Characterization of the oxycomplex of lignin peroxidases from Phanerochaete chrysosporium: equilibrium and kinetics studies

The oxycomplexes (compound III, oxyperoxidase) of two lignin peroxidase isozymes, H1 (pI = 4.7) and H8 (pI = 3.5), were characterized in the present study. After generation of the ferroperoxidase by photochemical reduction with deazoflavin in the presence of EDTA, the oxycomplex is formed by mixing ferroperoxidase with O2. The oxycomplex of isozyme H8 is very stable, with an autoxidation rate at 25 degrees C too slow to measure at pH 3.5 or 7.0. In contrast, the oxycomplex of isozyme H1 has a half-life of 52 min at pH 4.5 and 29 min at pH 7.5 at 25 degrees C. The decay of isozyme H1 oxycomplex follows a single exponential. The half-lives of lignin peroxidase oxycomplexes are much longer than those observed with other peroxidases. The binding of O2 to ferroperoxidase to form the oxycomplex was studied by stopped-flow methods. At 20 degrees C, the second-order rate constants for O2 binding are 2.3 X 10(5) and 8.9 X 10(5) M-1 s-1 for isozyme H1 and 6.2 X 10(4) and 3.5 X 10(5) M-1 s-1 for isozyme H8 at pH 3.6 and pH 6.8, respectively. The dissociation rate constants for the oxycomplex of isozyme H1 (3.8 Z 10(-3) s-1) and isozyme H8 (1.0 X 10(-3) s-1) were measured at pH 3.6 by CO trapping. Thus, the equilibrium constants (K, calculated from kon/koff) for both isozymes H1 (7.0 X 10(7) M-1) and H8 (6.2 X 10(7) M-1) are higher than that of myoglobin (1.9 Z 10(6) M-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

On the water and proton permeabilities across membranes from erythrocyte ghosts

The diffusional permeability of water across membranes from bovine and human erythrocyte ghosts was measured by a recently developed method which is based on the different indices of refraction of H2O and 2H2O. Resealed erythrocyte ghosts were prepared by a gel-filtration technique. Pd (2H2O/H2O) values of 1.2 X 10(-3) cm/s (human) and 1.7 X 10(-3) cm/s (bovine) were calculated at 20 degrees C. The activation energies of the water exchange were 23.5 kJ/mol (human) and 25.4 kJ/mol (bovine). Treatment of the ghosts with p-chloromercuribenzenesulfonic acid (PCMBS) led to a 60-70% inhibition of the diffusional water exchange. The pH equilibration across membranes of erythrocyte ghosts was measured by intracellular carboxyfluorescein. The rates of proton flux after pH-jumps (pH 7.3 to pH 6.1) were about 100-fold lower than those of the water exchange and dependent on the kind of anions present (Cl-, NO-3, SO2-4). The activation energies of proton flux were 60-70 kJ/mol. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) inhibited the exchange by 97-98% and lowered the activation energy. The inhibitor of water exchange, PCMBS, increased the proton-permeation rate by a factor of 4-5. It is assumed that the rate-limiting step for the proton permeation is determined by the anion exchange. Under this condition our results are not in accord with one channel as a common pathway for both the passive water and anion transport.     

Activation of lecithin-cholesterol acyltransferase by apolipoprotein D: comparison of proteoliposomes containing apolipoprotein D, A-I or C-I

To study the activation of lecithin-cholesterol acyl transferase (LCAT) (phosphatidylcholine:sterol O-acyltransferase, EC 2.3.1.43) by apolipoprotein D in comparison to apolipoproteins A-I and C-I, proteoliposomes with a phosphatidylcholine/free cholesterol molar ratio of 24:1, containing 10-300 micrograms/ml of apolipoproteins were used. The proteoliposomes were prepared by the cholate dialysis technique. In all proteoliposome preparations we found rouleaux structures and stacked discs. The particles formed with apolipoprotein A-I were the most homogeneous, followed by apolipoprotein D- and apolipoprotein C-I-containing particles. Apolipoprotein A-I was the most potent LCAT activator in our system followed by apolipoproteins C-I and D. The fractional esterification rate observed with apolipoprotein D-containing substrates amounted to 15-48% that of apolipoprotein A-I-containing ones. Neither apolipoprotein A-I- nor C-I-containing proteoliposomes gave linear reaction kinetics with LCAT. Even during the first 15-30 min of incubation, the kinetics deviated strikingly from linearity at all apolipoprotein concentrations. In contrast, proteoliposomes containing apolipoprotein D exhibited linear reaction kinetics up to 60-90 min. At low apolipoprotein A-I concentrations (5 micrograms/ml), the addition of apolipoprotein D to the incubates resulted in significantly higher esterification rates as compared to substrates containing apolipoprotein A-I only. This was not the case using substrates with high apolipoprotein A-I concentrations (50 micrograms/ml). From our results we speculate that apolipoprotein D may have some stabilizing effect on the enzyme LCAT.     

Studies on phospholipases from Streptomyces. III. Purification and properties of Streptomyces hachijoensis phospholipase C.

1. Phospholipase C [EC 3.1.4.3] found in the growth medium of Streptomyces hachijoensis was purified about sixty-fold by dialysis and column chromatography on Sephadex G-50. 2. The active fraction was separated by isoelectric focusing into two fractions, phospholipase C-I (pI 6.0) and phospholipase C-II (pI 5.6). 3. Both purified phospholipases C were homogeneous by immunodiffusion and were not differentiated as regards antigencity. 4. Phospholipase C-I had maximal activity at pH 8.0 and the optimal temperature was 50degree. Phospholipase C-I was stable at 50degrees for 30 min and was stable at neutral pH. 5. The activity of phospholipase C-I was inhibited by high concentrations of various detergents such as Triton X-100, sodium, cholate, SDS and was also inhibited by Ca2+, Ba2+, Al3+, and EDTA, but was stimulated by Mg2+, and ethyl ether. 6. The Km value of phospholipase C-I was 0.9 mM, using phosphatidylcholine as a substrate. 7. By the gel filtration procedure, the molecular weights of phospholipase C-I and -II were both determined to be 18,000. 8. Phosphatidylcholine, phosphatidylinositol, cardiolipin, sphingomyelin, and lysophosphatidylcholine were hydrolyzed by phospholipase C-I, but phosphatidylethanolamine and phosphatidylserine were hydrolyzed with difficulty under the same conditions, Phospholipase C-I also hydrolyzed phosphatidic acid.     

Fast protein chromatofocusing of human very-low-density lipoproteins

Using fast protein chromatofocusing, a high-efficiency column chromatography method with a self-generated pH gradient and focusing effects, soluble human very-low-density lipoprotein (VLDL) apolipoproteins were fractionated between pH 6.3 and 4.0. In the presence of 6 mol/l urea and with a flow rate of 1 ml/min, one run (up to 10 mg of protein) took 30 min. VLDL apolipoproteins were separated in seven peaks. As revealed by SDS-polyacrylamide gel electrophoresis, isoelectric focusing and double-immunodiffusion against mono-specific antisera, fractions corresponded to the following proteins: apolipoprotein C-I, albumin, apolipoproteins A-I, E, C-II plus C-III0, C-III1 and C-III2, respectively. Apolipoproteins were eluted in sharp, well-resolved peaks. The recovery of proteins was 78% of the starting material. With fast protein chromatofocusing, an efficient isolation of single apolipoproteins is possible from small amounts of VLDL apolipoprotein preparations. This technique is superior to the commonly used, time-consuming methods for apolipoprotein isolation.     

Characterization of lipoprotein particles isolated by immunoaffinity chromatography. Particles containing A-I and A-II and particles containing A-I but no A-II

Two populations of A-I-containing lipoprotein particles: A-I-containing lipoprotein with A-II (Lp (A-I with A-II], and A-I-containing lipoprotein without A-II (Lp (A-I without A-II] have been isolated from plasma of 10 normolipidemic subjects by immunoaffinity chromatography and characterized. Both types of particles possess alpha-electrophoretic mobility and hydrated density in the range of plasma high-density lipoproteins (HDL). Lp (A-I without A-II) and Lp (A-I with A-II) are heterogeneous in size. Lp (A-I without A-II) comprised two distinct particle sizes with mean apparent molecular weight and Stokes diameter of 3.01 X 10(5), and 10.8 nm for Lp (A-I without A-II)1, and 1.64 X 10(5), and 8.5 nm for Lp (A-I without A-II)2. Lp (A-I with A-II) usually contained particles of at least three distinct molecular sizes with mean apparent molecular weight and Stokes diameter of 2.28 X 10(5) and 9.6 nm for Lp (A-I with A-II)1, 1.80 X 10(5) and 8.9 nm for Lp (A-I with A-II)2, and 1.25 X 10(5) and 8.0 nm for Lp (A-I with A-II)3. Apoproteins C, D, and E, and lecithin:cholesterol acyltransferase (LCAT) were detected in both Lp (A-I without A-II) and Lp (A-I with A-II) with most of the apoprotein D, and E, and LCAT (EC 2.3.1.43) in Lp (A-I with A-II) particles. Lp (A-I without A-II) had a slightly higher lipid/protein ratio than Lp (A-I with A-II). Lp (A-I with A-II) had an A-I/A-II molar ratio of approximately 2:1. The percentage of plasma A-I associated with Lp (A-I without A-II) was highly correlated with the A-I/A-II ratio of plasma (r = 0.96, n = 10). The variation in A-I/A-II ratio of HDL density subfractions therefore reflects different proportions of two discrete types of particles: particles containing A-I and A-II in a nearly constant ratio and particles containing A-II but no A-II. Each type of particle is heterogeneous in size and in apoprotein composition.     

Discoidal complexes of A and C apolipoproteins with lipids and their reactions with lecithin: cholesterol acyltransferase

Micellar, discoidal complexes of human apolipoproteins A-I, A-II, C-I, C-II, C-III-1, and C-III-2 with egg phosphatidylcholine (egg-PC) and cholesterol were prepared by the cholate dialysis method. The complexes, isolated by gel filtration, had similar lipid and protein contents by weight, on the average: 1.77:0.083:1.0, egg-PC/cholesterol/apolipoprotein (w/w). The diameters of the discs, visualized by electron microscopy and estimated by gel filtration, ranged from 100 to 200 A. The alpha-helix content of the apolipoproteins in the complexes was from 50-72%, and their fluorescence properties indicated nonpolar, but quite varied environments for the tryptophan residues in the various complexes. Initial reactions of purified human lecithin: cholesterol acyltransferase with the complexes, adjusted to equal egg-PC concentrations, indicated that all the apolipoproteins activate the enzyme from 6-fold to 400-fold over control vesicles of egg-PC and cholesterol. In decreasing order of reactivity were the complexes with A-I, C-I, C-III-1, C-III-2, C-II, and A-II. These results indicate that aside from lipid-binding capacity and high amphipathic alpha-helix content, other structural features are required for optimal enzyme activation by apolipoproteins. Concentration and temperature dependence experiments gave similar apparent Km values, markedly different apparent Vmax, and very similar activation energies (about 19 kcal/mol), for the various complexes. These observations suggest that the rate-limiting enzymatic step of the reaction is common to all the complexes but that the activated enzyme levels differ from complex to complex. We propose that enzyme activation occurs upon binding to complexes via apolipoproteins. Addition of excess (5-fold) free apolipoprotein A-I or A-II to complexes resulted in the exchange of bound for free apolipoproteins and in loss of reactivity with the enzyme.     

Kinetics of formation and dissociation of metallocarboxypeptidases.

The rates of formation of a number of metallocarboxypeptidases from metal ions and bovine apocarboxypeptidase A (CPA) have been measured directly and by a competitive method. Rates were determined with pH = 6-8 by utilising the pH change attending metal-ion incorporation, employing indicator and stopped-flow. Second-order rate constants Kf, M-1 s-1 at 25 degrees C, I = 1 M NaCl, pH = 7, Tris = 25 micrometer) were 1.7 X 10(5) (Mn2+), 3 X 10(4) (Co2+), 5 X 10(3) (Ni2+), 7 X 10(5) Zn2+), and 9 X 10(5) (Cd2+). Relative incorporation rate constants were determined at 25 degrees, pH = 7.0, Tris = 0.1 M, by competing two metal ions for a deficiency of apoprotein and analyzing the products by differential enzyme activity. Agreement between the two methods was reasonable. Rate constants for dissociation of CoCPA, NiCPA, and ZnCPA were measured by loss of enzyme activity on addition of the metal ion scavenger EDTA. Values of kd at 25 degrees, I = 1.0 M NaCl, pH = 7.0 were 8 X 10(-3), 3 X 10(-5), and 4 X 10(-4) s(-1), respectively. Values of K obtained kinetically (kf/kd) were in good agreement with those determined by activity measurements of equilibrated solutions. Results are compared with those of bovine apocarbonic anhydrase, where generally significantly slower rates are encountered.     

Induction of Thioguanine- and Ouabain-Resistant Mutants and Single-Strand Breaks in the DNA of Chinese Hamster Ovary Cells by 3H-Thymidine

Cultured Chinese hamster cells were labeled with 6-³H-thymidine or 5-methyl-³H-thymidine and allowed to accumulate damage from ³H decays for various periods of time while frozen. The frequencies of cells resistant to 6-thioguanine or ouabain and the amount of DNA damage (i.e., number of single-strand breaks) were determined and compared with the mutation frequencies resulting from X and ultraviolet light irradiation. Whereas ³H decays and X rays made only 6-thioguanine-resistant mutants, ultraviolet light made both 6-thioguanine- and ouabain-resistant mutants. ³H decays originating at the 6 position were two to three times as effective as decays at the 5-methyl position in making drug-resistant mutants, but decays at both sites were equally effective in making single-strand breaks. Mutants and strand breaks produced by beta irradiation of the nucleus probably are the same irrespective of the site of the decay in thymine; these results indicate that the local transmutation effects of ³H decay produce more mutations when they occur at the 6 position than at the 5-methyl position.     

Characterization and separation of the arachidonic acid 5-lipoxygenase and linoleic acid omega-6 lipoxygenase (arachidonic acid 15-lipoxygenase) of human polymorphonuclear leukocytes

The cytosolic fraction of human polymorphonuclear leukocytes precipitated with 60% ammonium sulfate produced 5-lipoxygenase products from [14C]arachidonic acid and omega-6 lipoxygenase products from both [14C]linoleic acid and, to a lesser extent, [14C]- and [3H]arachidonic acid. The arachidonyl 5-lipoxygenase products 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) derived from [14C]arachidonic acid, and the omega-6 lipoxygenase products 13-hydroperoxy-9,11-octadecadienoic acid (13-OOH linoleic acid) and 13-hydroxy-9,11-octadecadienoic acid (13-OH linoleic acid) derived from [14C]linoleic acid and 15-hydroxyperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), and 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) derived from [14C]- and [3H]arachidonic acid were identified by TLC-autoradiography and by reverse-phase high-performance liquid chromatography (RP-HPLC). Products were quantitated by counting samples that had been scraped from replicate TLC plates and by determination of the integrated optical density during RP-HPLC. The arachidonyl 5-lipoxygenase had a pH optimum of 7.5 and was 50% maximally active at a Ca2+ concentration of 0.05 mM; the Km for production of 5-HPETE/5-HETE from arachidonic acid was 12.2 +/- 4.5 microM (mean +/- S.D., n = 3), and the Vmax was 2.8 +/- 0.9 nmol/min X mg protein (mean +/- S.D., n = 3). The omega-6 linoleic lipoxygenase had a pH optimum of 6.5 and was 50% maximally active at a Ca2+ concentration of 0.1 mM in the presence of 5 mM EGTA. When the arachidonyl 5-lipoxygenase and the omega-6 lipoxygenase were separated by DEAE-Sephadex ion exchange chromatography, the omega-6 lipoxygenase exhibited a Km of 77.2 microM and a Vmax of 9.5 nmol/min X mg protein (mean, n = 2) for conversion of linoleic acid to 13-OOH/13-OH linoleic acid and a Km of 63.1 microM and a Vmax of 5.3 nmol/min X mg protein (mean, n = 2) for formation of 15-HPETE/15-HETE from arachidonic acid.     

Na/H exchange in cultured chick heart cells. pHi regulation

The purpose of this study was to establish the existence of Na/H exchange in cardiac muscle and to evaluate the contribution of Na/H exchange to pHi regulation. The kinetics of pHi changes in cultured chick heart cells were monitored microfluorometrically with 6-carboxyfluorescein and correlated with Nai content changes analyzed by atomic absorption spectrophotometry; transmembrane H+ movements were evaluated under pH stat conditions. After induction of an intracellular acid load by pretreatment with NH4Cl, a regulatory cytoplasmic alkalinization occurred with a t1/2 of 2.9 min. pHi regulation required external Na+ and was concomitant with transmembrane H+ extrusion as well as a rapid rise in Nai content in an Na/H ratio of 1:1. Microelectrode recordings of membrane potential demonstrated directly the electroneutral character of pHi regulation. Acid-induced net Na+ uptake could be either stimulated by further decreasing pHi or inhibited by decreasing pHo; Na+ uptake was unaffected by tetrodotoxin (10 micrograms/ml), quinidine (10(-3) M), DIDS (10(-4) M), Clo-free solution, or HCO3-free solution. Amiloride (10(-3) M) maximally inhibited both pHi regulation and Na+ uptake; the ID50 for amiloride inhibition of Na+ uptake was 3 microM. Nao-dependent H+ extrusion showed half-maximal activation at 15 mM Nao; Li+, but not K+ or choline+, could substitute for Na+ to support H+ extrusion. Cao-free solution also stimulated acid-induced Na+ uptake. We conclude that pHi regulation following an acid load in cardiac muscle cells is by an amiloride-sensitive, electroneutral Na/H exchange. Stimulation of Na/H exchange up to 54 pmol/cm2 X s indicates the rapidity of this exchange across cardiac cell membranes. Na/H exchange may also participate in steady state maintenance of pHi.     

Chemotactic factor-induced activation of Na+/H+ exchange in human neutrophils. II. Intracellular pH changes

The intracellular pH (pHi) changes resulting from chemotactic factor-induced activation of Na+/H+ exchange in isolated human neutrophils were characterized. Intracellular pH was measured from the equilibrium distribution of [14C]-5,5-dimethyloxazolidine-2,4-dione and from the fluorescence of 6-carboxyfluorescein. Exposure of cells to 0.1 microM N-formyl-methionyl-leucyl-phenylalanine (FMLP) in 140 mM Na+ medium at extracellular pH (pHo) 7.40 led to a rise in pHi along an exponential time course (rate coefficient approximately 0.55 min-1). By 10 min, a new steady-state pHi was reached (7.75-7.80) that was 0.55-0.60 units higher than the resting pHi of control cells (7.20-7.25). The initial rate of H+ efflux from the cells (approximately 15 meq/liter X min), calculated from the intrinsic intracellular buffering power of approximately 50 mM/pH, was comparable to the rate of net Na+ influx (approximately 17 meq/liter X min), an observation consistent with a 1:1 stoichiometry for Na+/H+ exchange. This counter-transport could be inhibited by amiloride (apparent Ki approximately 75 microM). When either the external ([Na+]o) or internal Na ([Na+]i) concentrations, pHo, or pHi were varied independently, the new steady-state [Na+]i and pHi values in FMLP-stimulated cells were those corresponding to a chemical equilibrium distribution of Na+ and H+ across the cell membrane. By analogy to other activated cells, these results indicate that an alkalinization of pHi in human neutrophils is mediated by a chemotactic factor-induced exchange of internal H+ for external Na+.     

Effect of chemical perturbation with NaSCN on receptor-estradiol interaction. A new exchange assay at low temperature

When 0.5 M sodium thiocyanate is added to uterine cytosol previously labeled with excess [3H]-17 beta-estradiol, no change can be detected in the steady-state cytosol concentration of [3H]estradiol-receptor complex for at least 20 h at 4 degrees C. However, the rate of exchange of bound estradiol in the presence of NaSCN was found to be substantially higher than that in the absence of the chaotropic salt. In the presence of NaSCN, the dissociation rate of the complex increases about 10-fold (K-1 SCN = 1.10 x 10(-2) min-1 vs. K-1 = 1.07 X 10 (-3)min-1) while the rate of association increases about 2-fold (K1 SCN = 1.2 X 10(7) min-1M-1 vs.K1= 7.4 X 10(6) min-1 M-1). The Kd changes 6.4-fold (Kd SCN = 9 X 10(-10) M vs. Kd = 1.4 x 10(-10 M) with no decrease in the number of binding sites as shown by Scatchard plots of saturation experiments. This effect of NaSCN can be exploited to assay preformed estrogen-receptor complex by exchange with [3H]estradiol at low temperature. When the sample containing preformed complex is incubated overnight (16 h) at 4 degrees C with excess [3H]estradiol in the presence of 0.5 M NaSCN, there is a quantitative exchange of nonlabeled for estradiol without loss of binding sites. Hormonal steroids other than estrogens do not interfere, and the exchange estradiol is bound with high affinity. Precision, accuracy, and linearity of the method are highly satisfactory.     

Catalysis by cobalt(II)-substituted carbonic anhydrase II of the exchange of oxygen-18 between CO2 and H2O

We have measured the catalysis by Co(II)-substituted bovine carbonic anhydrase II from red cells of the exchange of 18O between CO2 and H2O using membrane-inlet mass spectrometry. We chose Co(II)-substituted carbonic anhydrase II because the apparent equilibrium dissociation constant of HCO3- and enzyme at pH 7.4, KHCO3-eff approximately equal to 55 mM, was within a practicable range of substrate concentrations for the 18O method. For the native, zinc-containing enzyme KHCO3-eff is close to 500 mM at this pH. The rate constant for the release from the active site of water bearing substrate oxygen kH2O was dependent on the fraction of enzyme that was free, not bound by substrate HCO3- or anions. The pH dependence of kH2O in the pH range 6.0-9.0 can be explained entirely by a rate-limiting, intramolecular proton transfer between cobalt-bound hydroxide and a nearby group, probably His-64. The rate constant for this proton transfer was found to be 7 X 10(5) S-1 for the Co(II)-substituted enzyme and 2 X 10(6) S-1 for the native enzyme. These results are applied to models derived from proton-relaxation enhancement of water exchanging from the inner coordination shell of the cobalt in carbonic anhydrase. The anions iodide, cyanate, and thiocyanate inhibited catalysis of 18O exchange by Co(II)-substituted carbonic anhydrase II in a manner competitive with total substrate (CO2 and HCO3-) at chemical equilibrium and pH 7.4. These results are discussed in terms of observed steady-state inhibition patterns and suggest that there is no significant contribution of a ternary complex between substrate, inhibitor, and enzyme.     

Isolation of glutamic acid methyl ester from an Escherichia coli membrane protein involved in chemotaxis.

We have isolated glutamic acid 5-methyl ester from an Escherichia coli protein that is involved in chemotaxis. The bacteria were first incubated with [methyl-3H]methionine under conditions which are known to result in methylation of the protein. The protein, isolated by gel electrophoresis, was then digested by successive treatment with three proteolytic enzymes. One of the products was [methyl-3H]glutamic acid 5-methyl ester, identified by comparison with an authentic sample in the following studies: (a) chromatography on an automatic amino acid analyzer, (b) chromatography on paper in two solvent systems, (c) chromatography on paper of the N-acetyl derivatives, and (d) stability of the ester bond to various pH conditions. No aspartic acid 4-methyl ester was found in the enzymatic digest. Treatment of the methylated protein with alkali released the radioactivity as [3H]methanol, which was identified by gas chromatography and by preparation of the 3,5-dinitrobenzoate.     

A procedure for the separation and quantitation of tryptophan and amino sugars on the amino acid analyzer

Tryptophan, 5-methyl tryptophan, glucosamine, and galactosamine can be separated from each other and hydrolysis products including lysinoalanine by chromatography on a 6 × 260-mm column of W-3H resin. The column is developed at 70°C for 20 min with pH 3.95 (0.4 Na⁺) buffer, followed by pH 6.4 (1 Na⁺) buffer for 55 min using a Beckman 119 CL amino acid analyzer. The recovery of the internal standards, 5-methyl tryptophan and galactosamine, can then be used to correct for tryptophan and glucosamine losses, respectively. The procedure uses the column and buffers normally employed for protein hydrolysate analysis and does not require additional resin columns, special buffers, or flow rate changes.     

Heavy riboflavin synthase from Bacillus subtilis. Quaternary structure and reaggregation

Heavy riboflavin synthase of Bacillus subtilis was purified by a simplified procedure. The enzyme is a complex protein containing about 3 alpha-subunits (23.5 X 10(3) Mr) and 60 beta-subunits (16 X 10(3) Mr). The 10(6) Mr protein dissociates upon exposure to pH values above neutrality. Phosphate ions increase the stability at neutral pH. The dissociation induced by exposure of the enzyme to elevated pH is reversible in phosphate buffer at neutral pH. The stability of the enzyme at elevated pH values is greatly enhanced by the substrate analogue, 5-nitroso-6-ribitylamino-2,4(1H, 3H)-pyrimidinedione. Electron micrographs of negatively stained enzyme specimens show spherical particles with a diameter of 15.6 nm. Various immunochemical methods show that the alpha-subunits are not accessible to antibodies in the native molecule. The native enzyme is not precipitated by anti-alpha-subunit serum, and riboflavin synthase activity is not inhibited by the serum. However, these tests become positive at pH values that lead to dissociation of the enzyme. Subsequent to dissociation of the native enzyme at elevated pH values, the beta-subunits form high molecular weight aggregates. These aggregates form a complex mixture of different molecular species, which sediment at velocities of about 48 S and 70 S. The average molecular weight was approximately 5.6 X 10(6). Homogeneous preparations have not been obtained. Electron micrographs show hollow, spherical vesicles with diameters of about 29 nm. The substrate analogue 5-nitroso-6-ribitylamino-2,4(1H, 3H)-pyrimidinedione can induce the reaggregation of isolated beta-subunits with formation of smaller molecules, which are structurally similar to native riboflavin synthase. A homogeneous preparation of reaggregated molecules was obtained by renaturation of beta-subunits from 6.4 M-urea in the presence of the ligand. The sedimentation velocity of this aggregate is about 7% smaller than that of the native enzyme. The molecular weight is 96 X 10(4). Electron micrographs show spherical particles with a diameter of about 17.4 nm. Inspection of the micrographs tentatively suggests the presence of a central cavity. It appears likely that these molecules, which are devoid of alpha-subunits, have the same number and spatial arrangement of beta-subunits as the native enzyme. All data are consistent with the hypothesis that the native enzyme consists of a central core of alpha-subunits surrounded by a capsid-like arrangement of beta-subunits. The number of beta-subunits and the shape of the protein suggest a capsid-like arrangement of beta-subunits.(ABSTRACT TRUNCATED AT 400 WORDS)     

The modulation of cytochrome c electron self-exchange by site-specific chemical modification and anion binding

The site-specific chemical modification of horse heart cytochrome c at Lys-13 and -72 using 4-chloro-3,5-dinitrobenzoic acid (CDNB) increases the electron self-exchange rate of the protein. In the presence of 0.24 M cacodylate (pH* 7.0) the electron self-exchange rate constants, kex, measured by a 1H NMR saturation transfer method at 300 K, are 600, 6 X 10(3) and 6 X 10(4) M-1 X s-1 for native, CDNP-K13 and CDNP-K72 cytochromes c respectively. Repulsive electrostatic interactions, which inhibit cytochrome c electron self-exchange, are differentially affected by modification. Measurements of 1H NMR line broadening observed with partially oxidised samples of native cytochrome c show that ATP and the redox inert multivalent anion Co(CN)3-6 catalyse electron self-exchange. At saturation a limiting value of approximately 1.4 X 10(5) M-1 X s-1 is observed for both anions.     

Oxygen exchange between phosphate and water accompanies calcium-regulated ATPase activity of skinned fibers from rabbit skeletal muscle

The extent of oxygen exchange between phosphate and water has been measured for the calcium-regulated magnesium-dependent ATPase activity of chemically skinned fibers from rabbit skeletal muscle. The oxygen exchange was determined for isometrically held fibers by measuring with a mass spectrometer the distribution of 18O atoms in the product inorganic phosphate when ATP hydrolysis was carried out in H2(18)O. The extent of exchange was much greater in relaxed muscle (free Ca2+ less than 10(-8) M) than in calcium-activated muscle (free Ca2+ approximately equal to 3 X 10(-5) M). Activated fibers had an ATPase activity at least 30-fold greater than the relaxed fibers. These results correlate well with the extents of oxygen exchange accompanying magnesium-dependent myosin and unregulated actomyosin ATPase activities, respectively. In relaxed fibers, comparison of the amount of exchange with the ATPase activity suggests that the rate constant for the reformation of myosin-bound ATP from the myosin products complex is about 10 s-1 at 20 degrees C and pH 7.1. In each experiment the distribution of 18O in the Pi formed was incompatible with a single pathway for ATP hydrolysis. In the case of the calcium-activated fibers, the multiple pathways for ATP hydrolysis appeared to be an intrinsic property of the actomyosin ATPase in the fiber. These results indicate that in muscle fibers, as in isolated actomyosin, cleavage of protein-bound ATP is readily reversible and that association of the myosin products complex with actin promotes Pi release.     

17O and 14N n.m.r. studies of the Co (II) interaction with cyclo(Ala*-Ala) in aqueous solution

The complexation of cyclo(Ala*-Ala) with the cobaltous ions in aqueous solution was investigated by 17O and 14N n.m.r. spectroscopy. The 17O and 14N transverse relaxation time (T2p) and chemical shift (delta omega a) of cyclo(Ala*-Ala) were measured as a function of the temperature at pH = 7.03 +/- 0.02, and pH = 6.45 +/- 0.02, and as a function of pH at room temperature. No effects of pH on the transverse relaxation time and chemical shift were observed. Complementary 17O studies of the solvent water molecules were also carried out. The hyperfine coupling constant and the entropy and enthalpy of activation for the exchange of cyclo(Ala*-Ala) and water molecules between the coordinated and noncoordinated states were determined by least-square fit of theoretical equation for the chemical shift delta omega a to experimental data. The hyperfine coupling constant of the peptide bound oxygen was determined to be (-1.6 +/- 0.1) X 10(5) Hz and the entropy and enthalpy (32.0 +/- 3.0) kJ/mol and (-12.0 +/- 1.0) e.u, respectively. Information obtained from 17O n.m.r. study allows some inferences concerning the probable coordination sphere of the cobaltous ion. There are three types of complexes: Co(H2O)6(2+), CoL X 5H2O and CoL2 X 4H2O, with relative concentrations 19.9%, 2.9%, and 77.2%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)