Infrared evidence of cyanide binding to iron and copper sites in bovine heart cytochrome c oxidase. Implications regarding oxygen reduction

Cyanide binding to bovine heart cytochrome c oxidase at five redox levels has been investigated by use of infrared and visible-Soret spectra. A C-N stretch band permits identification of the metal ion to which the CN- is bound and the oxidation state of the metal. Non-intrinsic Cu, if present, is detected as a cyanide complex. Bands can be assigned to Cu+CN at 2093 cm-1, Cu2+CN at 2151 or 2165 cm-1, Fe3+CN at 2131 cm-1, and Fe2+CN at 2058 cm-1. Fe2+CN is found only when the enzyme is fully reduced whereas the reduced Cu+CN occurs in 2-, 3-, and 4-electron reduced species. A band for Fe3+CN is not found for the complex of fully oxidized enzyme but is for all partially reduced species. Cu2+CN occurs in both fully oxidized and 1-electron-reduced oxidase. CO displaces the CN- at Fe2+ to give a C-O band at 1963.5 cm-1 but does not displace the CN- at Cu+. Another metal site, noted by a band at 2042 cm-1, is accessible only in fully reduced enzyme and may represent Zn2+ or another Cu+. Binding of either CN- or CO may induce electron redistribution among metal centers. The extraordinary narrowness of ligand infrared bands indicates very little mobility of the components that line the O2 reduction site, a property of potential advantage for enzyme catalysis. The infrared evidence that CN- can bind to both Fe and Cu supports the possibility of an O2 reduction mechanism in which an intermediate with a mu-peroxo bridge between Fe and Cu is formed. On the other hand, the apparent independence of Fe and Cu ligand-binding sites makes a heme hydroperoxide (Fe-O-O-H) intermediate an attractive alternative to the formation an Fe-O-O-Cu linkage.     

Characterization of jack-bean alpha-D-mannosidase as a zinc metalloenzyme.

1. Two methods were used to obtain alpha-mannosidase free from unbound Zn2+, (a) by removal of excess of metal ion from preparations purified in the presence of Zn2+ and (b) by purification under conditions that eliminate the need to add Zn2+. 2. The purified enzyme is homogeneous on ultracentrifugation, polyacrylamide-gel electrophoresis and gel chromatography. 3. The molecular weight is estimated to be 230 000. 4. The enzyme contains between 470 and 565 mug of zinc/g of protein, corresponding to between 1.7 and 2 atoms of zinc/enzyme molecule. The contents of other metals are much lower. 5. The enzyme is inactivated by chelating agents and activity is restored by Zn2+. 6. No other metal ion was found to replace Zn2+ with retention of activity. Some bivalent metal ions, e.g. Cu2+, rapidly inactivate the enzyme. 7. The results indicate that jack-bean alpha-mannosidase exists naturally as a zinc-protein complex and may be considered as a metalloenzyme.     

The effect of divalent cations on bovine spermatozoal adenylate cyclase activity.

The effect of divalent cations on bovine sperm adenylate cyclase activity was studied. Mn2+, Co2+, Cd2+, Zn2+, Mg2+ and Ca2+ were found to satisfy the divalent cation requirement for catalysis of the bovine sperm adenylate cyclase. These divalent cations in excess of the amount necessary for the formation of the metal-ATP substrate complex were found to stimulate the enzyme activity to various degrees. The magnitude of stimulation at saturating concentrations of the divalent cations was strikingly greater with M2+ than with either Ca2+, Mg2+, Zn2+, Cd2+ or Co2+. The apparent Km was lowest for Zm2+ (0.1 - 0.2 mM) than for any of the other divalent cations tested (1.2 - 2.3 mM). The enzyme stimulation by Mn2+ was decreased by the simultaneous addition of Co2+, Cd2+, Ni2+ and particularly Zn2+ and Cu2+. The antagonism between Mn2+ and Cu2+ or Zn2+ appeared to have both competitive and non-competitive features. The inhibitory effect of Cu2+ on Mn2+-stimulated adenylate cyclase activity was prevented by 2,3-dimercaptopropanol, but not by dithiothreitol, L-ergothioneine, EDTA, EGTA or D-penicillamine. Ca2+ at concentrations of 1-5 mM was found to act synergistically with Mg2+, Zn2+, Co2+ and Mn2+ in stimulating sperm adenylate cyclase activity. The Ca2+ augmentation of the stimulatory effect of Zn2+, Co2+, Mg2+ and Mn2+ appeared to be specific.     

Kinetics and equilibria for the formation of a new DNA metal-intercalator: the cyclic polyamine Neotrien/copper(II) complex

A study has been performed of the kinetics and equilibria involved in complex formation between the macrocyclic polyamine 2,5,8,11-tetraaza[12]-[12](2,9)[1,10]-phenanthrolinophane (Neotrien) and Cu(II) in acidic aqueous solution and ionic strength 0.5 M (NaCl), by means of the stopped-flow method and UV spectrophotometry. Spectrophotometric titrations and kinetic experiments revealed that the binding of Cu(II) to Neotrien gives rise to several 1:1 complexes differing in their degree of protonation. Under the experimental hydrogen ion concentration range investigated, complexation occurs by two parallel paths: (a) M2+ + (H4L)4+ <==> (MH4L)6+ and (b) M2+ + (H3L)3+ <==> (MH3L)5+. The rate constants values found for complex formation, by paths (a) and (b), are much lower than the values expected from water exchange at copper(II) and other amine/Cu(II) complexation kinetic constants. Kinetic experiments at different NaCl concentrations indicated that this finding was not due to chloride ion competition in complex formation with Neotrien, but it was related to a ring rigidity effect. As the phenanthroline moiety could, in principle, interact with nucleic acids by intercalation or external binding, some preliminary measurements concerned with the possible interactions occurring between the Cu(II)/Neotrien complex and calf thymus DNA (CT-DNA) have also been carried out. The absorption spectra of the Cu(II)/Neotrien complex change upon addition of CT-DNA at pH 7.0, revealing the occurrence of complex-nucleic acid interactions. Moreover, fluorescence titrations, carried out by adding the Cu(II)/Neotrien complex to CT-DNA, previously saturated with ethidium bromide (EB), show that the Cu(II)/Neotrien complex is able to displace EB from DNA, suggesting the complex is able to intercalate into the polynucleotide and then to cleave the phosphodiester bond of DNA.     

The inhibitor protein of the cAMP-dependent protein kinase-catalytic subunit interaction. Parameters of complex formation

The formation of a complex between the catalytic subunit of the cAMP-dependent protein kinase and the Inhibitor Protein of this enzyme has been examined by means of nondenaturing gel electrophoresis. Two forms of complex were identified, both containing a 1:1 molar ratio of the component proteins. The formation of the major of the two forms is markedly enhanced by the presence of nucleotide triphosphate and divalent cation. Either Mg2+ or Mn2+ serves to promote complex formation. With Mg2+, only ATP is effective for enhancing complex formation, whereas with Mn2+ complex formation occurs to an equal extent with ATP, GTP, ITP, and adenyl-5'-yl imidodiphosphate. The formation of the two complexes is only minimally dependent upon nucleotide triphosphate. It is suggested that the two types of complex are a result of different species of catalytic subunit. Two principal forms of the complex have been detected occurring maximally in approximately a 2.5:1 ratio. In the accompanying paper (Fletcher, W.H., Van Patten, S.M., Cheng, H-C., and Walsh, D.A. (1986) J. Biol. Chem. 261, 5504-5513), we have described the use of a fluoresceinated derivative of catalytic subunit as a cytochemical probe to localize the Inhibitor Protein and the regulatory subunit of the protein kinase. The integrity of this fluorophore has been further characterized using the method of examining catalytic subunit-Inhibitor Protein interaction delineated here.     

Affinity chromatography of phosphofructokinase.

The behavior of mammalian phosphofructokinase on immobilized adenine nucleotides was investigated. Three different insolubilized ligands were compared using a pure rabbit muscle phosphofructokinase. N⁶-[(6-aminohexyl)-carbamoyl-methyl]-ATP-Sepharose bound at least 90 times more enzyme than either N⁶-(6-aminohexyl)-AMP-agarose or ATP-adipic acid hydrazide-Sepharose. The elution of phosphofructokinase from the ATP-Sepharose with various metabolites and combinations of metabolites was investigated. The enzyme is eluted specifically from N⁶-[(6-aminohexyl)-carbamoyl]-ATP-Sepharose with a mixture of 25 μm each of fructose 6-phosphate and ADP (±Mg²⁺). The enzyme is not eluted either with ATP (25 μm), fructose 1,6-diphosphate (1 mm), ADP (25 μm), fructose 6-phosphate (1 mm) alone, or with a mixture of fructose 1,6-diphosphate (25 μm) and ATP (25 μm). The recovery of bound enzyme was usually greater than 90%. A mixture of glucose 6-phosphate and ADP or a mixture of IDP and fructose 6-phosphate also elutes the enzyme, but the recovery with these eluants was only about 40%. It was concluded that the “dead-end” complex is the most effective in the elution. Using this method, phosphofructokinase has been prepared in an essentially homogeneous form from muscle and brain of rabbit and rat. The overall isolation procedure involves a high speed centrifugation of crude extracts which sediments phosphofructokinase as a pellet, followed with adsorption on N⁶-[(6-aminohexyl)-carbamoyl-methyl]-ATP-Sepharose and specific elution with the mixture of fructose 6-phosphate and ADP.     

Magnetic resonance studies on the copper site of dopamine beta-monooxygenase in the presence of cyanide and azide anions

In order to elucidate the coordination state of water molecules in the Cu(II) site of dopamine [( 3,4-dihydroxyphenyl)ethylamine] beta-monooxygenase, measurements of the paramagnetic 1H nuclear magnetic relaxation rate of solvent water in the enzyme solution containing cyanide or azide as an exogenous ligand were carried out to obtain the values of intrinsic paramagnetic relaxation rate decrements Rp1 and Rp2 for the ligand-enzyme 1:1 and 2:1 complexes, respectively. Rp1 (percent) values were 53 (pH 5.5) and 52 (pH 7.0) for cyanide and 38 (pH 5.5) and 32 (pH 7.0) for azide, while Rp2 (percent) values were 98 (pH 5.5) and 96 (pH 7.0) for azide. Although no Rp2 values for cyanide were obtained because of its reducing power at the Cu(II) site, the Rp1 and Rp2 values obtained above prove that the Cu(II) center has two coordinated water molecules that are exchangeable for exogenous ligands at either pH. Supporting evidence was provided by electron paramagnetic resonance (EPR) titration, in which the enzyme solution containing cyanide-enzyme (1:1) complex in an equal proportion to uncomplexed enzyme gave an observed paramagnetic relaxation rate decrement, Rp, of 23%. Another characteristic of the Rp1 and Rp2 values was their invariability with respect to pH, indicating that the three-dimensional structure of the Cu(II) site is pH-invariant within the range examined. Binding constants of ligand to enzyme Kb1 and Kb2 for 1:1 and 2:1 complex formation, respectively, were also determined through an analysis of the Rp values; it was found that Kb1 was larger than Kb2 irrespective of pH. (ABSTRACT TRUNCATED AT 250 WORDS)     

Reconstitution of Cu,Zn-superoxide dismutase by the Cu(I).glutathione complex

The reconstitution of Cu,Zn-superoxide dismutase from the copper-free protein by the Cu(I).GSH complex was monitored by: (a) EPR and optical spectroscopy upon reoxidation of the enzyme-bound copper; (b) NMR spectroscopy following the broadening of the resonances of the Cu(I).GSH complex after addition of Cu-free,Zn-superoxide dismutase; and (c) NMR spectroscopy of the Cu-free,Co(II) enzyme following the appearance of the isotropically shifted resonances of the Cu(I), Co enzyme, Cu(I).GSH was found to be a very stable complex in the presence of oxygen and a more efficient copper donor to the copper-free enzyme than other low molecular weight Cu(II) complexes. In particular, 100% reconstitution was obtained with stoichiometric copper at any GSH:copper ratio between 2 and 500. Evidence was obtained for the occurrence of a Cu(I).GSH.protein intermediate in the reconstitution process. In view of the inability of copper-thionein to reconstitute Cu,Zn-superoxide dismutase and of the detection of copper.GSH complexes in copper-over-loaded hepatoma cells (Freedman, J.H., Ciriolo, M.R., and Peisach, J. (1989) J. Biol. Chem. 264, 5598-5605), Cu(I).GSH is proposed as a likely candidate for copper donation to Cu-free,Zn-superoxide dismutase in vivo.     

Cobalt(II) and copper(II) binding of Bacillus cereus trinuclear phospholipase C: a novel 1H NMR spectrum of a 'Tri-Cu(II)' center in protein.

The phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PC-PLC(Bc)) is a tri-Zn enzyme with two 'tight binding' and one 'loose binding' sites. The Zn2+ ions can be replaced with Co2+ and Cu2+ to afford metal-substituted derivatives. Two Cu2+-substituted derivatives are detected by means of 1H NMR spectroscopy, a 'transient' derivative and a 'stable' derivative. The detection of sharp hyperfine-shifted 1H NMR signals in the 'transient' derivative indicates the formation of a magnetically coupled di-Cu2+ center, which concludes that the Zn2+ ions in the dinuclear (Zn1 and Zn3) sites are more easily replaced by Cu2+ than that in the Zn2 site. This might possibly be the case for Co2+ binding. Complete replacement of the three Zn2+ ions can be achieved by extensive dialysis of the enzyme against excess Cu2+ to yield the final 'stable' derivative. This derivative has been determined to have five-coordinated His residues and an overall S'=1/2 spin state with NMR and EPR, consistent with the formation of a tri-Cu2+ center (i.e. a di-Cu2+/mono-Cu2+ center) in this enzyme. The binding of substrate to the inert tri-Cu2+ center to form an enzyme-substrate (ES) complex is clearly seen in the 1H NMR spectrum, which is not obtainable in the case of the native enzyme. The change in the spectral features indicates that the substrate binds directly to the trinuclear metal center. The studies reported here suggest that 1H NMR spectroscopy can be a valuable tool for the characterization of di- and multi-nuclear metalloproteins using the 'NMR friendly' magnetically coupled Cu2+ as a probe.     

Partial purification of 6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydropterin triphosphate synthetase from chicken liver.

An enzyme that catalyzes the formation of 6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydropterin triphosphate (D-erythrodihydroneopterin triphosphate) and formic acid from GTP has been purified about 3700-fold from homogenates of chicken liver. The molecular weight of the enzyme, D-erythrodihydroneopterin triphosphate synthetase (GTP cyclohydrolase), has been estimated to be 125,000 by gel filtration on Ultrogel AcA-34. The enzyme functions optimally between pH 8.0 and 9.2 and is considerably heat-stable. No cofactors or metal ions have been demonstrated to be required for activity; however, the reaction is strongly inhibited by Cu2+ and Hg2+. GTP is the most efficient substrate, with GDP being 1/17 as active and guanosine, GMP, and ATP being inactive. The Km for GTP has been found to be 14 micrometer. Although the overall reaction catalyzed by D-erythrodihydroneopterin triphosphate synthetase from chicken liver is identical with that from Escherichia coli GTP cyclohydrolase, immunological studies show no apparent homology between the two enzymes.     

Evaluation of the metal-ion-coordinating differences between the 2'-, 3'- and 5'-monophosphates of adenosine

The stability constants of the 1:1 complexes formed between Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+ or Cd2+ and 2'AMP2-, 3'AMP2- or 5'AMP2- were determined by potentiometric pH titration in aqueous solution (I = 0.1 M, NaNO3; 25 degrees C). The experimental conditions were carefully selected such that self-association of the nucleotides and their complexes is negligibly small; i.e. it was made certain that the properties of the monomeric divalent-metal-ion--AMP [M(AMP)] complexes were studied. Based on recent measurements with simple phosphate monoesters, R-MP2- where R is a non-coordinating residue [Massoud, S. S. & Sigel, H. (1988) Inorg. Chem. 27, 1447-1453], it is shown that all the M(AMP) complexes of the alkaline earth ions, with the possible exception of Mg(5'AMP), have exactly the stability expected for a sole-phosphate coordination of the metal ion. The same property is revealed for the complexes with Mn2+, Co2+, Zn2+ or Cd2+ and 3'AMP2-; in case of Ni(3'AMP) and Cu(3'AMP) a slight stability increase just at the edge of the experimental-error limits is indicated. This slight stability increase is attributed to the formation of a macrochelate (possibly with N-3); in fact, additional information confirms macrochelation for Cu(3'AMP). About 45% of Cu(2'AMP) exists in aqueous solution as a macrochelate (probably involving N-3); the other M(2'AMP) complexes (M2+ = Mn2+, Co2+, Ni2+, Zn2+, Cd2+) form (if at all) only traces of a base-backbound species. Most pronounced is macrochelate formation with 5'AMP2-: all mentioned 3d ions and Zn2+ or Cd2+ form to some extent macrochelates via N-7 (the structures of these closed species are indicated). In case of M(5'AMP) the base-binding site is certain: replacement of N-7 by a CH unit (tubercidin 5'-monophosphate) eliminates any increased complex stability, whereas formation of the 1,N6-etheno bridge to form 1,N6-ethenoadenosine 5'-monophosphate results in the phenanthroline-like N-6,N-7 site which facilitates macrochelation significantly.     

Interaction of Cu2+ ion with milk xanthine oxidase

The interaction of Cu2+ ion with milk xanthine oxidase (XO) has been studied by optical spectroscopy, circular dichroism, ESR and transient kinetic techniques. It is observed that XO forms optically observable complexes with Cu2+ ion. The pH dependence studies of the formation of Cu2+-XO complex by optical spectroscopy and circular dichroism show that at least one ionizable group may be responsible for the formation of the complex. The EPR studies show that Cu2+ ion binds to XO with sulfur and nitrogenous ligands. The transient kinetic study of the interaction of Cu2+ with XO shows the existence of two Cu2+ bound XO complexes formed at two different time scales of the interaction, one at < or =5 ms and the other one at around 20 s. The complex formed at longer time scale may be responsible for the inhibition of the enzyme activity.     

Interaction of RNA polymerase with promoters from bacteriophage fd.

Replicative form DNA of bacteriophage fd, which had been fragmented with the restriction endonuclease II from Hemophilus parainfluenzae (endo R- HpaII), was reacted with Escherichia coli RNA polymerase; the resulting stable preinitiation complexes were analysed using the filter binding assay followed by gel electrophoresis. At 120mM KCL the first-order rate constants for complex decay were determined to be 10(-2)-10(-6)s-1. The second-order rate constants for complex formation were found to be about 10(6) -10(7) M-1 s-1. From these values association constants for the individual promoters were calculated to be 2 x 10(-8) -2 x 10(-11) M-1. The rate of formation and the stability of promoter complexes was enhanced in superhelical DNA. No evidence was found for stable promoter-specific closed complexes consisting of enzyme and helical DNA. This and the kinetic data suggest that the unwinding of base pairs is already important early in promoter selection, and not only for the formation of the final open complex. The initiation of RNA synthesis form the preinitiation complex was faster than complex dissociation and complex formation for all promoters. Consequently, the initiation efficiency of a promoter is determined by the rate of complex formation, and not by its 'affinity' for the enzyme. No correlation was found between the relative order of the fd promoters for the binding and the dissociation reaction. This is explained by different structural determinants, for the two reactions, which are located in different parts of the promoter DNA.     

Preparation of an alcohol-dehydrogenase--NAD(H)--sepharose complex showing no requirement of soluble coenzyme for its activity.

1. Horse liver alcohol dehydrogenase and an NADH analogue, N6-[(6-aminohexyl)carbamoylmethyl]-NADH, have been co-immobilized to Sepharose 4B under conditions permitting binary complex formation between the enzyme and the cofactor. 2. The enzyme-coenzyme-matrix preparations were assayed with a coupled oxidoreduction reaction and showed activities, prior to addition of coenzyme, that were up to 40% of that obtained in excess of free coenzyme. 3. A molar ratio of 1:1 between the amount of bound enzyme was sufficient to obtain high activities in the absence of free coenzyme. 4. The highest recycling rate obtained for the immobilized nucleotide was 3400 cycles per hour. 5. Both thermal and storage stability of alcohol dehydrogenase was increased when the enzyme was co-immobilized with the NADH analogue. 6. The efficiency of the immobilized preparations (measured as product formation per minute and per assay volume) was higher (1.4 to 5 times in our assays) than the corresponding systems of free enzyme (in total enzyme units) and nucleotide in an identical assay volume.     

Electron paramagnetic resonance of copper ion and manganese ion complexes with the ionophore A23187.

The binding of Cu2+ and Mn2+ to the ionophore A23187 in chloroform, 90% ethanol, and sonicated phospholipid dispersions in aqueous mediums has been investigated with electron paramagnetic resonance (epr). The spectra indicated axial symmetry for the Cu2+ complexes and distorted octahedral for the Mn2+ complexes. The coordination between metal ion and its ligands is predominantly ionic in character. The stoichiometry, at the concentrations employed, was found to be 1:2 M2+/ionophore except in 90% ethanol where evidence existed for the 1:1 Cu-A23187 complex, as well. Through competition with Mn2+, the sequence of relative affinities in 90% ethanol was measured to be: Mn2+ greater than La3+ greater than Cu2+ greater than Ca2+ greater than Mg2+ greater than Sr2+. The K A of Mn-A23187 binding is greater than 10 10 M-2. In phospholipid dispersions the spectral characteristics of the Cu complex, particularly g, were observed to be a sensitive function of the hydrocarbon chain mobility. This allowed a calculation of the rotational correlation time of the complex to be made. In sonicated dipalmitoyllecithin was computed to be 10-9 sec, reflecting a local viscosity similar to that sensed by the nitroxide spin-label 2,2,6,6-tetramethylpiperidin-1-oxyl. In a (1:1) lecithin-cholesterol dispersion the complex was significantly more immobilized.     

The initial binding of Cu(II) to some amino acids and dipeptides: a 13C nuclear-magnetic-resonance study.

The initial binding of Cu2+ ot L-lysine, L-histidine, glycyl-histidine and histidyl-glycine in aqueous solutions was examined by 13C nuclear magnetic resonance spectroscopy. The measurements were carried out in a substantially improved way employing the pulse Fourier transform technique. Spectra of both high quality and resolution were obtained. Cu2+ complex formation with L-lysine occurred with the alpha-amino and carboxyl group attributable to the well expressed broadening effect of the 13C signals of the alpha-carbon atom and the carboxyl atom. The epsilon-amino group was not involved. Measurements of the Cu chelates using L-histidine and glycyl-histidine and histidyl-glycine confirmed the ambidentate nature of the histidine residue. It was concluded that an equilibrium exists between two Cu-complex species designated as histamine-like and histamine-like/glycine-like species. In the homogeneous histamine-like Cu complex, the Cu2+ is exclusively bound with 4 nitrogens, while in the other species one oxygen of the glycyl carboxyl group is involved in the Cu2+ binding. Blocking of this carboxyl groups by peptide bonding as found in histidyl-glycine favoured the formation of a Cu complex where the imidazole carbons of the histidyl residue were the most influenced species.     

Binding of divalent cations with low density lipoproteins. A study by ESR

The binding of bivalent metal ions Cu2+, Zn2+, Ca2+, Mg2+ to low-density lipoproteins (LDL) was investigated by the ESR technique. The monitoring of ESR spectra of paramagnetic Mn2+ ions in the presence of above-listed cations made it possible to evaluate the dissociation constants of their complexes with LDL. The effective dissociation constant of the complex Mn(2+)-LDL used for calculations was KD = (1.1 +/- 0.4) x 10(-4) M according to literature data. The investigated cations may be classified into two groups: 1) low dissociation constants were characteristic for Cu2+ ions [KD = (1.3 +/- 0.5) x 10(-4) M], which demonstrated a high oxidative ability, and for Zn2+ [KD = (0.95 +/- 0.45) x 10(-4) M] and Mn2+ ions, which could strongly influence the copper-induced LDL oxidation; 2) Ca2+ and Mg2+ were characterized by higher values of KD [(6 +/- 1) x 10(-4) M and (7.5 +/- 1.5) x 10(-4) M, accordingly] and slightly affected the Cu(2+)-induced oxidation of LDL. The results of the present work reinforced our earlier conjecture that cations may influence the process of lipid peroxidation, binding only to particular binding sites on the surface of LDL.     

Calmodulin affinity chromatography yields a functional purified erythrocyte (Ca+ + Mg2+)-dependent adenosine triphosphatase.

The (Ca2+ + Mg2+)-dependent ATPase of human erythrocyte membranes was solubilized with deoxycholate and purified by calmodulin affinity chromatography to yield a functional enzyme. The method gave an enzyme purified 207-fold as compared with that of the erythrocyte membranes. The molecular weight of the ATPase was in the range 135 000-150 000, as revealed by a single major band after electrophoresis on dodecyl sulphate/polyacrylamide gels. The isolated enzyme was highly sensitive to calmodulin, since the activity was increased about 9-fold. At 37 degrees C and in the presence of calmodulin the purified ATPase had a specific activity of 10.1 mumol/min per mg of protein. Triton X-100 or deoxycholate stimulated the calmodulin-deficient enzyme in a concentration-dependent fashion whereby the calmodulin-sensitivity was lost. The purification method is suitable for studying the lipid-sensitivity of the ATPase, since the lipids can easily be exchanged without a significant loss of activity. A purification procedure described by Niggli, Penniston & Carafoli [(1979) J. Biol. Chem. 254, 9955-9958] resulted in an enzyme that indeed was pure but was lacking a predominant feature, namely the modulation by calmodulin.     

Calcium ion-flux across phosphatidylcholine membranes mediated by ionophore A23187.

The antibiotic A23187 carries Ca2+ across Müller-Rudin membranes made from 1,2-dierucoyl-sn-glycero-3-phosphocholine and n-decane. The conductance of the membranes is not increased by the Ca2+-transport. The flux depends linearly on Ca2+ concentration and ionophore concentration (above pH 6). It increases with increasing pH, approximately by a factor of 4-5 between pH 6 and pH 8. Maximal Ca2+-fluxes of about 10(-10) mol-cm-2-s-1 were found. A counter transport of H+ could not be detected. The complex formation between A23187 and Ca2+ in egg phosphotidylcholine vesicles was studied spectroscopically. The results are consistent with the formation of a 2:1 complex. Optical absorption measurements on single phophatidylcholine membranes were used to calculate the concentration of membrane-bound ionophore A23187.     

Membrane-bound F1 ATPase from Micrococcus Sp. ATCC 398E. Purificationa and characterization ny affinity chromatography.

A chemically reactive ATP analogue, 6-[(3-carboxy-4-nitrophenyl)thio]-9-beta-D-ribofuranosylpurine 5'-triphosphate (Nbs6ITP) has been synthesized. It has the ability to form stable thioether bonds between the 6-position of the purine ring and aliphatic mercapto groups. The nucleotide moiety of the reagent has been covalently bound to agarose, via iminobispropylamine and N-acetyl-homocysteine as space with the purpose of producing an affinity chromatography material. The affinity matrix binds solubilized F1 ATPase from a crude extract of Micrococcus sp. membranes. Afterwards the enzyme can be selectively eluted from the column at a defined ATP concentration. This method is superior to the conventional purification with respect to speed and convenince of the preparation. The affinity chromatography leads in a one-step process to the same purity to enzyme, substituting several steps of the conventional method. In addition, the affinity matrix was used for binding studies. Although the presence of Mg2+ ions is a prerequisite for the hydrolysis of nucleoside 5'-triphosphates, evidence is presented indicating that the binding of the nucleoside triphosphates to highly purified F1 ATPase from Micrococcus sp. appears not to be influenced by Mg2+ ion concentrations so far examined.