Tyrosine substitution of a conserved active‐site histidine residue activates Plasmodium falciparum peroxiredoxin 6

Peroxiredoxins efficiently remove hydroperoxides and peroxynitrite in pro‐ and eukaryotes. However, isoforms of one subfamily of peroxiredoxins, the so‐called Prx6‐type enzymes, usually have very low activities in standard peroxidase assays in vitro. In contrast to other peroxiredoxins, Prx6 homologues share a conserved histidyl residue at the bottom of the active site. Here we addressed the role of this histidyl residue for redox catalysis using the Plasmodium falciparum homologue PfPrx6 as a model enzyme. Steady‐state kinetics with tert‐butyl hydroperoxide (tBuOOH) revealed that the histidyl residue is nonessential for Prx6 catalysis and that a replacement with tyrosine can even increase the enzyme activity four‐ to six‐fold in vitro. Stopped‐flow kinetics with reduced PfPrx6^WT, PfPrx6^C128A, and PfPrx6^H39Y revealed a preference for H₂O₂ as an oxidant with second order rate constants for H₂O₂ and tBuOOH around 2.5 × 10⁷ M^?1 s^?1 and 3 × 10⁶ M^?1 s^?1, respectively. Differences between the oxidation kinetics of PfPrx6^WT, PfPrx6^C128A, and PfPrx6^H39Y were observed during a slower second‐reaction phase. Our kinetic data support the interpretation that the reductive half‐reaction is the rate‐limiting step for PfPrx6 catalysis in steady‐state measurements. Whether the increased activity of PfPrx6^H39Y is caused by a facilitated enzyme reduction because of a destabilization of the fully folded enzyme conformation remains to be analyzed. In summary, the conserved histidyl residue of Prx6‐type enzymes is non‐essential for catalysis, PfPrx6 is rapidly oxidized by hydroperoxides, and the gain‐of‐function mutant PfPrx6^H39Y might provide a valuable tool to address the influence of conformational changes on the reactivity of Prx6 homologues.     

A proton nuclear magnetic resonance investigation of histine-binding protein J of salmonella typhimurium: A model for transport of L-histidine across cytoplasmic membrane

Genetic evidence suggests that the high-affinity L-histidine transport in Salmonella typhimurium requires the participation of a periplasmic binding protein (histidine-binding protein J) and two other proteins (P and Q proteins). The histidine-binding protein J binds L-histidine as the first step in the high-affinity active transport of this amino acid across the cytoplasmic membrane. High-resolution proton nuclear magnetic resonance spectroscopy at 600 MHz is used to investigate the conformations of this protein in the absence and presence of substrate. Previous nuclear magnetic resonance results reported by this laboratory have shown that there are extensive spectral changes in this protein upon the addition of L-histidine. When resonances from individual amino acid residues of a protein can be resolved in the proton nuclear magnetic resonance spectrum, a great deal of detailed information about substrate-induced structural changes can be obtained. In order to gain a deeper insight into the nature of these structural changes, deuterated phenylalanine or tyrosine has been incorporated into the bacteria. Proton nuclear magnetic resonance spectra of selectively deuterated histidine-binding protein J were obtained and compared to the normal protein. Several of the proton resonances have been assigned to the various aromatic amino acid residues of this protein. A model for the high-affinity transport of L-histidine across the cytoplasmic membrane of S typhimurium is proposed. This model, which is a version of the pore model, assumes that both P and Q proteins are membrane-bound and that the interface between these two proteins forms the channel for the passage of substrate. The histidine-binding protein J serves as the “key” for the opening of the channel for the passage of L-histidine. In the absence of substrate, this channel or gate is closed owing to a lack of appropriate interactions among these three proteins. The channel can be opened upon receiving a specific signal from the “key”; namely, the substrate-induced conformational changes in the histidine-binding protein J molecule. This model is consistent with available experimental evidence for the high-affinity transport of L-histidine across the cytoplasmic membrane of S typhimurium.     

Schistosoma mansoni: Decarboxylation of 5-hydroxytryptophan, l-dopa,and l-histidine in adult and larval schistosomes

Homogenates of adult and larval Schistosoma mansoni, decarboxylate 5-hydroxytryptophan (5-HTP) and l-dopa in vitro, but not l-histidine. The enzyme responsible, similar to mammalian aromatic-l-amino acid carboxy-lyase (EC 4.1.1.28), has a K_m of 5.1 × 10^?5M, a V_max of 1.1 nmole/30 min/mg protein, and high activity at pH 7.9. No evidence for a specific l-histidine decarboxylase (EC 4.1.1.22) was found in either stage of the schistosome. In vivo, schistosomules form serotonin from 5-HTP, detectable by thin-layer chromatography, within 3–4 hr after addition of ¹⁴C-labeled 5-HTP to the medium. Addition of ¹⁴C-labeled tryptophan under similar conditions results in no detectable formation of 5-HTP.     

The binding of cationic surfactants by DNA

Isotherms for the binding of dodecyltrimethylammonium (DTA⁺) and tetradecyltrimethylammonium (TTA⁺) ions by DNA in aqueous solution at 30°C are reported. The binding isotherms were determined using a potentiometric technique with cationic surfactant-selective electrodes. The DNA concentrations used are 5 × 10^?4 and 10^?3 equiv./kg, Surfactant concentrations varying from 3 × 10^?6M to the critical micelle concentration. The influence of added NaCl (0.01 M) on the binding process is studied. The binding process is shown to be highly cooperative. Applying the binding theory of Schwarz and of Satake and Yang, binding constants and cooperativity parameters can be calculated. The binding constant K is found to be 1.2kT larger for TTA⁺ than for DTA⁺ in salt-free solution, and 1.4kT larger for TTA⁺ than for DTA⁺ in 0.01 M NaCl. The cooperativity parameter u is about 1.4kT larger for TTA⁺ in salt-free solution and 1.2kT larger in 0.01 M NaCl. It is concluded that the hydrophobic part of the bound surfactant is not completely immersed in the hydrophobic DNA core, but also interacts with other surfactant molecules. This situation is compared to the case of micelle formation.     

Vanadyl(IV) conalbumin. II. Mixed metal and anion binding studies

Electron paramagnetic resonance (epr) studies demonstrate that at low levels of conalbumin (CA) saturation with Fe³⁺ or VO²⁺, a ph-dependent preference of the metal exists for different protein binding-site configurations,A, B, and C. The vanadyl ion epr spectra of mixed VO²⁺, Fe³⁺-conalbumin in which Fe³⁺ is preferentially bound to the N- or C-terminal binding site are consistent with all three configurations being formed at both metal sites. At high pH the spectra suggest interaction between binding sites. In the absence of HCO₃^?, VO²⁺ is bound almost exclusively in B configuration; a full binding capacity of 2 VO²⁺ per CA is retained. Stoichiometric amounts of HCO₃^? convert the epr spectrum from B to an A, B, C type. Addition of oxalate to bicarbonate-free preparations converts the B spectrum to an A′, B, C′ type where the B resonances have lost intensity to the A′ and C′ resonances but have not changed position. The data suggest that configuration B is anion independent and that only one equivalent of binding sites at pH 9 responds to the presence of HCO₃^1? or oxalate by changing configuration but not metal binding capability. The form of the bound anion may be HCO₃^? rather than CO₃^2?. The formation rate of the colored ferric conalbumin complex by oxidizing Fe²⁺ to Fe³⁺ in limited HCO₃^? at pH 9 is also consistent with one equivalent of sites having different anion requirements than the remaining sites. Increased NaCl or NaClO₄ concentration or substitution of D₂O for water as solvent affect the environment of bound VO²⁺, but the mechanisms of action are unknown.     

Determination of the molecular weight of proteins in heterogeneous mixtures: use of an air-driven ultracentrifuge for the analysis of protein--protein interactions.

A high-speed air-driven ultracentrifuge (Airfuge) has been used to study the molecular weights of proteins in heterogeneous mixtures. The method is based on previous studies (M. A. Bothwell, G. J. Howlett, and H. K. Schachman, 1978, J. Biol. Chem., 253, 2073–2077) which showed that at sedimentation equilibrium in the Airfuge the fraction of a protein remaining in an upper fraction of the Airfuge tube is almost linearly related to the exponential of the reduced molecular weight of the protein. In this study the total fraction of each particular protein remaining in an upper fraction of the Airfuge tube is determined by quantitative sodium dodecyl sulfate-gel electrophoresis. This procedure allows a wide range of proteins to be analyzed in a single Airfuge experiment. The method yields the “native” molecular weights of the protein components and is independent of the shape of the macromolecules being studied. Interactions occurring between the components in solution can be detected from the Airfuge data, and procedures are described which allow the experimental data for such interactions to be analyzed in terms of an equilibrium constant for the interaction. Results obtained for the electrostatic interaction at neutral pH between lysozyme and ovalbumin (K = 1.1 × 10⁵, m^?1) and lysozyme and bovine serum albumin (K = 1.0 × 10⁵, m^?1) agree well with literature values.     

Conformational study of calf brain tubulin

The conformation of calf brain tubulin has been monitored by circular dichroism, optical rotatory dispersion, and spectrophotometric titration as a function of pH, temperature, ligand concentrations, and denaturants. At pH 7, calf brain tubulin maintains its structural integrity between 5 and 37 °C as determined by circular dichroism. Furthermore, the presence of MgCl ₂ up to 1.6 × 10^?2m does not induce any observable changes in the circular dichroism spectra, nor does 10^?4m CaCl₂. With increasing pH, the spectral data can best be described as a gradual loosening of the secondary structure between pH 7 and 9. Both spectral and titrimetric data suggest a major unfolding of tubulin between pH 9 and 10. The apparent pK of tyrosine shifts from 10.85 to 9.98 upon transferring from buffer to 6 m guanidine hydrochloride, indicating that at least 14 of the 15 tyrosine groups are not fully accessible to protons in the native protein. The single disulfide bridge in calf brain tubulin helps to maintain a domain which is highly resistant to unfolding by denaturants.     

The influence of anions and inhibitors on the catalytic metal ion in Co(II)-substituted horse liver alcohol dehydrogenase

¹H-NMR and electronic spectroscopic data are reported for the interaction of the effector molecule imidazole and the inhibitor molecule pyrazole with horse liver alcohol dehydrogenase whose catalytic zinc ions were replaced by Co(II). In addition ¹³C-NMR and optical data are given for the binding of acetate to this enzyme species. For the binary complex with imidazole an assignment of the protons of the metal-coordinated imidazole has been made and it was found that the rate of exchange of the effector molecule is slow on the NMR time scale. In the presence of NADH which is bound to the open conformation of the binary complex, the most pronounced change is a shift of the -CH₂ protons of the metal-coordinated cysteine residues which is attributed to hydrogen bonding interactions between the carboxamide group of the nicotinamide moiety with cysteine 46. The ¹H-NMR spectra of the binary complex of Co(II)-HLADH with pyrazole show resonances assigned to the protons in the 3-and 4-positions of the bound inhibitor, the NH proton resonance is not detectable. In the ternary complex with pyrazole and NAD⁺ only the resonances of the -CH₂ protons (beyond 150 ppm) are changed whereas the protons of histidine 67 and the bound inhibitor are unchanged. The data demonstrate that the coordination environment of the catalytic metal ion is changed very little when the protein changes from the open to the closed conformation. The only changes observed are the -CH₂ proton resonances of the metal-coordinating cysteines which are sensitive to local conformational changes within the ternary complex Co(II)-HLADH · Imidazole · NADH in the open conformation or global changes in the ternary complex Co(II)-HLADH · Pyrazole · NAD⁺ in the closed conformation. Acetate which can be regarded as a substrate model was shown to induce a similar change in the optical spectra of the Co(II) enzyme as all other anions observed so far. From the optical changes a dissociation constant of acetate at the catalytic metal site of 200±50 mM was calculated and from the changes of the ¹³C-NMR linewidth of ¹³C acetate direct bonding of the anion to the catalytic Co(II) ion can be demonstrated to occur under the conditions of rapid exchange. The implications of these data for the assessment of tetracoordination around the catalytic metal ion as well as the chemical nature of intermediates occurring along the catalytic pathway are discussed.This work has been performed with contribution of the project Projetto Strategico Biotechnologie CNR and with financial support from the Deutsche Forschungsgemeinschaft, NATO, Bundesminister für Forschung und Technologie, and the Universität des Saarlandes     

Purification and characterization of selenium-glutathione peroxidase from hamster liver

Hamster liver glutathione peroxidase was purified to homogeneity in three chromatographic steps and with 30% yield. The purified enzyme had a specific activity of approximately 500 μmol cumene hydroperoxide reduced/min/mg of protein at 37 °C, pH 7.6, and 0.25 mm GSH. The enzyme was shown to be a tetramer of indistinguishable subunits, the molecular weight of which was approximately 23,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single isoelectric point of 5.0 was attributed to the active enzyme. Amino acid analysis determined that selenocysteine, identified as its carboxymethyl derivative, was the only form of selenium. One residue of cysteine was found to be present in each glutathione peroxidase subunit. The presence of tryptophan was colorimetrically determined. Pseudo-first-order kinetics of inactivation of the enzyme by iodoacetate was observed at neutral pH with GSH as the only reducing agent. An optimal pH of 8.0 at 37 °C and an activation energy of 3 kcal/mol at pH 7.6 were found. A ter-uni-ping-pong mechanism was shown by the use of an integrated-rate equation. At pH 7.6, the apparent second-order rate constants for reaction of glutathione peroxidase with hydroperoxides were as follows: k₁ (t-butyl hydroperoxide), 7.06 × 10⁵ mm min^?1; k₁ (cumene hydroperoxide), 1.04 × 10⁶ mm^?1 min^?1; k₁ (p-menthane hydroperoxide), 1.2 × 10⁶ mm^?1 min^?1; k₁ (diisopropylbenzene hydroperoxide), 1.7 × 10⁶ mm^?1 min^?1; k₁ (linoleic acid hydroperoxide), 2.36 × 10⁶ mm^?1 min^?1; k₁ (ethyl hydroperoxide), 2.5 × 10⁶ mm^?1 min^?1; and k₁ (hydrogen peroxide), 2.98 × 10⁶ mm^?1 min^?1. It is concluded that for bulky hydroperoxides, the more hydrophobic the substrate, the faster its reduction by glutathione peroxidase.     

A study of the histidine residues of human carbonic anhydrase B using 270 MHz proton magnetic resonance

Nine resonances in the 270 MHz proton magnetic resonance spectrum of human carbonic anhydrase B have been identified with imidazole C₍₂₎ protons of histidine residues, six of which are observed to titrate with pK_a values in the range 4.7 to 7.4. The behaviour of the nine resonances has been studied in the presence of the inhibitors, iodide, cyanide, acetate, hexacyanochromate, and imidazole. Measurements have also been made of the enzyme in its apo, cobalt, and mono-alkylated forms. Used in conjunction with the crystal structure, these results have enabled the tentative assignment of all nine resonances to particular histidine residues in the amino-acid sequence. Three of the active-site histidines at positions 64, 67, and 200 have low pK_a values and cannot be directly linked to the activity of the enzyme. However, the resonances assigned to the three metal-liganding histidines do exhibit changes on anion binding and with pH, which parallel changes in the esterase activity. These results are consistent with the model of an ionizable water molecule bound to the zinc ion.Linewidth measurements of the resonances of the histidine residues on the enzyme surface are used to estimate pseudo-first-order rate constants of the order of 4 × 10³ s^?1 for D⁺ exchange between imidazole N and solvent in the absence of buffer. These rates are observed to increase in the presence of small amounts of the buffers Tris and imidazole.     

Biochemical analysis of a Chinese cabbage phytocystatin-1

The phytocystatins are inhibitors of papain-like cysteine proteinases that are implicated in defense mechanisms and the regulation of protein turnover. BCPI-1, a Brassica rapa (Chinese cabbage) phytocystatin isolated from flower buds, contains an extended C-terminal region that contains a single Cys residue at position 102. In an effort to investigate the role of the C-terminus and this Cys residue in BCPI-1 activity, purified recombinant proteins of BCPI-1, including wild-type BCPI-1 (wtBCPI-1), N-terminus BCPI-1 (BCPI-1??C), C-terminus BCPI-1 (BCPI-1??N), and BCPI-1 with a single Cys residue exchange to Ser (BCPI-1C102S), were generated and their inhibitory activities against papain were investigated. Kinetic analysis revealed that the monomeric forms of wtBCPI-1 (K _i = 6.84 ± 0.3 × 10^?8 M) inhibited papain more efficiently than the dimeric forms of wtBCPI-1 (K _i = 1.01 ± 0.5 × 10^?7 M). Experiments with recombinant BCPI-1C102S demonstrated that the dimerization of wtBCPI-1 caused by the formation of an intermolecular disulfide bond at the cysteine residue. The inhibitory activity of the recombinant proteins, except BCPI-1??N, was reduced in the pH range of 7.0?C11.5 and was highly stable over a wide range of temperatures. Thus, dimerization mediated by the cysteine residue in the extended C-terminal region and alkaline conditions reduced the inhibitory activity of BCPI-1.     

Hydrogen-deuterium exchange of the tryptophan residues in bovine α-lactalbumin

Effects of deuteration on the Raman spectrum of a tryptophan residue have been examined. The 1386 cm^?1 line of deuterated tryptophan residue has been found to be useful for tracing the hydrogen-deuterium exchange reaction of this residue in a protein. An examination on bovine α-lactalbumin at pH 6.4 and at 20°C indicates that two of the four tryptophan residues exchange with a rate constant much greater than 9 × 10^?4 sec^?1, while the other two exchange with a rate constant of 4 × 10^?5 sec^?1. The latter two have been assigned to Trp 28 and Trp 108 of this protein. The kinetics of hydrogen-deuterium exchange reaction of completely “free” tryptophan residue have been examined by a proton magnetic resonance study on tryptophan itself. By taking the result of this examination into account, the chance of exposure to the solvent for Trp 28 or Trp 108 has been estimated to be 3 × 10^?6 at pH 6.4 and at 20°C.     

Reversible trifluoroacetic acid-induced conformational changes in glycophorin as detected by proton nuclear magnetic resonance spectroscopy

High-field (270 MHz) ¹H-NMR has been employed to study the solution conformation of glycophorin A, a sialoglycoprotein which spans the human erythrocyte membrane. Glycophorin A is one of the most fully characterized integral membrane proteins known, making it an excellent model for the study of membrane-bound proteins. This protein consists of three distinct domains: a glycosylated extracellular N-terminus, a hydrophobic intramembranous segment, and a polar cytoplasmic C-terminus. These domains contain aromatic residues which serve as convenient ¹H-NMR conformational probes. The aromatic region of the NMR spectrum of glycophorin A in ²H₂O shows single, well-resolved His and Tyr resonances. No resonances are observed, however, for the Phe residues which are located in or near the hydrophobic domain. These observations suggest that considerable heterogeneity with respect to segmental motions exists within the protein. This is consistent with circular dichroism data showing the intramembranous segment to be completely helical with the extremities of the protein being predominantly random coils. The helix of the hydrophrobic domain is remarkably resistant to conventional denaturing conditions including variations in pH, and temperature, and treatment with guanidine hydrochloride. However, in trifluoroacetic acid, which strongly solvates peptide backbones, there is extensive reversible unfolding of the helical structure as evidenced by the appearance of Phe resonances. Solvent titration experiments indicate that approximately a 1 : 1 volume ratio of trifluoroacetic acid to ²H₂O is required to initiate unfolding of the helix.     

Differential hydrogen ion titrations of the histidine residues in Helix pomatia haemocyanin

The properties of the histidine residues in Helix pomatia haemocyanin have been studied by differential hydrogen ion titrations. In oxy-and deoxyhaemocyanin 31 × 10^?5 histidine residues per g protein are titrated in contrast to 35 × 10^?5 residues in apohaemocyanin. The difference corresponds to a stoichiometry of one histidine residue per copper atom bound. Even in apohaemocyanin about 6 × 10^?5 histidine residues per g protein are not titrated in their normal pH region.In the presence of sufficient calcium to displace the dissociation completely out of the titration region the titration curve of apohaemocyanin could he linarized according to the model of Linderstrøm—Lang. In oxy- and deoxyhaemocyanin, however, a distinct deviation from linearity was found under the same conditions. In the absence of calcium the effect of the dissociation adds up to this deviation.The electrostatic interaction factors were determined for the protein at 0.1 M KCl and for the dissociation products: halves and tenths at 1.0 M KCl. The electrostatic interaction factor for the wholes and the halves are much smaller than the values calculated from the Linderstrøm—Lang equation, using the radius of the equivalent sphere either obtained from electron microscopy or from the partial specific volume. This is probably due to solvent penetration. For the tenths at 1.0 M KCl. this effect is small,     

Influence of stearic acids on resveratrol-HSA interaction

The interaction between the natural polyphenol resveratrol and human serum albumin (HSA), the most abundant transport protein in plasma, has been studied in the absence and in the presence of up to six molecules of stearic acids (SA) pre-complexed with the protein. The study has been carried out by using the intrinsic fluorescence of both HSA and resveratrol. Protein and polyphenol fluorescence data indicate that resveratrol binds to HSA with an association constant k _a ?=?(1.10?±?0.14)?×?10⁵?M^?1 and (1.09?±?0.02)?×?10⁵?M^?1, respectively, whereas Job plot evidences the formation of an equimolar protein/drug complex. Low SA content associated with HSA does not affect significantly the structural conformation of the protein and its interaction with resveratrol, whereas high SA content induces conformational changes in the protein, and reduces resveratrol binding affinity. The photostability of resveratrol in the different samples changes in the order: buffer <?(high [SA]/HSA)?<?HSA?<?(low [SA]/HSA). The results on (SA/HSA)-resveratrol samples highlight the ability of the protein to bind hydrophobic and amphiphilic ligands and to protect from degradation an important antioxidant molecule under biologically relevant conditions.     

X-ray crystal structure of maltitol (4-O-α-d-glucopyranosyl-d-glucitol

Maltitol, crystallised from aqueous solution, has m.p. 146.5–147°, [α]_d + 106.5° (water), and is orthorhombic with the space group P2₁2₁2₁ and Z = 4, and with cell dimensions a = 8.166(5), b = 12.721(9), and c = 13.629(6) Å. The molecule shows a fully extended conformation with no intramolecular hydrogen-bonds. All nine hydroxyl groups are involved in intermolecular hydrogen-bond networks and in bifurcated, finite chains. The d-glucopyranosyl moiety has the ⁴C₁ conformation, and the conformation about the C-5–C-6 bond is gauche-gauche. The d-glucitol residue has the bent [ap, Psc, Psc (APP)] conformation. The empirical formula for the solubility in water is C = 119.1 + 1.204 T + 4.137 × 10^?2 T² ? 7.137 × 10^?4 T³ + 7.978 × 10^?6 T⁴. The thermal properties are as follows: ΔH_f = 13.5 kcal.mol^?1, and Q = ?5.57 kcal.mol^?1.     

Muscarinic cholinergic binding sites in the developing avian heart.

The potent muscarinic cholinergic antagonist 3-quinuclidinyl benzylate (QNB) has been used to detect and quantify muscarinic receptors in the developing chick heart. Specific binding in microsomal pellets prepared from hearts ranging in age from 70 hr in ovo to adulthood was examined and was found to increase from 4 × 10^?13 moles of [³H]QNB bound/mg of protein at the earliest stage tested to 5 × 10^?12 moles of [³H]QNB/mg of protein at birth and then to drop slightly to 2 × 10^?12 moles of [³H]QNB/mg of protein at the latest age tested. The developmental significance of these results is discussed.     

The presence of LHRH-like receptors in Dunning R3327H prostate tumors

Quantitative analyses of LH-RH-like membrane receptors were performed in five tumors from the transplantable Dunning R3372H rat prostatic adenocarcinoma. The binding of D-Trp⁶-LH-RH, an agonist of LH-RH, was observed in all 5 tumors. The antagonist [Ac-Dp-Cl-Phe^1,2,D-Trp³,D-Lys⁶,D-Ala¹⁰]-LH-RH was bound to 4 tumors. The apparent equilibrium dissociation constant (K_d) for D-Trp⁶-LH-RH receptor was from 2.6–3.9 × 10^?10 M. The apparent equilibrium B_max values (maximum number of binding sites) were from 17.2–86.0 fmol/mg membrane protein for D-Trp⁶-LH-RH receptor. The K_d for the antagonist was from 2.4–2.7 × 10^?10 M and the B_max values were from 35.5–66.0 fmol/mg membrane protein. Similar binding studies performed in 6 normal rat prostates showed no binding capacities.     

S-adenosylmethionine: Protein-carboxyl methyltransferase from erythrocyte

Protein methylase II (S-adenosylmethionine:protein—carboxyl methyltrans-ferase), which modifies free carboxyl residues of protein, was purified from both rat and human blood, and properties of the enzymes were studied. The pH optima for the reaction were dependent on the substrate proteins used; pH 7.0 was found with endogenous substrate, 6.1 with plasma, 6.5 with γ-globulin, and 6.0 with fibrinogen. The molecular weight of the enzymes from both rat and human erythrocytes were identical (25,000 daltons) determined by Sephadex G-75 chromatography. Partially purified enzyme from rat erythrocytes showed three peaks on electrofocusing column at pH 4.9, 5.5 and 6.0. The K_m values of the enzymes from rat and human erythrocytes showed 3.1 × 10^?6m and 1.92 × 10^?6m at pH 6.0, 1.96 × 10^?6m and 1.78 × 10^?6m at pH 7.2, respectively, for S-adenosyl-l-methionine. It is also found that S-adenosyl-l-homocysteine is a competitive inhibitor for protein methylase II with K_i value of 1.6 × 10^?6m.     

Inactivation of Copper,Zinc superoxide dismutase by H2O2 : Mechanism of protection

Cu,Zn SOD is known to be inactivated by HO₂⁻ and to be protected against that inactivation by a number of small molecules including formate, imidazole, and urate. This inactivation has been shown to be due to oxidation of a ligand field histidine residue by a bound oxidant formed by reaction of the active site Cu(II) with HO₂⁻. We now report that protective actions of both formate and NADH increase as the pH was raised in the range 8.0–9.5. This is taken to indicate increased accessibility of the Cu site with rising pH and/or increased reactivity of the bound oxidant toward exogeneous substrates at high pH. Formate appears to act as a sacrificial substrate that protects by competing with the endogenous histidine residue for reaction with the bound oxidant, or that repairs the damage by reducing the histidyl radical intermediate. The same is likely also true of NADH.