiCl extracts, as with the one obtained from spinach thylakoids (Avital, S., and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067-7072), could form active hybrid ATPases when reconstituted into inactive beta-less Rhodospirillum rubrum chromatophores. Pure CF1 beta fractions that have been isolated from these extracts could not form such active hybrids by themselves, but could do so when supplemented with trace amounts (less than 5%) of CF1 alpha. A mitochondrial F1-ATPase alpha subunit was recently reported to be a heat-shock protein, having two amino acid sequences that show a highly conserved identity with sequences found in molecular chaperones (Luis, A. M., Alconada, A., and Cuezva, J. M. (1990) J. Biol. Chem. 265, 7713-7716). These sequences are also conserved in CF1 alpha isolated from various plants, but not in F1 beta subunits. The above described reactivation of CF1 beta by trace amounts of CF1 alpha could thus be due to a chaperonin-like function of CF1 alpha, which involves the correct, active folding of isolated pure CF1 beta.     

Structure function relationship of vanadate bound to a single site in chloroplast CF1-ATPase as determined by X-ray absorption

The structure of vanadate, a phosphate analogue which was suggested to function in the presence of tightly bound ADP and divalent cations as a transition state inhibitor of CF1-ATPase, was investigated by X-ray absorption spectroscopy. Analysis of the vanadium K-edge was used for determination of the structure of vanadate bound to a single site in CF1-ATPase containing a single tightly bound ADP. There was a decrease in the intensity of the 1s-3d pre-edge transition and a change in the shape of two other shoulders at the edge region upon binding of vanadate to CF1 in the presence of Mg2+ ions. The changes are due to alteration in the structure of vanadium from tetrahedral to a five-coordinated trigonal bipyramidal geometry. Comparison of the pre-edge peak intensity of ADP-vanadate complex, and model compound resolved by crystallography support the proposed structure of CF1-bound vanadate. ⁵¹V NMR measurements were used to verify the pentacoordinated structure of ADP-vanadate complex used as a model in the X-ray absorption studies. The inhibition of a single and multiple site activity by vanadate and by MgADP was measured. Vanadate inhibition of CF1-ATPase activity decreased more than 90 fold in the presence of MgADP. A differential specificity of the inhibition in single and multiple mode of activity was observed. It is suggested that ADP-vanadate binds to the active sites of the enzyme as a pentacoordinated vanadium having approximate trigonal bipyramidal geometry. This structure is analogous to the proposed transition state of the phosphate during the synthesis and the hydrolysis of ATP by CF1.     

Functions and localization of nucleotide-binding sites of CF1-ATPase using dialdehyde derivatives of ADP and ATP

The covalent binding of dialdehyde derivatives of ATP and ADP (o-ATP and o-ADP) results in inactivation of chloroplast CF1-ATPase, the degree of inactivation being increased at a rise in temperature and pH. o-ADP causes predominant inhibition of the Mg2+-dependent, while o-ATP--of both Mg2+- and Ca2+-dependent activities of CF1-ATPase. The substrates and reaction products prevent the enzyme inactivation, whereas the stimulators of the Mg2+-dependent ATPase activity enhance it. The effect of these stimulators is correlated with predominant incorporation of [3H] o-nucleotide into the beta-subunit of CF1. In the absence of the stimulators o-ADP is predominantly bound to the alpha-subunit of CF1. The binding of o-ADP and o-ATP to the beta-subunit is increased in the presence of Mg2+. A comparative analysis of the labelled nucleotides incorporation into individual subunits and the changes in the catalytic and regulatory properties of the enzyme demonstrated that the catalytic and stimulator-sensitive "regulatory" sites of the enzyme are located on the beta-subunits.     

N-Ethylmaleimide inhibition of the catalytic activities of the Dunaliella salina coupling factor 1 (CF1) and the restoration of the inhibition of the CF1 ATPase activity by N-ethylmaleimide

The sensitivity of the catalytic activities of the D. salina chloroplast coupling factor 1 (CF1) to chemical modification by N-ethylmaleimide has been investigated. When D. salina thylakoid membranes are treated with N-ethylmaleimide, both photophosphorylation and the inducible CF1 ATPase activity are partially (approx. 60%) inhibited. The inhibition of both activities does not require the presence of a proton-motive force, and the inhibition of photophosphorylation is directly related to the N-ethylmaleimide-covalent modification of CF1 as shown by the time-course for the inhibition and the maximal extent of inhibition. Treatment of the purified, latent, D. salina CF1 with low concentrations of N-ethylmaleimide also results in the partial (approx. 60%) inhibition of the inducible ATPase activity (I50 approximately 50 microM). The inhibition does not require the presence of the chemical modifier during the activation of the enzyme. N-ethylmaleimide-induced inhibition of the ATPase activity of either membrane-bound or solubilized CF1 is partially reversed by either prolonged incubation at low concentrations of N-ethylmaleimide or short incubation times at high concentrations of N-ethylmaleimide. The results are interpreted as indicating multiple binding sites on the D. salina CF1 that have different rates of reactivity with N-ethylmaleimide. Those sites (or site) that react rapidly with N-ethylmaleimide cause(s) an inhibition of both ATP synthase and ATPase activities, whereas those sites (or site) that react more slowly partially restore(s) the original ATPase activity. The effects of N-ethylmaleimide on the catalytic activity of D. salina CF1 are probably mediated by N-ethylmaleimide-induced conformational changes of the enzyme.     

Directed mutagenesis of the beta-subunit of F1-ATPase from Escherichia coli

Oligonucleotide-directed mutagenesis was used to generate six mutant strains of Escherichia coli which had the following specific amino acid substitutions in the beta-subunit of F1-ATPase: (i) Lys-155----Gln; (ii) Lys-155----Glu; (iii) Gly-149----Ile; (iv) Gly-154----Ile; (v) Tyr-297----Phe;(vi) Tyr-354----Phe. The effects of each mutation on growth of cells on succinate plates or limiting (3 mM) glucose and on cell membrane ATPase activity and ATP-driven pH gradient formation were studied. The results showed Lys-155 to be essential for catalysis, as has been predicted previously from sequence homology and structural considerations; however, the results appear to contradict the hypothesis that Lys-155 interacts with one of the substrate phosphate groups because the Lys-155----Glu mutation was less detrimental than Lys-155----Gln. Gly-149 and Gly-154 have been predicted to be involved in essential conformational changes in F1-ATPase by virtue of their position in a putative glycine-rich flexible loop structure. The mutation of Gly-154----Ile caused strong impairment of catalysis, but the Gly-149----Ile mutation produced only moderate impairment. The two tyrosine residues chosen for mutation were residues which have previously received much attention due to their being the sites of reaction of the inactivating chemical modification reagents 4-chloro-7-nitrobenzofurazan (Tyr-297) and p-fluorosulfonylbenzoyl-5'-adenosine (Tyr-354). We found that mutation of Tyr-297----Phe caused only minor impairment of catalysis, and mutation of Tyr-354----Phe produced no impairment. Therefore, a direct role for either of these tyrosine residues in catalysis is unlikely.     

Coupling factor activity of the purified pea mitochondrial F1-ATPase

The pea cotyledon mitochondrial F1-ATPase was released from the submitochondrial particles by a washing procedure using 300 mM sucrose/2 mM Tricine (pH 7.4). The enzyme was purified by DEAE-cellulose chromatography and subsequent sucrose density gradient centrifugation. Using polyacrylamide gel electrophoresis under non-denaturing conditions, the purified protein exhibited a single sharp band with slightly lower mobility than the purified pea chloroplast CF1-ATPase. The molecular weights of pea mitochondrial F1-ATPase and pea chloroplast CF1-ATPase were found to be 409 000 and 378 000, respectively. The purified pea mitochondrial F1-ATPase dissociated into six types of subunits on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Most of these subunits had mobilities different from the subunits of the pea chloroplast CF1-ATPase. The purified mitochondrial F1-ATPase exhibited coupling factor activity. In spite of the observed differences between CF1 and F1, the mitochondrial enzyme stimulated ATP formation in CF1-depleted pea chloroplast membranes. Thus, the mitochondrial F1 was able to substitute functionally for the chloroplast CF1 in reconstituting photophosphorylation.     

Na_2SO_3对不同状态CF_1－ATPase活力的促进作用

Ｎａ２ＳＯ３对ＣＦ１－ＡＴＰａｓｅ活力的促进作用与酶所处状态有关。ＣＦ０降低ＣＦ１对Ｎａ２ＳＯ３的亲和力和Ｎａ２ＳＯ３促进的最大反应速率。在Ｎａ２ＳＯ３作用下,膜上ＣＦ１－ＡＴＰａｓｅ的活化能高于游离的。膜上和游离ＣＦ１－ＡＴＰａｓｅ的γ亚基上二硫键的还原可以提高Ｎａ２ＳＯ３对酶活力的促进作用。Ｎａ２ＳＯ３对甲醇活化的ＣＦ１－ＡＴＰａｓｅ活力的促进作用只有在甲醇活化的亚适浓度下才能充分表现出来。Ｎａ２ＳＯ３对Ｍｇ２＋抑制的解除作用因ＣＦ１－ＡＴＰａｓｅ处于不同活化状态而不同。     

Presteady-state kinetics of ATP hydrolysis by chloroplast CF1-ATPASE

The kinetics of reversible inactivation of chloroplast CF1-ATPase by Mg2+ and ADP was studied. The rate of inactivation obeys the first-order equation and is independent of ADP concentration. An analysis of the dependence of the inactivation rate on Mg2+ concentration demonstrated that the limiting step of inactivation is other than Mg2+ binding, i.e. the subsequent steps which include, in all probability, the conformational changes of the enzyme. The original Mg2+-dependent activity of CF1-ATPase is close to that observed under steady-state conditions in the presence of sulphate and methanol and exceeds the Ca2+-dependent activity approximately 6-fold. Preincubation of CF1-ATPase with Mg2+ results in inhibition of the original activity of the enzyme. This effect is not removed by addition of the ATP-regenerating system (pyruvate kinase + phosphoenol pyruvate) to the preincubation medium but is diminished by sulphite and the non-hydrolyzed analog of ATP--beta, gamma-methyladenosine-5-triphosphate. After addition of AMPPCP to the reaction mixture the initial reaction rate is decreased, while the steady-state rate is increased. It may be concluded that the Mg2+-dependent inactivation of CF1-ATPase is induced by the tightly bound ADP. The latter can be replaced by ATP, which in contrast to ADP does not form an inactive complex with the enzyme. A comparison of experimental results with literature data suggests that the mechanism of "alternating sites" proposed by Boyer et al. for ATP hydrolysis by soluble CF1-ATPase is not realized under the given experimental conditions.     

ATP hydrolysis catalyzed by a beta subunit preparation purified from the chloroplast energy transducing complex CF1.CF0

Washing thylakoid membranes with 1 M LiCl causes the release of the beta subunit from the chloroplast energy transducing complex (CF1.CF0) in spinach chloroplasts. This protein purifies by size exclusion chromatography as a 180-kDa aggregate and, thus, is probably composed of a trimer of beta polypeptides. The purified aggregate binds ADP to a high and a low affinity site with dissociation constants of 15 and 202 microM, respectively. Mg2+ is required for ADP to bind to both sites. Manganese binds to the protein in a cooperative manner to at least two sites with high affinity. The beta subunit preparation catalyzes Mg2+-dependent ATP hydrolysis at rates which are comparable to other subunit-deficient CF1 preparations and is increased by treatments known to activate the Mg2+-ATPase activity of CF1. However, Ca2+ is not an effective cofactor for this reaction and treatments which activate the Ca2+-ATPase of CF1 are either ineffective or inhibitory.     

Effect of photooxidation on biological properties of botulinum neurotoxin A

Photooxidation of botulinic neurotoxin A in the presence of methylene blue is associated with a decrease in toxicity down to complete detoxication. During neurotoxin photooxidation, when the toxicity makes up to 1 to 3% of the original one, the conformation of the neurotoxin molecule and its antigenic properties remain unchanged. Under these conditions, using diethylpyrocarbonate, a specific reagent for histidine, the photooxidized neurotoxin was found to contain 5-6 oxidized histidine residues per molecule of neurotoxin; this was accompanied by changes in the UV absorbance spectrum around 280 nm. It was assumed that the main decrease in neurotoxin toxicity during photooxidation is probably due to oxidation of tryptophane, since the differential UV spectra suggest that the higher the extremum around 280 nm, the greater the decrease of toxicity; chemical modification of histidine residues alone causes no noticeable detoxication.     

Kinetics of Mg2+-dependent CF1-ATPase in the presence of stimulators

A kinetic study of ATP hydrolysis by CF1-ATPase from chloroplasts in the presence of optimal concentrations of the stimulators, sodium sulfite and ethyl alcohol, has been carried out. At MgCl2/ATP ratios more than 1 the reaction kinetics obey the Michaelis--Menten equation. At ATP excess the kinetics are of the second order with respect to Mg2+. The data obtained are consistent with the hypothesis on the formation of an enzyme substrate Mg.CF1-MgATP complex containing beside Mg-ATP substrate Mg2+. The dependence of the maximal rate of the reaction on pH was studied. Two active groups with pK of 6.3 and 8.9 were revealed. The group responsible for Mg+2 binding to the enzyme has a pK of 8.3. The possible nature of the active groups of the enzyme is discussed.     

Evidence for an essential histidine residue in the Neurospora crassa plasma membrane H+-ATPase

The Neurospora crassa plasma membrane H+-ATPase is rapidly inactivated in the presence of diethyl pyrocarbonate (DEP). The reaction is pseudo-first-order showing time- and concentration-dependent inactivation with a second-order rate constant of 385-420 M-1.min-1 at pH 6.9 and 25 degrees C. The difference spectrum of the native and modified enzyme has a maximum near 240 nm, characteristic of N-carbethoxyhistidine. No change in the absorbance of the inhibited ATPase at 278 nm or in the number of modifiable sulfhydryl groups is observed, indicating that the inhibition is not due to tyrosine or cysteine modification, and the inhibition is irreversible, ruling out serine residues. Furthermore, pretreatment of the ATPase with pyridoxal phosphate/NaBH4 under the conditions of the DEP treatment does not inhibit the ATPase and does not alter the DEP inhibition kinetics, indicating that the inactivation by DEP is not due to amino group modification. The pH dependence of the inactivation reaction indicates that the essential residue has a pKa near 7.5, and the activity lost as a result of H+-ATPase modification by DEP is partially recovered after hydroxylamine treatment at 4 degrees C. Taken together, these results strongly indicate that the inactivation of the H+-ATPase by DEP involves histidine modification. Analyses of the inhibition kinetics and the stoichiometry of modification indicate that among eight histidines modified per enzyme molecule, only one is essential for H+-ATPase activity. Finally, ADP protects against inactivation by DEP, indicating that the essential residue modified may be located at or near the nucleotide binding site.     

Directed mutagenesis of the dicyclohexylcarbodiimide-reactive carboxyl residues in beta-subunit of F1-ATPase of Escherichia coli

Previous studies in which dicyclohexylcarbodiimide (DCCD) was used to inactivate F1-ATPase enzymes have suggested that two glutamate residues in the beta-subunit are essential for catalysis. In the Escherichia coli F1-ATPase, these are residues beta-Glu-181 and beta-Glu-192. Oligonucleotide-directed mutagenesis was used to change these residues to beta-Gln-181 and beta-Gln-192. The beta-Gln-181 mutation produced strong impairment of oxidative phosphorylation in vivo and also of ATPase and ATP-driven proton-pumping activities in membranes assayed in vitro. A low level of each activity was detected and an F1-ATPase appeared to be assembled normally on the membranes. Therefore, it is suggested that the carboxyl side chain at residue beta-181 is important, although not absolutely required, for catalysis in both directions on E. coli F1-ATPase. The beta-Gln-192 mutation produced partial inhibition of oxidative phosphorylation in vivo and membrane ATPase activity was reduced by 78%. These results contrast with the complete or near-complete inactivation seen when E. coli F1-ATPase is reacted with DCCD and imply that DCCD-inactivation is attributable more to the attachment of the bulky DCCD molecule than to the derivatization of the carboxyl side chain of residue beta-Glu-192. M. Ohtsubo and colleagues (Biochem. Biophys. Res. Commun. (1987) 146, 705-710) described mutagenesis of the F1-beta-subunit of thermophilic bacterium PS3. Mutations (Glu----Gln) of the residues homologous to Glu-181 and Glu-192 of E. coli F1-beta-subunit both caused total inhibition of ATPase activity. Therefore, there was a marked difference in results obtained when the same residues were modified in the PS3 and E. coli F1-beta-subunits.     

Chromatographic purification of the chloroplast ATP synthase (CF0-CF1) and the role of CF0 subunit IV in proton conduction

Chromatographic procedures were developed to purify chloroplast ATP synthase (CF0-CF1) in large amounts and to resolve subunits from this enzyme. The ATP synthase thus obtained has high ATP-Pi exchange and Mg2(+)-ATPase activities upon incorporation into asolectin liposomes. The purity of this preparation was about 95%. By modifications of this chromatographic procedure, we purified subunit IV-deficient CF0-CF1, subunit IV-deficient CF0, and subunit IV. Both ATP-Pi exchange and Mg2(+)-ATPase activities were impaired by depletion of subunit IV from CF0-CF1. Partial restoration of these activities was obtained by reconstituting subunit IV-deficient CF0-CF1 with subunit IV. The impairment of these activities was likely caused by a loss in proton conductivity of CF0 upon removal of subunit IV. The dicyclohexylcarbodiimide-sensitive Mg2(+)-ATPase of subunit IV-deficient CF0-CF1 was not as sensitive to the depletion of subunit IV as ATP-Pi exchange. Nearly 90% of subunit IV could be removed, but Mg2(+)-ATPase activity was inhibited by only 40-60%. Thus subunit IV of CF0-CF1 may not participate directly in proton transfer but may have a role in organizing and/or stabilizing CF0 structure.     

Study of the amino acid residue topography of the adenosine triphosphatase center of (Ca--Mg)-dependent sarcoplasmic reticulum adenosine triphosphatase by kinetic and spectral methods

The topography of HS- and NH2-groups and tryptophane residues in ATPase centre of (Ca--Mg)-ATPase on sarcoplasmic reticulum (SR) was investigated by kinetics, electron spectroscopy and spectrofluorimetry method. Both o-phthalaldehyde interacting with lysine or arginine residue or with end amino acid and fluorescein dimercuric acetate interaction with cysteine residue of HS-groups make (Ca--Mg)-ATPase both in SR and the pure enzyme completely inactive at molar ratio enzyme: inhibitor equal to 1 : 1. A 500 molar ATP surplus reduces drastically the enzyme inactivation rate by both inhibitors. The data supplied by the spectrofluorimetry and the induction-resonance theory were used to calculate the distances between nearest tryptophane residues and chromophore (o-FTC) generated by o-phthalaldehyde interaction with NH2-group the protein amino acid residue (17 A) and o-FTC and fluorescein dimercuric acetate (19 A) attached to enzyme HS-group. Because o-FTC is inside the protein pocket it is not accessible to J- ions up to 2.5 M KJ. However some tryptophane resudies and fluorescein dimercuric acetate attached to HS-group are near to the macromolecule surface. Lysine (or arginine residues) or end amino acid NH2-group and cysteine residues HS-group, and some tryptophane residues are at ATPase centre of (Ca--Mg)-ATPase from sarcoplasmic reticulum. Possible topography of the centre is discussed.