Isolation of bovine cytochrome c1 as a single non-denatured subunit using gel filtration or high pressure liquid chromatography in deoxycholate

The non-denatured cytochrome $\text{c}{}_1$ subunit of bovine ubiquinone-cytochrome $\text{c}$ reductase was isolated using either gel filtration or high pressure liquid chromatography in 1% deoxycholate. The preparation was a single band on polyacrylamide gel electrophoresis in dodecyl sulfate, had a heme content of 31 nmol heme/mg protein, had an absorbance ratio $\text{A}{}_{417}\text{A}{}_{278}\text{= 2.65}$, a visible spectrum with maxima at 553, 530, 523.5, 417, 317, and 277 nm for the reduced protein, and an amino acid analysis identical to that previously reported for the isolated denatured protein. The Stokes' radius of this non-denatured deoxycholate solubilized protein was 34Å, indicating that the protein either is a dimer in deoxycholate, is asymmetric, or binds large amounts of detergent.     

Electron transport components from methanogenic bacteria: The ferredoxin from Methanosarcinabarkeri (strain Fusaro)

A ferredoxin has been isolated from the methanogenic organism $\text{Methanosarcina}\text{barkeri}$ (strain Fusaro). The protein appears to be constituted by two identical subunits of molecular weight approx. 6000 daltons. The UV-visible spectrum of the protein is characterized by two broad absorption peaks centered at 410 and 300 nm and an absorbance ratio $\text{A}{}_{410}\text{A}{}_{300}\text{= 0.8}$. The molar extinction coefficients at 410 and 300 nm are 36,500 and 45,625 M^?1 cm^?1, respectively. The amino acid compsition of $\text{M.}\text{barkeri}$ ferredoxin shows a preponderance of acidic residues and lacks five amino acids. The protein contains 8 cysteine residues and approx. 7 iron atoms and 7–8 acid-labile sulfide groups per molecule which are indicative of the presence of two iron-sulfur clusters in the molecule. The N-terminal sequence shows a high degree of homology with the sequences of ferredoxins from $\text{Clostridium}\text{pasteurianum}\text{,}\text{Desulfovibrio}\text{gigas}$ and $\text{Desulfovibrio}\text{africanus}\text{.}\text{M.}\text{barkeri}$ ferredoxin functions as an electron carrier in the pyruvate dehydrogenase system. Its possible role in a variety of electron transfer reactions is discussed.     

Isolation and properties of somatic and testicular cytochromes c from rat tissues

Somatic (c_s) and a testis-specific (c_{t I}) cytochromes c were purified to homogeneity from rat tissues (heart, liver, kidney, and testis). The purification procedure involved (1) homogenization of tissues at pH 4.5, (2) treatment with methanol-chloroform solvents, (3) hydroxylapatite column chromatography, (4) carboxymethyl-cellulose column chromatography, and (5) Sephacryl S-200 gel filtration. The isolated cytochromes c were free from polymeric and other “modified” forms, and did not bind CO, azide, or cyanide. The absorption maxima and the molecular weights of both cytochromes c_s and c_{t I} were identical. The ratio of $\text{A}{}_{\mathrm{<mtext>549.5\; </mtext><mtext>nm</mtext>}}\text{(}\text{reduced}\text{)}\text{A}{}_{\mathrm{<mtext>280\; </mtext><mtext>nm</mtext>}}\text{(}\text{oxidized}\text{)}$ for cytochromes c_s averaged 1.28. The unique properties of cytochrome c_{t I}, compared to somatic cytochrome c, were as follows: (1) different elution profiles from hydroxylapatite and carboxymethyl-cellulose column chromatography experiments, (2) less basic intrinsic molecular charge shown by the slow mobility in native polyacrylamide gel electrophoresis, (3) probable asymmetric molecular shape as evidenced from gel filtration experiments, (4) significantly higher millimolar extinction coefficient values (33.6 at 549.5 nm), (5) a low ratio (1.04) of $\text{A}{}_{\mathrm{<mtext>549.5\; </mtext><mtext>nm</mtext>}}\text{(}\text{reduced}\text{)}\text{A}{}_{\mathrm{<mtext>280\; </mtext><mtext>nm</mtext>}}\text{(}\text{oxidized}\text{)}$, and (6) difference of about 20 amino acid residues per mole.     

Stoichiometry versus coupling ration in the cotransport of Na and different neutral amino acids

The stoichiometry of Na coupling to amino acid movement across the brush border membrane of the rabbit distal ileum has been determined under initial rate conditions.The coupling ratio, defined as the amino acid-dependent Na influx/the Na-dependent amino acid influx, was equal to unity for alanine, measured over a 10-fold range of Na and alanine concentrations. Coupling ratio values determined under a single set of conditions for a number of amino acids varied from 1 for serine to 4.6 for methionine. Reducing the methionine concentration from 12.5 to 1.5 mM caused the coupling ratio value to fall from 4.6 to 1.2.These results are explained by assuming a fixed stoichiometry of 1 : 1 under all conditions, with initial binding of the amino acid (A) to the Na-dependent carrier (E) but with some amino acids being able to cross on the Na-dependent carrier in the absence of Na.The variation in coupling ratio values can be used to calculate K_A, the apparent dissociation constant of amino acid from the Na-dependent carrier in the absence of Na, and the ratio $\text{k}{}_1\text{k}{}_2$, where k₁ and k₂ are first-order rate constants for translocation of the complexes EA and EANa, respectively. This method of processing results has been defined as delta analysis. The value of K_A for methionine is 3.6 ± 1.1 mM and the $\text{k}{}_1\text{k}{}_2$ ratio is 1.01 ± 0.07. The constant coupling ratio value of 1 for alanine indicates that the value for K_A is extremely high or that the k₁ value is extremely low.     

NMR characterization of three forms of ferredoxin from Desulphovibrio gigas, a sulphate reducer

A NMR and magnetic susceptibility study of the oxidized and reduced states of three different oligomers (forms) of a [4Fe-4S] ferredoxin protein from Desulphovibrio gigas, FdI, FdI′, and FdII was carried out. FdI and FdI′ are different trimers and FdII a tetramer of the same basic subunit. A probable assignment of the contact shifted resonances is indicated. Since the temperature dependences of the contact shifted resonances associated with each [4Fe-4S] are not all similar a delocalized model for the spin densities on the 4Fe does not apply. The exchange rate between oxidized and reduced states is slow on the NMR time scale. The three oligomers are not magnetically equivalent. Using the “three state hypothesis” terminology it is shown that FdI_ox is predominantly in the C^2? state and changes upon reduction into the C^3? state, while FdII_ox is in the C^? state and changes into the C^2? state. FdI′ does not easily fit into this classification. This study shows a similarity of magnetic behaviour between FdI and bacterial ferredoxins (e.g. Bacillus polymyxa) and between FdII and HiPIP from Chromatium sp.. The influence of the quaternary structure on the stabilization of the different oxidation states of ferredoxins as well as on their redox potentials is discussed.     

Isolation and characterization of isocitrate lyase of castor endosperm

Isocitrate lyase (threo-D_S-isocitrate glyoxylate-lyase, EC 4.1.3.1) has been purified to homogeneity from castor endosperm. The enzyme is a tetrameric protein (molecular weight about 140,000; gel filtration) made up of apparently identical monomers (subunit molecular weight about 35,000; gel electrophoresis in the presence of sodium dodecyl sulfate). Thermal inactivation of purified enzyme at 40 and 45 °C shows a fast and a slow phase, each accounting for half of the intitial activity, consistent with the equation: , where A₀ and A_t are activities at time zero at t, and k₁ and k₂ are first-order rate constants for the fast and slow phases, respectively. The enzyme shows optimum activity at pH 7.2–7.3. Effect of [S]on enzyme activity at different pH values (6.0–7.5) suggests that the proton behaves formally as an “uncompetitive inhibitor.” A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pK_a equal to 6.9. Successive dialysis against EDTA and phosphate buffer, pH 7.0, at 0 °C gives an enzymatically inactive protein. This protein shows kinetics of thermal inactivation identical to the untreated (native) enzyme. Full activity is restored on adding Mg²⁺ (5.0 mm) to a solution of this protein. Addition of Ba²⁺ or Mn²⁺ brings about partial recovery. Other metal ions are not effective.     

Crystallographic studies of immunoglobulins: crystallization of the Fc fragment of rabbit IgG with and without cleavage of the inter-chain disulphide bridge

The Fc fragment was prepared from rabbit immunoglobulin G by digestion with papain, both with the inter-chain disulphide bond intact, and after reduction and alkylation. These two types of Fc crystallized in different, yet related forms, each with one dimer in the asymmetric unit. The covalently linked dimer crystallized in space group $\text{P2}{}_1\text{; a = 68.85 ± 0.05}\text{A}\text{?}\text{, b = 72.50 ± 0.05}\text{A}\text{?}\text{, c = 60.40 ± 0.05}\text{A}\text{?}$ and β = 105.1 ± 0.2 °. The reduced, non-covalently linked dimer also crystallized in space group $\text{P2}{}_1\text{; a = 81.55 ± 0.05}\text{A}\text{?}\text{, b = 55.65 ± 0.05}\text{A}\text{?}\text{, c = 68.85 ± 0.05}\text{A}\text{?}$ and β = 1051 ± 0.2 °. A non-crystallographic 2-fold axis relating the two identical polypeptide chains is clearly visible in the h0l projection of the second crystal form.     

Isolation and characterization of a rubredoxin and an (8Fe-8S) ferredoxin from Desulfuromonas acetoxidans

A two cluster (4Fe4S) ferredoxin and a rubredoxin have been isolated from the sulfur-reducing bacterium Desulfuromonas acetoxidans. Their amino acid compositions are reported and compared to those of other iron-sulfur proteins.The ferredoxin contains 8 cysteine residues, 8 atoms of iron and 8 atoms of labile sulfur per molecule; its minimum molecular weight is 6163. The protein exhibits an absorbance ratio of $\text{A}{}_{385}\text{A}{}_{283}\text{= 0.74}$. Storage results in a bleaching of the chromophore; the denatured ferredoxin is reconstitutable with iron and sulfide. The instability temperature is 52°C.The rubredoxin does not differ markedly from rubredoxins from other anaerobic bacteria.     

A cobalt containing protein isolated from Desulfovibrio gigas, a sulfate reducer

A protein which contains a cobalt porphyrin was isolated from the sulfate reducer Desulfovibrio gigas. This protein has a molecular weight of approximately 16,700 daltons and is acidic, having an iso-electric point at 3.7. The N-terminal residue was shown to be threonine, and a cobalt analysis gave 0.8 cobalt atoms/molecule, suggesting the presence of a single prosthetic group. The protein has a violet color with absorption bands typical of a metal porphyrin center with maxima at 420 nm, 580 nm with a shoulder at 550 nm. The ratio $\text{A}{}_{420}\text{(γ)}\text{A}{}_{588}\text{(α)}$ is 2.1. The protein has no electron paramagnetic resonance (e.p.r.) spectrum, and as the visible spectrum suggests, it probably contains diamagnetic Co^III porphyrin. However the cobalt centre appears to be protected from reduction by sodium dithionite or sodium borohydride. Att$\text{e}$mpts at ligand substitution with strong nucleophiles such as CN, causes a slight spectral shift to higher wavelenghts. The cobalt porphyrin can be extracted from the protein with an acidified acetone solution, indicating that it is not covalently bound to the protein.     

New experimental approach to the estimation of rate of electron transfer from the primary to secondary acceptors in the photosynthetic electron transport chain of purple bacteria

A method for calculating the rate constant ($\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$) for the oxidation of the primary electron acceptor (A₁) by the secondary one (A₂) in the photosynthetic electron transport chain of purple bacteria is proposed.The method is based on the analysis of the dark recovery kinetics of reaction centre bacteriochlorophyll (P) following its oxidation by a short single laser pulse at a high oxidation-reduction potential of the medium. It is shown that in Ectothiorhodospira shaposhnikovii there is little difference in the value of $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ obtained by this method from that measured by the method of Parson ((1969) Biochim. Biophys. Acta 189, 384–396), namely: $\text{(4.5±1.4) · 10}{}^3\text{s}{}^{\mathrm{?1}}$ and $\text{(6.9±1.2) · 10}{}^3\text{s}{}^{\mathrm{?1}}$, respectively.The proposed method has also been used for the estimation of the $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ value in chromatophores of Rhodospirillum rubrum deprived of constitutive electron donors which are capable of reducing P⁺ at a rate exceeding this for the transfer of electron from A₁ to A₂. The method of Parson cannot be used in this case. The value of $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ has been found to be $\text{(2.7±0.8) · 10}{}^3\text{s}{}^{\mathrm{?1}}$.The activation energies for the A₁ to A₂ electron transfer have also been determined. They are 12.4 kcal/mol and 9.9 kcal/mol for E. shaposhnikovii and R. rubrum, respectively.     

Cytoskeletal proteins used as marker of differentiation in mouse terato;carcinoma cells

The cyclic AMP receptor protein (CRP) of Escherichia coli has been crystallized. The crystals are orthorhombic, space group $\text{P2}{}_1\text{2}{}_1\text{2}{}_1\text{, a = 46.5}\text{A}\text{?}$, b = 97.1 Å, $\text{c = 105.4}\text{A}\text{?}$, with one dimeric CRP molecule per asymmetric unit.     

The composition-dependence of the extent of reaction between a multivalent acceptor and a bivalent ligand: Implications of self-interaction of the ligand in precipitin effects

Theory is presented relating to the reversible interaction of an f-valent acceptor, A, with a bivalent ligand, B, which leads to the formation of a series of complexes comprising networks of alternating A and B molecules. An explicit expression is derived for the overall extent of reaction in terms of the total molar concentrations of reactants $\text{(}\text{m}{}_{\mathrm{A}}$ and $\text{m}{}_{\mathrm{B}}\text{)}$, the valency of the acceptor and the site-binding constant, k, governing the equilibria. It is shown by differentiation of this expression holding $\text{m}{}_{\mathrm{A}}$ (or $\text{m}{}_{\mathrm{B}}$) fixed that relations are available for the independent evaluation of f and k from a combination of precipitin and radioimmunoassay experiments. Moreover, it is established that dilution with solvent ($\text{m}{}_{\mathrm{A}}\text{/}\text{m}{}_{\mathrm{B}}$ fixed) cannot lead to the appearance of a precipitate with this type of crosslinking system. The latter observation forms the background for the development of theory pertaining to the joint operation of ligand dimerization, 2B?B₂, and crosslinking of the multivalent acceptor with bivalent B₂. The theoretical examination of this system is developed in terms of site-probability functions and involves the delineation of unique solutions for the extent of crosslinking reaction aided by the definition of the extent of binding in defined limits. It is shown with the use of numerical examples that the system involving self-associating ligand may result in the appearance of a precipitate on dilution with solvent and the conditions for the operation of this phenomenon are elucidated. It is noted that other types of ligand self-interaction may lead to similar effects in crosslinking systems, and the general principles emerging from this study are discussed in terms of systems in which antibody ligands are known to be involved in association reactions or are suspected to be so involved on the basis of precipitation effects observed on dilution with solvent.     

Some observations on a new type of point average molecular weight

A new type of (reduced) point average molecular weight A¹, is described. Several interesting properties are developed: (i) $\text{A}{}^1\text{=}\text{reduced}$ weight average molecular weight over the whole cell, $\text{A}{}_{\mathrm{<mtext>w</mtext>}}{}^{\mathrm{o}}\text{A}{}^1\text{(}\text{meniscus}\text{) = A}{}_{\mathrm{<mtext>w</mtext>}}\text{(}\text{meniscus}\text{); (iii) A}{}^1\text{(}\text{zero concentration}\text{) =}\text{reduced}$ number average molecular weight, A_n (meniscus). In addition, its usefulness in extracting the meniscus concentration, J(a), and in examining heterogeneous systems such as mucus glycoproteins, are discussed. The evaluation and application of $\text{A}{}^1$ requires only simple computational facilities, without the use for large-scale multiple data acquisition and recycling techniques.     

Neutral amino acid transport in Leishmania promastigotes

Neutral amino acid transport was investigated in Leishmania promastigotes. Proline and alanine transport occur against their concentration gradient although there is a very rapid (40% at 30 min) conversion of proline to alanine. Uptake of these amino acids occurs by a sodium-independent route which is completely eliminated by addition of CCCP or KCN. $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for proline and alanine are 80 μM and 63 μM with $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ values of 6.4 and 7.2 nmol/min per mg dry weight, respectively. Countertransport of proline, alanine and phenylalanine was measured by loading the cells with a variety of neutral amino acids and proline analogs, followed by CCCP addition. The effect of aminooxyacetic acid, an inhibitor of alanine aminotransferase (EC 2.6.1.2), on proline and alanine countertransport was also examined. The results obtained are consistent with the presence of at least two systems for neutral amino acid transport in Leishmania promastigotes.     

Oxidation of anionic nitroalkanes by flavoenzymes,and participation of superoxide anion in the catalysis

The reactivities of anionic nitroalkanes with 2-nitropropane dioxygenase of Hansenula mrakii, glucose oxidase of Aspergillus niger, and mammalian d-amino acid oxidase have been compared kinetically. 2-Nitropropane dioxygenase is 1200 and 4800 times more active with anionic 2-nitropropane than d-amino acid oxidase and glucose oxidase, respectively. The apparent K_m values for anionic 2-nitropropane are as follows: 2-nitropropane dioxygenase, 1.61 mm; glucose oxidase, 16.7 mm; and d-amino acid oxidase, 11.1 mm. Anionic 2-nitropropane undergoes an oxygenase reaction with 2-nitropropane dioxygenase and glucose oxidase, and an oxidase reaction with d-amino acid oxidase. In contrast, anionic nitroethane is oxidized through an oxygenase reaction by 2-nitropropane dioxygenase, and through an oxidase reaction by glucose oxidase. All nitroalkane oxidations by these three flavoenzymes are inhibited by Cu and Zn-superoxide dismutase of bovine blood, Mn-superoxide dismutases of bacilli, Fe-superoxide dismutase of Serratia marcescens, and other $\text{O}{}_2{}^{\mathrm{?}}$ scavengers such as cytochrome c and NADH, but are not affected by hydroxyl radical scavengers such as mannitol. None of the $\text{O}{}_2{}^{\mathrm{?}}$ scavengers tested affected the inherent substrate oxidation by glucose oxidase and d-amino acid oxidase. Furthermore, the generation of $\text{O}{}_2{}^{\mathrm{?}}$ in the oxidation of anionic 2-nitropropane by 2-nitropropane dioxygenase was revealed by ESR spectroscoy. The ESR spectrum of anionic 2-nitropropane plus 2-nitropropane dioxygenase shows signals at g₁ = 2.007 and g₁₁ = 2.051, which are characteristic of $\text{O}{}_2{}^{\mathrm{?}}$. The $\text{O}{}_2{}^{\mathrm{?}}$ generated is a catalytically essential intermediate in the oxidation of anionic nitroalkanes by the enzymes.     

The presence of multiple cytochrome b proteins in succinate-cytochrome c reductase.

Two cytochrome $\text{b}$ proteins were isolated from succinate-cytochrome $\text{c}$ reductase and the cytochrome $\text{b-c}{}_1$ complex. Their molecular weights were determined to be 37,000 and 17,000 daltons by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Spectral properties and amino acid composition of these two proteins are reported in the paper.     

Damping of oscillations in the semiquinone absorption in reaction centers after successive flashes. Determination of the equilibrium between QA−QB and QAQB−

A quantitative model for the damping of oscillations of the semiquinone absorption after successive light flashes is presented. It is based on the equilibrium between the states Q_A^?Q_B and Q_AQ_B^?. A fit of the model to the experimental results obtained for reaction centers from Rhodopseudomonas sphaeroides gave a value of $\text{α =}\text{[}\text{Q}{}_{\mathrm{<mtext>A</mtext>}}{}^{\mathrm{?}}\text{Q}{}_{\mathrm{<mtext>B</mtext>}}\text{]}\text{([}\text{Q}{}_{\mathrm{<mtext>A</mtext>}}{}^{\mathrm{?}}\text{Q}{}_{\mathrm{<mtext>B</mtext>}}\text{] + [}\text{Q}{}_{\mathrm{<mtext>A</mtext>}}\text{Q}{}_{\mathrm{<mtext>B</mtext>}}{}^{\mathrm{?}}\text{])}\text{= 0.065 ± 0.005}$ (T = 21°C, pH 8).     

Electron diffraction study on single crystals of l-type and dl-type lecithins

An electron diffraction study was carried out on thin single micro-crystals of l-type and dl-type dipalmitoyl lecithins grown in xylene suspensions and fine net patterns were obtained and the mechanism of the thermotropic phase transitions of them was clarified.From the apparent structure of diffraction patterns in low temperature, it is confirmed that the two dimensional lattices have p mm symmetry in l-type and in dl-type lecithins. Lattice parameters from the [001] projection are $\text{d}{}_{100}\text{= 9.9}\text{A}\text{?}$ and $\text{d}{}_{010}\text{= 8.8}\text{A}\text{?}$ in l-type, and $\text{d}{}_{100}\text{= 17.2}\text{A}\text{?}$ and $\text{d}{}_{010}\text{= 8.9}\text{A}\text{?}$ in dl-type.With anisotropic variation of dimensions along a and b axes, i.e. contraction for a and expansion for b, induced by temperature rise by electron irradiation during the observation, these diffraction patterns of the lattices of l-type and dl-type were transformed into those characterized by the six diffraction spots having nearly the same spacings. Four of them are observed on slightly outer and two are slightly inner positions as compared with their mean spacings of about (4.1 Å)^?1 in l-type and about (4.2 Å)^?1 in dl-type. The changes in the patterns observed indicate that at low temperatures the hydrocarbon chains are nearly perpendicular to the layer in dl-type lipid, and tilted with a more complicated packing in l-type ones. The dimension along a in dl-type is twice as large as that in l-type.