Calcium and potassium ion binding by tobacco mosaic virus ribonucleic acid

Calcium and potassium ion titration experiments were performed on solutions of tobacco mosaic virus RNA using ion-specific electrodes. The data obtained were analyzed using Scatchard and Klotz plots for the number of binding sites per nucleotide (n), and the apparent stability constant for complex formation, beta Me. The experimental design also allowed for the determination of the number of protons released per metal ion bound, chi. The calcium ion titration in water yielded values of 0.45 for n, 6.03 for log beta Ca and 0.24 for chi. When this titration was repeated in 0.01 M-KCl, the values were found to be 0.11 for n, 5.08 for log beta Ca and zero for chi. An aqueous potassium titration was also performed, with values for n, log beta K and chi of 0.25, 2.96 and less than 0.10, respectively.     

The Donnan effect in tobacco mosaic virus and its components

Osmotic pressure studies were carried on tobacco mosaic virus (TMV) and its components, protein and RNA, as well as on bis(3,3′-aminopropyl)amine, reported to be present in TMV preparations. Solvents were phosphate and barbital buffers at different values of pH and ionic strength. Measurements were made at room temperature. The Donnan effect was exhibited by TMV protein in phosphate buffer of 0.01 ionic strength at pH values ranging between 5.8 and 7.5. The observed values of the Donnan effect at pH 5.8 and 5.97 were in reasonable agreement with theoretical values calculated from the charge obtained by hydrogen ion titration. TMV-RNA in phosphate buffer at pH 7.5 and ionic strength 0.01 did not exhibit more than 1% of the expected Donnan effect. This is explained tentatively as the result of firm binding of metal ions. Negative values of osmotic pressure were observed with bis(3,3′-aminopropyl)amine. Similar anomalous osmosis was sometimes observed with TMV protein and with TMV. In agreement with earlier observations, TMV did not exhibit the Donnan effect in phosphate buffer of 0.01 ionic strength at pH values ranging from 5.5 to 8.0. However, TMV dialysed extensively in the presence of EDTA at pH 8.5 and TMV produced by reconstitution of purified protein and RNA did exhibit the Donnan effect in both phosphate and barbital buffers. The magnitude was of the same order as that calculated from the net charge determined by hydrogen ion titration. When reconstituted TMV, which did exhibit Donnan effect, was treated with calcium ions, the effect was abolished.     

Stabilizing influence of calcium and magnesium ions on the decameric structure of chiton hemocyanins

The stabilizing effects of Ca2+ and Mg2+ ions on the decameric structure of hemocyanins from two representative chitons, Stenoplax conspicua and Mopalia muscosa were investigated by light-scattering molecular weight measurements, ultracentrifugation, absorbance, and circular dichroism methods. The dissociation profiles at any given pH resulting from the decrease in divalent ion concentration, investigated at a fixed protein concentration of 0.1 g.liter-1, could be fitted by a decamer-to-dimer-to monomer scheme of subunit dissociation. The initial decline in the light-scattering molecular weight curves required one or two apparent binding sites per hemocyanin dimer formed as intermediate dissociation product, with apparent dissociation constants (kD,2) for Ca2+ ions of 0.7 to 7 X 10(-4) M, not very different from the value of 2.5 X 10(-4) M obtained by Makino by equilibrium dialysis for the hemocyanin of the opistobranch, Dolabella auricularia. The binding of Mg2+ ion to S. conspicua and M. muscosa hemocyanins appears to be both weaker than the binding of Ca2+ and more pH dependent, with kD,2 values ranging from the 3 X 10(-4) to 4 X 10(-2) M at pH 8.5 to 9.5. The dissociation the decameric hemocyanin species (sedimentation coefficient ca. 60 S) is also observed in the ultracentrifugation with the initial appearance of 18-20 S dimers, followed by a shift in equilibrium to monomeric species of lower sedimentation rates of 11-12 S as the divalent ion concentration is reduced below 1 X 10(-4) M Ca2+ and Mg2+. The dissociation of dimers to monomers in the second step of the reaction is characterized by one or two binding sites per subunit and a somewhat stronger affinity for divalent ions, indicated by apparent dissociation constants (kD,1) of 0.7 X 10(-4) to 3 X 10(-3) M. Circular dichroism and absorbance measurements at 222 and 346 nm suggest no significant changes in the conformation of the hemocyanin subunits produced by the different stages of subunit dissociation.     

Calcium binding of troponin C. I. A potentiometric titration study.

Structural changes of troponin C on calcium binding were studied by hydrogen ion titration, circular dichroism, and fluorescence measurements. The potentiometric titration curves in the carboxyl region are shifted towards lower pH with calcium binding. The intrinsic pK of the carboxyl groups at the calcium binding sites decreases by 0.8 pK unit on calcium binding; on the other hand, magnesium ions have little effect on the intrinsic pK of the carboxyl groups. The intrinsic pK of the imidazole group is not affected by calcium binding. The value of w, an electrostatic interaction factor, is identical for calcium-free and calcium-bound troponin C and is about half of the value calculated assuming a compact sphere. The results of difference titration on the calcium binding indicate that the pH of troponin C solution increases on addition of CaCl2 up to 2 mol of Ca2+ per mol of troponin C and then decreases on further addition of CaCl2. The pH increase is depressed in the presence of MgCl2, in the low pH region, or at high ionic strength. The pH increase is also observed on addition of MgCl2. The ellipticity at 222 nm was measured under the same conditions as the difference titration measurements, and the relation between the pH change and the conformational change of troponin C on calcium binding is discussed based on the results obtained. The number of calcium binding sites and the binding constants estimated by analysis of these difference titration curves were in agreement with the results of Potter and Gergely (22). No magnesium binding site was observed. The tyrosine fluorescence measurements indicated that the binding site near tyrosine-109 is one of the high affinity sites.     

Divalent cation binding to reduced and octanoyl acyl-carrier protein

Mn(II) EPR binding studies with reduced acyl-carrier protein (ACP-SH) strongly suggest the presence of two relatively high-affinity manganese-binding sites (average Kd/site approximately 80 microM) at physiological pH. Lowering the pH or titrating with sodium chloride reduces the average number of bound divalent cations and decreases the binding affinity. This is consistent with the idea that anionic ligand(s), e.g. the carboxylate of glutamic or aspartic acid, on the protein are involved in manganese ion coordination. At pH values above 8.0, binding affinity is also reduced, whereas the average number of bound metal ions increases to about five at pH 8.5. By interacting weakly with divalent cations (average Kd/site approximately 1 mM), octanoyl acyl-carrier protein (OcoACP) exhibits dramatically different metal-ion-binding properties compared to ACP-SH. Calcium and magnesium can compete in either ACP species for manganese binding. Photochemically-induced dynamic nuclear polarisation 1H-NMR experiments strongly suggest that ACP-SH and OcoACP undergo at pH-induced conformational change between pH 5.5 and pH 7.0, and that divalent cations stabilize the protein against such pH-induced structural perturbations.     

Physical investigations of the hemocyanin of the chiton, Cryptochiton stelleri (Middendorff)

The hemocyanin of the giant Pacific chiton, Cryptochiton stelleri has a molecular weight of 4.2 +/- 0.3 X 10(6), determined by light-scattering, and a sedimentation coefficient of 60S. The fully dissociated subunits in nondenaturing solvents, at pH 10.6, 1 X 10(-2)M EDTA and in 8.0 M urea, pH 7.4 have molecular weights of 4.10 X 10(5) and 4.35 X 10(5), close to one-tenth of the molecular mass of the parent hemocyanin decamers. In the pH region from about 3.5 to 11 the molecular weight (Mw), determined at constant protein concentration of 0.10 g1(-1) exhibits a bell-shaped molecular weight profile centering about the physiological pH of the hemolymph of 7.2. The pH-Mw profile is best accounted for in terms of a three state, decamer-dimer-monomer dissociation scheme. Analysis of the Mg2+ and Ca2+ effects on the molecular weight transitions suggest stabilization of the hemocyanin decamers through one bound divalent ion per hemocyanin monomer or dimer. Urea, GdmCl, and the higher members of the chaotropic salt series are effective dissociating agents for Cryptochiton stelleri hemocyanin. The dissociation profile obtained with urea at pH 8.5, 0.01 M Mg2+, 0.01 M Ca2+ has been analyzed in terms of both the two- and three-species schemes of subunit-dissociation. Hydrophobic stabilization of the subunit contacts is suggested by the large number of apparent amino acid groups (Napp), of the order of 30 between dimers stabilizing the decamers, and 120 apparent amino acid groups between each monomer forming the constituent dimers.     

The structure of the zinc sites of Escherichia coli DNA-dependent RNA polymerase.

X-ray absorption spectroscopy is ideally suited for the investigation of the electronic structure and the local environment (approximately 5 A) of specific atoms in biomolecules. While the edge region provides information about the valence state of the absorbing atom, the chemical identity of neighboring atoms, and the coordination geometry, the extended x-ray absorption fine structure region contains information about the number and average distance of neighboring atoms and their relative disorder. The development of sensitive detection methods has allowed studies using near physiological concentrations (as low as approximately 100 microM). RNA polymerase from Escherichia coli contains two zinc atoms: one tightly bound in the beta' subunit, the subunit that participates in template binding, and the other loosely bound in the beta subunit, the subunit that participates in substrate binding. X-ray absorption studies of these zinc sites in the native protein and of the zinc site in the beta' subunit after removal of the zinc in the beta subunit site by p-(hydroxymercuri)benzenesulfonate (Giedroc, D. P., and Coleman, J. E. (1986) Biochemistry 25, 4969-4978) indicate that both zinc sites have octahedral coordination. The zinc in the beta' subunit site has four sulfur ligands at an average distance of 2.36 +/- 0.02 A and two oxygen (or nitrogen) ligands at an average distance of 2.23 +/- 0.02 A. The beta subunit zinc site has five sulfur ligands at an average distance of 2.38 +/- 0.01 A and one histidine nitrogen ligand at 2.14 +/- 0.02 A. These results are in general agreement with earlier biochemical and spectroscopic studies.     

On the location and function of the noncatalytic sites on the bovine heart mitochondrial F1-ATPase

Modification of Tyr-345 at a catalytic site in a single beta subunit of the bovine heart mitochondrial F1-ATPase (MF1) by 5'-p-fluorosulfonylbenzoylinosine did not affect subsequent labeling of noncatalytic sites at Tyr-368 and His-427 in three copies of the beta subunit by 5'-p-fluorosulfonylbenzoyladenosine (FSBA). These results clearly show that the beta subunit contains at least parts of the catalytic and noncatalytic nucleotide binding sites. Inactivation of MF1 by 96% with FSBA was accompanied by a decrease in the endogenous ADP content from 1.86 to 0.10 mol per mol of MF1. Decrease in the endogenous ADP content during the inactivation of the enzyme with FSBA paralleled loss in activity in a manner which suggests that the reaction of FSBA with an open noncatalytic site promoted release of ADP from another noncatalytic site until the third site reacted with FSBA. Two pKa values of about 5.9 and 7.6 were observed on the acid side of the pH optimum in the pH-rate profile for ATP hydrolysis catalyzed by MF1 in neutral acid buffers. In contrast, a single pKa of 5.9 was present in the pH-rate profile for ITP hydrolysis catalyzed by the enzyme in the same buffers. The augmented rate observed for ATP hydrolysis at pH 8.0, over that observed at pH 6.5, was lost as the enzyme was inactivated by FSBA in a manner suggesting that modulation is lost as the third noncatalytic site is modified. This suggests that ATP hydrolysis by MF1 is modulated in a pH-dependent manner by ATP binding to an open noncatalytic site. Two other modulations associated with binding of adenine nucleotides to noncatalytic sites, ADP-induced hysteretic inhibition and apparent negative cooperativity reflected by the Hill coefficient for the hydrolysis of 50-3000 microM ATP at pH 8.0, also disappeared as the third noncatalytic site reacted with FSBA.     

Effects of pH on the interaction of ligands with the (H+ + K+)-ATPase purified from pig gastric mucosa

The effects of K+, Na+ and ATP on the gastric (H+ + K+)-ATPase were investigated at various pH. The enzyme was phosphorylated by ATP with a pseudo-first-order rate constant of 3650 min-1 at pH 7.4. This rate constant increased to a maximal value of about 7900 min-1 when pH was decreased to 6.0. Alkalinization decreased the rate constant. At pH 8.0 it was 1290 min-1. Additions of 5 mM K+ or Na+, did not change the rate constant at acidic pH, while at neutral or alkaline pH a decrease was observed. Dephosphorylation of phosphoenzyme in lyophilized vesicles was dependent on K+, but not on Na+. Alkaline pH increased the rate of dephosphorylation. K+ stimulated the ATPase and p-nitrophenylphosphatase activities. At high concentrations K+ was inhibitory. Below pH 7.0 Na+ had little or no effect on the ATPase and p-nitrophenylphosphatase, while at alkaline pH, Na+ inhibited both activities. The effect of extravesicular pH on transport of H+ was investigated. At pH 6.5 the apparent Km for ATP was 2.7 microM and increased little when K+ was added extravesicularly. At pH 7.5, millimolar concentrations of K+ increased the apparent Km for ATP. Extravesicular K+ and Na+ inhibited the transport of H+. The inhibition was strongest at alkaline pH and only slight at neutral or acidic pH, suggesting a competition between the alkali metal ions and hydrogen ions at a common binding site on the cytoplasmic side of the membrane. Two H+-producing reactions as possible candidates as physiological regulators of (H+ + K+)-ATPase were investigated. Firstly, the hydrolysis of ATP per se, and secondly, the hydration of CO2 and the subsequent formation of H+ and HCO3-. The amount of hydrogen ions formed in the ATPase reaction was highest at alkaline pH. The H+/ATP ratio was about 1 at pH 8.0. When CO2 was added to the reaction medium there was no change in the rate of hydrogen ion transport at pH 7.0, but at pH 8.0 the rate increased 4-times upon the addition of 0.4 mM CO2. The results indicate a possible co-operation in the production of acid between the H+ + K+-ATPase and a carbonic anhydrase associated with the vesicular membrane.     

Aspartate transcarbamylase of Escherichia coli. Heterogeneity of binding sites for carbamyl phosphate and fluorinated analogs of carbamyl phosphate.

Some preparations of both native aspartate transcarbamylase from Escherichia coli and catalytic subunit have fewer tight binding sites per oligomer for carbamyl-P than the number of catalytic peptide chains. In contrast, the number of sites for the tight-binding inhibitor N-(phosphonacetyl)-L-aspartate does equal the number of catalytic chains in each case. Binding of the labile carbamyl-P was determined using rapid gel filtration, with conversion to stable carbamyl-L-aspartate during collection. Native enzyme (six catalytic chains) obtained from cells grown under the conditions of J.C. Gerhart and H. Holoubek (J. Biol. Chem. (1967) 242, 2886-2892) has 5.4 tight sites for carbamyl-P at pH 8.0 (KD = 9.9 muM), whereas native enzyme from cells grown with higher concentrations of glucose, uracil, and histidine (to yield more enzyme per unit volume of culture) has only 1.9 tight sites at pH 8.0 (KD = 4.6 muM) and only 2.3 tight sites at pH 7.0 (KD = 2.6 muM). At pH 8.0, catalytic subunit (three catalytic chains) obtained from the former native enzyme has 2.2 tight sites for carbamyl-P (KD = 2.4 muM) and the number of sites is 2.3 in the presence of 35 mM succinate, whereas catalytic subunit obtained from the latter native enzyme has 1.8 tight sites (KD = 3.6 muM) in the absence of succinate and 2.3 tight sites in its presence. The number of tight binding sites is also less than the number of subunit peptide chains in 19F nuclear magnetic resonance experiments performed with catalytic subunit and two fluorinated analogs of carbamyl-P at comparable concentrations of analogs and active sites. A model is proposed in which incomplete removal of formylmethionine from the NH2 termini of the enzyme under conditions of extreme depression affects affinity for ligands.     

A specific effect of calcium ion on the polymerization-depolymerization of tobacco mosaic virus protein.

Tobacco mosaic virus protein stored in the cold at low ionic strength between pH 5 and 5.5 is highly polymerized. When such protein is brought to room temperature and mixed with acetate buffer and additional electrolyte to give a final pH of 6.5 and ionic strength of 0.1, the protein is still in the polymerized state. When the temperature is dropped to about 5 °C, the protein depolymerizes rapidly, in the normal manner, if the added electrolyte is barium chloride, magnesium chloride, or potassium chloride. However, if it is 0.01 m calcium chloride, the depolymerization is slow, requiring about 12 h to reach completion. When the temperature of this depolymerized solution is raised, the protein polymerizes rapidly; when the temperature is dropped, the protein depolymerizes rapidly, just as in solutions free of calcium.Ion-binding studies show that calcium is bound to the protein during the initial step when it is brought to pH 6.5 and room temperature in the presence of calcium. The calcium is released during the slow depolymerization when the temperature is dropped and is not bound again during polymerization at pH 6.5, brought about by an increase in temperature. This means that polymerized protein at pH 5.5 has a structure capable of binding calcium ions, probably a helical structure like that of the protein in the virus. When pH is raised to pH 6.5 at room temperature, this structure remains long enough for calcium to be bound when present. These calcium ions stabilize the polymer, resulting in slow depolymerization when the temperature is lowered. When the temperature is raised at pH 6.5, a different, looser polymer structure is obtained, one not capable of binding calcium.     

The Effect of pH on the Binding of Calcium to Pea Epicotyl Cell Walls and its Implications for the Control of Cell Extension

Cell walls were prepared from the epicotyls of dark-grown pea(Pisum sativum L.) seedlings. The walls were found to bind externally-added⁴⁵Ca²⁺, with a binding constant of 4 ? 10^–4 mol dm^–3and a maximum capacity of 1.5 ? 10^–8 g-ions of Ca²⁺ perg fresh weight of epicotyl. The binding capacity decreased asthe pH of the medium was decreased below 6.0, suggesting thatthe calcium was bound by an anionic group with an apparent pKof 4.7. More than half the calcium binding was due to polygalacturonicacid in the wall, since up to 60% of the calcium binding capacitywas removed by pre-incubation of the cell walls with polygalacturonase(E.C.3.2.1.15). Only small decreases in calcium binding wereseen following pre-incubation with protease, nucleases, phospholipaseand hemicellulase. These results indicate that calcium willbe displaced from the cell wall at hydrogen ion concentrationswhich are known to occur in the wall during wall extension.They are consistent with a mechanism by which calcium inhibitswall extension by forming ionic bridges between polygalacturonicacid molecules, and also with the hypothesis that calcium andhydrogen ions exert opposing influences on cell wall extensionby competing for the same binding sites on the polygalacturonicacid. Key words: Pea epicotyl, Cell wall, Calcium, pH     

Preparation and characterization of various Escherichia coli RNA polymerases containing one or two intrinsic metal ions

The Escherichia coli DNA-dependent RNA polymerase (RPase) holoenzyme (alpha 2 beta beta' sigma) possesses 2 mol equiv of Zn: beta and beta' subunits each contain one Zn ion. An in vitro metal-substitution method developed earlier (method I) was used to remove the two intrinsic Zn ions and then to reconstitute other metal ions into the beta subunit of RPase. One Cd or Hg ion was successfully reconstituted into half-active enzymes (rec-Cd1- or rec-Hg1-RPase), while Mn or Ni ion was not incorporated. A new, simplified in vitro metal-substitution method (method II), which omitted the low-pH treatment and subsequent urea dialysis in method I, was devised in this study. Consequently, Zn or Cd could be incorporated into both the beta and beta' subunits, resulting in rec-Zn2- or rec-Cd2-RPase, respectively. However, only one Hg was incorporated, probably due to steric hindrance by the large size of the Hg ion, while Mn, Ni, or Cr was not bound by the reconstituted enzyme, which instead incorporated only one Zn. Analysis of the metal content of various reconstituted RPases indicated that without low-pH treatment Zn bound to both the beta and beta' subunits when Zn concentrations were higher than 2 X 10(-6)M, but it bound only to the beta' subunit at lower concentrations. Moreover, low-pH treatment destroys the metal binding site in the beta' subunit. The metal sites on the beta and beta' subunits did not have significant affinity for the transition metals such as Mn, Ni, and Cr.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of an adenosine 3'':5''-cyclic monophosphate phosphodiesterase from baker''s yeast. Its binding to subcellular particles, catalytic properties and gel-filtration behaviour.

1. The 3':5'-cyclic AMP phosphodiesterase in the microsomal fraction of baker's yeast is highly specific for cyclic AMP, and not inhibited by cyclic GMP, cyclic IMP or cyclic UMP. Catalytic activity is abolished by 30 micrometer-EDTA. At 30 degrees C and pH8.1, the Km is 0.17 micrometer, and theophylline is a simple competitive inhibitor with Ki 0.7 micrometer. The pH optimum is about 7.8 at 0.25 micrometer-cyclic AMP, so that over the physiological range of pH in yeast the activity changes in the opposite direction to that of adenylate cyclase [PH optimum about 6.2; Londesborough & Nurminen (1972) Acta Chem. Scand. 26, 3396-3398].2. At pH 7.2, dissociation of the enzyme from dilute microsomal suspensions increased with ionic strength and was almost complete at 0.3 M-KCl. MgCl2 caused more dissociation than did KCl or NaCl at the same ionic strength, but at low KCl concentrations binding required small amounts of free bivalent metal ions. In 0.1 M-KCl the binding decreased between pH 4.7 and 9.3. At pH 7.2 the binding was independent of temperature between 5 and 20 degrees C. These observations suggest that the binding is electrostatic rather than hydrophobic. 3. The proportion of bound activity increased with the concentration of the microsomal fraction, and at 22 mg of protein/ml and pH 7.2 was 70% at I0.18, and 35% at I0.26. Presumably a substantial amount of the enzyme is particle-bound in vivo. 4. At 5 degrees C in 10 mM-potassium phosphate, pH 7.2, the apparent molecular weight of KCl-solubilized enzyme decreased with enzyme concentration from about 200 000 to 40 000. In the presence of 0.5M-KCl, a constant mol.wt. of about 55 000 was observed over a 20-fold range of enzyme concentrations.     

A direct fluorescence study of the transient steps induced by calcium binding to sarcoplasmic reticulum ATPase

The sarcoplasmic reticulum intrinsic fluorescence level was closely correlated with the ATPase functional state, from pH 5.5 to 8.5. The fluorescence signal was used in stopped flow measurements for direct study of transient pump kinetics after calcium binding or removal. The signal change time course, which depends solely on the free calcium concentration in the observation chamber, was analyzed as a single exponential. Rate constants (kobs) were relatively slow (5 to 20 s-1), indicating multistep interaction between calcium and the transport protein. At pH 7 and 20 degrees C, and in the presence of 100 mM potassium and 1 to 20 mM MgCl2, kobs first decreased, and then increased as the calcium concentration rose. Similar experiments were performed at pH 6. Data were analyzed according to a scheme in which sarcoplasmic reticulum . calcium complex formation is controlled by a slow isomerization step occurring either before or after the rapid calcium binding to the high affinity site. The results are discussed with reference to published rapid quenching experiments. Under our conditions, i.e. in the absence of a calcium gradient across the membrane, the calcium pump cycle step in which reorientation of the calcium binding sites occurs cannot be identified with the isomerization step mentioned above.     

Electrochemical proton gradient in Micrococcus lysodeikticus cells and membrane vesicles.

Using the distribution of weak acids to measure the pH gradient (delta pH; interior alkaline) and the distribution of the lipophilic cation [3H]tetraphenylphosphonium+ to monitor the membrane potential (delta psi; interior negative), we studied the electrochemical gradient or protons (delta mu- H+) across the membrane of Micrococcus lysodeikticus cells and plasma membrane vesicles. With reduced phenazine methosulfate as electron donor, intact cells exhibited a relatively constant delta mu- H+ (interior negative and alkaline) of -193 mV to -223 mV from pH 5.5 to pH 8.5. On the other hand, in membrane vesicles under the same conditions, delta mu- H+ decreased from a maximum value of -166 mV at pH 5.5 to -107 mV at pH 8.0 and above. This difference is related to a differential effect of external pH on the components of delta mu- H+. In intact cells, delta pH decreased from about -86 mV (i.e., 1.4 units) at pH 5.5 to zero at pH 7.8 and above, and the decreases in delta pH was accompanied by a reciprocal increase in delta psi from -110 mV at pH 5.5 to -211 mV at pH 8.0 and above. In membrane vesicles, the decrease in delta pH with increasing external pH was similar to that described for intact cells; however, delta psi increased from -82 mV at pH 5.5 to only -107 mV at pH 8.0 and above.     

pH and temperature resolve the kinetics of two pools of calcium bound to the sarcoplasmic reticulum Ca2+ -ATPase

Calcium bound to the sarcoplasmic reticulum Ca2+ -ATPase was removed by chelating free calcium ion with EGTA. The kinetic calcium binding reaction to the calcium-unbound ATPase was studied by varying the pH (6.0-8.8) and temperature (0-20 degrees C) at a saturating concentration of 50-100 microM [Ca2+]. At pH 6.0 and 0 degrees C, calcium sites of the enzyme at a rate of t1/2 approximately 10 s. By increasing the pH from 6.0 to 8.8, about half of the total calcium sites were converted from a slow binding state to a rapid binding state (less than 2s). The maximum level was reached at about pH 7.4, and the midpoint of the conversion was observed at about pH 6.7. On the other hand, the slow binding reaction to the other sites was not significantly affected by the pH increase. At pH 7.0 and 20 degrees C, about 90% of the total calcium sites rapidly (less than 2s) bound calcium. The present results suggest that pH and temperature resolve the kinetics of two pools of calcium bound to the Ca2+-ATPase.     

The uptake of protons by heme-linked ionizable groups on azide binding to methemoglobin

When azide ion reacts with methemoglobin in unbuffered solution the pH of the solution increases. This phenomenon is associated with increases in the pK values of heme-linked ionizable groups on the protein which give rise to an uptake of protons from solution. We have determined as a functional of pH the proton uptake, delta h+, on azide binding to methemoglobin at 20 degrees C. Data for methemoglobins A (human), guinea pig and pigeon are fitted to a theoretical expression based on the electrostatic effect of these sets of heme-linked ionizable groups on the binding of the ligand. From these fits the pK values of heme-linked ionizable groups are obtained for liganded and unliganded methemoglobins. In unliganded methemoglobin pK1, which is associated with carboxylic acid groups, ranges between 4.0 and 5.5 for the three methemoglobins; pK2, which is associated with histidines and terminal amino groups, ranges from 6.2 to 6.7. In liganded methemoglobin pK1 lies between 5.8 and 6.3 and pK2 varies from 8.1 to 8.5. The pH dependences of the apparent equilibrium constants for azide binding to the three methemoglobins at 20 degrees C are well accounted for with the pK values calculated from the variation of delta h+ with pH.     

The structure and function of ribonuclease T1. XX. Specific inactivation of ribonuclease T1 by reaction with tosylglycolate.

1. Ribonuclease T1 [EC 3.1.4.8] was inactivated by reaction with tosylglycolate (carboxymethyl rho-toluenesulfonate). At pH 5.5 and 8.0, alkylation of the gamma-carboxyl group of glutamic acid-58 appeared to be the predominant reaction and the major cause of inactivation by tosylglycolate, as in the case of the iodoacetate reaction, although the rate of inactivation was slower than that by iodoacetate. At pH 8.0, histidine residues were also alkylated to some extent. 2. The maximal rate of inactivation was observed at around pH 5.5 and the pH dependence of the rate of inactivation suggested the implication of two groups in the reaction, with apparent pKa values of about 3-4 (possibly histidine residue(s)). 3. In the presence of substrate analogs, ribonuclease T1 was markedly protected from inactivation by tosylglycolate at pH 5.5. The extent of protection corresponded to the binding strength of the substrate analog, except for guanosine. Ribonuclease T1 was much less protected from inactivation by guanosine than by 3'-AMP or 3'-CMP, which has a lower binding strength toward ribonuclease T1. This may indicate that glutamic acid-58 is situated in the catalytic site, at which the phosphate moiety of these nucleotides directly interacts. 4. Enzyme which had been extensively inactivated with tosylglycolate at pH 5.5 scarcely reacted with iodoacetate at pH 5.5, suggesting that these reagents react at the same site, i.e. glutamic acid-58. On the other hand, enzyme which had been inactivated almost completely with tosylglycolate at pH 8.0 still reacted with iodoacetate to some extent at pH 8.0, and the modes of reaction of tosylglycolate and iodoacetate toward ribonuclease T1 appeared to be somewhat different.