Flash spectroscopic characterization of photosynthetic electron transport in isolated heterocysts

Electron transport was studied in heterocysts of the filamentous cyanobacterium Anabaena 7120 using spectral and kinetic analysis of absorbance transients elicited by single turnover flashes. Consistent photosynthetic turnovers were observed only in the presence of an exogenous source of reductant; therefore measurements were routinely made under a gas phase containing H2. Prominent absorbance changes corresponding to the oxidation of cytochrome c (554 nm) and the reduction of cytochrome b563 (563 nm) were observed. Under the most reducing conditions (99% H2/1% O2) cytochrome b563 was partially reduced between flashes in a slow, dark reaction. At 10-15% O2, the slow, dark reduction of cytochrome b563 was eliminated. Cytochrome turnover ceased entirely at high O2 concentrations (30%) but was restored by the addition of 25 microM KCN, demonstrating an interaction between the photosynthetic and respiratory electron transfer chains. Strobilurin A slowed the re-reduction of cytochrome c and eliminated the appearance of reduced cytochrome b563 by blocking electron transfer between reduced plastoquinone and the cytochrome b/f complex. Inhibition at a second site was apparent with 2-(n-heptyl)-4-hydroxyquinoline N-oxide, which blocked the reoxidation of cytochrome b563 but had little effect on cytochrome c relaxation. In uncoupled heterocysts, the rates of cytochrome c re-reduction and cytochrome b563 reduction were equal. Additional unassigned absorbance changes at 475 nm, 515 nm, and 572 nm were partially characterized. No absorbance change corresponding to an electrochromic shift was observed.     

A microassay for the determination of monoamine oxidase activity using electron capture gas chromatography

A sensitive and specific assay for determining monoamine oxidase (MAO) activity in serum and platelets is described. The procedure employs m-iodobenzylamine as substrate. The product, m-iodobenzaldehyde, is separated on an OV-17 column and measured by electron capture. Gas chromatography offers the advantage of analytical specificity without the need for additional procedural steps to eliminate potential interferences. Electron capture detection of the iodinated aldehyde is sufficiently sensitive to allow routine analysis on platelet samples of less than 15 μg of protein and serum aliquots of 50 μl or less. Analysis of approximately 80 samples per day may be accomplished by a single worker by employing an automatic sampling system for gas chromatographic injection. This fact, in addition to the small sample size, makes the method particularly suitable for the determination of large numbers of clinical samples.     

A dynamical study on the interactions between the cytoskeleton components in the human erythrocyte as detected by saturation transfer electron paramagnetic resonance of spin-labeled spectrin, ankyrin, and protein 4.1

Isolated human erythrocyte spectrin, ankyrin, and protein 4.1 have been labeled with the maleimide spin label, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl, and studied by saturation transfer electron paramagnetic resonance spectroscopy. The presence of the labels does not affect the reassociation of these proteins with erythrocyte membranes selectively depleted of either spectrin-actin or of all the extrinsic proteins. When maleimide spin-labeled spectrin is reassociated with the erythrocyte membrane in presence of all the cytoskeleton components, including endogeneous or purified muscle actin, spectrin still preserves its flexible character. The rotational mobilities of maleimide spin-labeled ankyrin and maleimide spin-labeled protein 4.1 are of the same order of magnitude (tau c (L"/L) approximately 5 X 10(-5) and 8 X 10(-5) s, respectively, at 2 degrees C), while protein 4.1 is almost three times smaller in size than ankyrin. This result indicates that the movements of membrane-bound maleimide spin-labeled protein 4.1 are more restricted than those of ankyrin. This suggests that their respective binding sites have different structural properties. The rotational movements of both proteins are slowed down on the addition of spectrin indicating that protein 4.1 as well as ankyrin also represents one of the links of the cytoskeleton to the membrane.     

A rapid procedure for the isolation and purification of photosynthetic reaction centers from Rhodopseudomonas sphaeroides R-26

A rapid purification procedure has been developed for the isolation of reaction centers From Rhodopseudomonas sphaeroides strain R-26. The procedure takes about 7 h and results in yields of 60–75%. The ratio of the optical absorbances at 280 and 800 nm is between 1.4 and 1.5, and preparations can be made with either one or two quinones per reaction center. EPR spectra show a sharp g 1.83 signal for the ubisemiquinone. The substitution of lauryl maltoside for lauryldimethylamine oxide suppresses reaction-center degradation in solution.     

A physico-chemical study of heparin: Evidence for a calcium-induced co-operative conformational transition

The conformations of heparin in aqueous solution in the presence of sodium, potassium, magnesium and calcium cations were studied using circular dichroism, optical rotation, nuclear magnetic resonance and equilibrium dialysis. Potassium and magnesium cations, when added to sodium heparinate solutions, cause small chiroptical changes. Binding of calcium ions gives rise to large changes in both optical rotation and circular dichroism. This is indicative of a major change in chain conformation, which is also manifest in ¹³C and ¹H n.m.r.⁴Equilibrium dialysis suggests one mole of calcium bound per mole of tetrasaccharide, which n.m.r. indicates to be appropriately sulphated iduronateglucosamine-iduronate-glucosamine. The calcium is chelated by two iduronate carboxyl groups. Proton-proton coupling constants, determined by convolution difference spectroscopy and Carr-Purcell sequences, indicate that, over the temperature range 285 to 353 K, the iduronate ring is best described as ¹C₄(l) and the glucosamine residue as ⁴C₁(d) for both sodium and calcium forms.The conformational change induced by calcium is ascribed to rotation around the glycosidic linkages. The binding process is co-operative and the binding constant of 10³ to 10⁴m^?1 is biologically significant. The findings are consistent with intramolecular binding. Hence, this study represents the first report of a polysaccharide undergoing a cation-induced intramolecular disorder-order process. The authors postulate that a function of the post-polymerization epimerization of d-glucuronate to l-iduronate is the attainment of the precise geometry required for co-operative calcium binding with consequent modulation of the flexibility of the tetrasaccharide units.     

A kinetic and spectral study of the alkaline transitions of chloroperoxidase

The optical spectrum of chloroperoxidase in the near ultraviolet and visible region was studied from pH 6 to 12. Chloroperoxidase undergoes a first transition which is irreversible at pH 7 and a second transition near pH 11. The second transition is reversible provided the incubation period above pH 11 is kept as short as possible. The spectral properties of the intermediates were studied in the Soret region by means of a rapid scan apparatus. The rates of the transitions were measured in a stopped-flow apparatus. The pH dependence of both the spectra and the rate constants indicate that at least three ionizations are involved in the first alkaline transition.     

Evidence for two sites of inhibition of photosynthetic electron transport by dibromothymoquinone

Two sites in the photosynthetic electron transport chain of spinach chloroplasts are sensitive to inhibition by the plastoquinone antagonist dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone). This compound imposes maximal inhibition on reactions involving electron transport from water to a terminal acceptor such as ferricyanide at concentrations of about 1 μm. At concentrations of about 10 μm, dibromothymoquinone also inhibits electron transport reactions catalyzed by photosystem II in the presence of p-phenylenediimines or p-benzoquinones. This inhibition is observed in both untreated and $\text{KCN}\text{Hg}$-inhibited chloroplast preparations. Thiol incubation of chloroplasts exposed to dibromothymoquinone relieves inhibition at both sites. This reversal of inhibition is, however, different for the two sites. Restoration of ferricyanide reduction, which is blocked by 1 μm dibromothymoquinone, required high thiol/inhibitor ratios and incubation times with thiol of up to 3 min. The reversal of inhibition of p-phenylenediimine reduction by photosystem II, on the other hand, requires a thiol/inhibitor ratio of 1, and incubation times as short as 5 s. Addition of bovine serum albumin to absorb dibromothymoquinone results in a partial restoration of photosystem II reactions, but ferricyanide reduction, which requires photosystem II and photosystem I, cannot be restored by this procedure.     

Spectroelectrochemical determination of the heterogeneous electron transfer kinetics of soluble spinach ferredoxin

Heterogeneous electron transfer rate parameters for soluble spinach ferredoxin are reported using a recently developed single potential step spectroelectrochemical technique. The reductive kinetics were measured by monitoring the $\text{decrease}$ in absorbance as a function of time for several overpotential steps at methyl viologen modified optically transparent gold minigrid electrodes. These measurements yielded an average formal heterogeneous electron transfer rate constant (k_f,h^o′ = 6.5 (±1.3) × 10^?5 cm/s) and electrochemical transfer coefficient (α = 0.60 ± (0.16)) at pH 7.5. These results are the first heterogeneous electron transfer rate parameters reported for this species.     

Binding of terbium to apoferritin: a fluorescence study

Luminescence measurements show that apoferritin binds three Tb(III) atoms per subunit in accordance with crystallographic evidence. Fe(II) competes with Tb(III) for at least some of the binding sites. This competition may be the molecular basis for the inhibition of iron incorporation into apoferritin brought about by Tb(III). Ca(II), which is generally replaced by Tb(III) in Ca(II) binding proteins, does not compete with the lanthanide for binding to apoferritin.     

Biosynthetic mechanism of ribulose-1,5-bisphosphate carboxylase in the purple photosynthetic bacterium, Chromatium vinosum. III. Absence of extrachromosomal DNA

Inducible formation of ribulose-1,5-bisphosphate (RuBP) carboxylase in the cells of Chromatium vinosum under autotrophic conditions was not affected by six different inhibitors of DNA synthesis. Photosynthetic CO2 fixation and RuBP carboxylase activities were not influenced by seven reagents known to eliminate plasmids. Plasmids were not detectable by agarose gel electrophoresis employing either the cleared lysate or alkaline sodium dodecyl sulfate method, nor were they detected by ethidium bromide-CsCl density gradient centrifugation. Overall experimental results tend to indicate that plasmids are absent in the Chromatium cells and that the induction of RuBP carboxylase is presumably not regulated in the DNA replication process.     

A systematic approach to the comparison of protein structures

A systematic method has been developed for comparing the backbone conformations of proteins (Remington & Matthews, 1978). Two proteins are compared by successively optimizing the agreement between all possible segments of a chosen length from one protein, and all possible segments of the same length from the other protein. The method reveals any similarities between the two proteins, and provides an estimate of the statistical significance of any given structure agreement that is obtained.The method has been tested in a number of cases, including comparisons of the dehydrogenases and of the pancreatic and bacterial serine proteases. These examples were chosen to test the ability of the comparison method to detect structural similarities in the presence of large insertions and deletions. The results suggest that the detection of the “nucleotide binding fold” in the dehydrogenases is at the limit of the capability of the comparison technique in its original form, although it may be possible to generalize the method to allow for insertions and deletions in proteins.The results of many protein comparisons, made with different probe lengths, are summarized. For medium and long probe lengths, the average value of the structural agreement does not depend very much on the type of protein being compared. The average value of the structure agreement increases with the square root of the probe length, but for probe lengths above about 40 residues, the standard deviation is independent of probe length. From these observations it is possible to construct a generalized probability diagram to evaluate the significance of any structure agreement that might be obtained in comparing two proteins.     

Irreversible inhibition of folate transport in Lactobacillus casei by covalent modification of the binding protein with carbodiimide-activated folate

Activated folate formed by reaction of folic acid and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide irreversibly inhibits the folate transport system of Lactobacillus casei. Complete inhibition of both folate binding to the carrier protein and folate transport was achieved by pretreatment of the cells at low temperature (4 °C) and at neutral pH with 200 nm activated folate. Fifty percent inhibition of binding and transport occurred at 35 and 40 nm activated folate, respectively. Specificity was demonstrated by the fact that excess nonactivated folate added during the pretreatment step afforded complete protection of the binding protein against inhibition, and that activated folate had no effect on the binding or transport of thiamine. Rapid measurements at 4 °C were employed to show that, prior to the appearance of irreversible inhibition, activated folate (K_i = 15 nM) interacted reversibly with the binding site for folate (K_d = 0.8 nM). Cells treated with activated [³H]folate incorporated 1 mol of folate per mole of binding protein. Purification of the labeled protein followed by digestion with Pronase led to the isolation of a compound identified as ?-N-folyl lysine. The ?-amino group of a lysyl residue thus appears to be the nucleophilic group at the binding site that reacts with activated folate.     

Radical ions derived from hydrides and methyls of aluminum,silicon and phosphorus: A semi-empirical SCFMO study

Semi-empirical SCFMO calculations were made of the energies, and geometric and electronics structures of a range of radical ions of type MR₃^± and M₂R₆^± where M = Al, Si or P, and R = H or CH₃. In each of the MH₃ radicals, methylation effects an increase in the HMH angle: the structure of Al₂Me₆^?, formed by γ-irradiation of Al₂Me₆, is found to have C_s symmetry and to resemble a weak complex of AlMe₂ and AlMe₄^?. Possible identities for the radical, other than AlH₃^?, formed on γ-irradiation of LiAlH₄ are suggested, and a considerable number of plausible identities are firmly ruled out.     

Calculation of protein volumes: An alternative to the Voronoi procedure

All the methods that have been applied to assessing local volume occupation or packing in proteins have particular defects. For example, the Voronoi method used by Richards (method A) and by Finney misallocates both non-bonded and covalent contacts in a geometrically rigorous, though chemically inconsistent, manner. Richards' method B, in which covalent and non-bonded contacts are partitioned in chemically sensible ways, is unfortunately not completely rigorous, in that every polyhedron vertex has associated with it a small vertex-error polyhedron, which is not allocated to any atom.We present here a generalization of the Voronoi method that is particularly suited to multicomponent assemblies such as proteins. This radical plane partitioning of volume is completely rigorous; it gives rise to no vertex error, and handles the more numerous non-bonded contacts realistically. Its application to RNAase-S is described and the results compared with both Voronoi's method and Richards' method B. A particular advantage of both the radical plane and Richards' methods is a relative insensitivity to the treatment of the surface, a problem that has plagued other approaches to describing packing in proteins. Although the radical plane is seen to misallocate volume chemically between covalently-bonded neighbours, this problem vanishes when groups of atoms in side-chain residues are considered.     

Rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase: A comparison and immunological study of purified solubilized preparations,and alteration of enzyme levels by cholestyramine feeding

Some properties of various preparations of solubilized 3-hydroxy-3-methylglutaryl CoA reductase from rat liver are described. One, prepared by solubilization with deoxycholate, has been brought to a level of purity such that only a sińgle component is detected by polyacrylamide gel electrophoresis. A second preparation, solubilized by high salt concentration and heat treatment, has also been purified to a high level of purity so that only minor contaminants are detected. The deoxycholate-solubilized 3-hydroxy-3-methylglutaryl CoA reductase has a molecular weight of 197,000–202,000. Electrophoresis of both preparations treated with mercaptoethanol on polyacrylamide gels in the presence of sodium dodecyl sulfate revealed one band with a molecular weight of 65,000. The data are consistent with the trimeric structure consisting of three polypeptide chains of apparently identical molecular weight. An antiserum to the deoxycholate-solubilized preparation has been prepared. Despite major differences among these preparations in specific activity, in stability to cold, and in the requirement of high salt concentration for preservation, both samples react in the same manner to the antibody and are immunologically indistinguishable. A preparation solubilized by freeze-thawing also reacts with the antiserum. Possible reasons for the variations in specific activity are considered, and it is concluded that specific activity changes cannot be reliably related to protein concentration unless the protein is isolated.Application of the immunological assay to an analysis of the effect of feeding cholestyramine to rats shows that compared to normals the diurnal cycle is unchanged but the rate of enzyme protein synthesis in the cholestyramine-fed rats is greatly accelerated. However, the first-order rate constant for degradatation of enzyme protein remains essentially unchanged throughout the falling phases of the cycle. The specific activity relationships of the enzyme protein of cholestyramine-fed rats appear to be altered when compared to that of normally fed controls.     

A polarimetric study of the reaction of bis(L-serinato)copper(II) with formaldehyde

Polarimetric data have shown that the base-catalyzed reaction of bis(L-serinato)copper(II) with excess formaldehyde proceeds via the initial dissociation of the proton on the nitrogen atom of the amino acid chelate. A bis(oxazolidine)copper(II) complex appears as an intermediate but this species is not detected polarimetrically at 50°C and above.     

Temperature dependent conformational changes at the active site of pyruvate kinase. A li nuclear magnetic resonance study.

The interaction of Li⁺, a weak activator of pyruvate kinase, with substrate and inhibitor complexes of the enzyme has been investigated by magnetic resonance techniques. Proton relaxation rate (PRR) titrations indicate that the dissociation constant of Li⁺ from the ternary enzyme-Mn(II)-phosphoenolpyruvate (P-enolpyruvate) complex is 15 mm at 5 °C and 17 mm at 30 °C. The electron paramagnetic resonance spectrum of the enzyme-Mn(II)-Li(I)-P-enolpyruvate complex is the superposition of spectra for two distinct species (Reed, G. H., and Cohn, M. (1973) J. Biol. Chem.248, 6436–6442). Low temperatures favor the form giving rise to the more nearly isotropic spectrum, whereas high temperatures favor the species giving rise to the anisotropic “K⁺-like” spectrum. ⁷Li nuclear magnetic resonance data are consistent with a model in which the two forms observed by epr correspond to differing Mn(II) to Li(I) distances. The form giving rise to the anisotropic spectrum is characterized by a Mn(II) to Li(I) distance of 4.7 Å, and in the more isotropic form this distance is approximately 9 Å. The 4.7 Å separation of the Mn(II) and Li(I) in the anisotropic form of the complex compares favorably with the 4.9 Å separation of Mn(II) and T1(I) (Reuben, J., and Kayne, F. J. (1971) J. Biol. Chem.246, 6227–6234) in the P-enolpyruvate complex, although T1⁺ is a much better activator of the pyruvate kinase reaction. Thus, a change in the distance between the monovalent and divalent cations does not account quantitatively for the lower activation by Li⁺, inasmuch as more than 50% of the enzyme-Mn(II)-Li(I)-P-enolpyruvate complex has the “active” conformation with respect to the separation of the cations and the epr spectrum of the complex. As reported previously (Reed, G. H., and Morgan, S. D. (1974) Biochemistry13, 3537–3541), the dissociation constant of oxalate and the epr spectrum for the ternary complex of pyruvate kinase with Mn(II) and oxalate are not influenced by the species of monovalent cation present. The nuclear relaxation rates of Li⁺ are increased in the presence of the ternary oxalate complex, although the separation of the Mn(II) and Li(I) appears to be much greater than for the “anisotropic” form of the P-enolpyruvate complex.     

Lysine to 13C-labeled homoarginine conversion in microbial cytochromes c. Electron transport rates and 13C nuclear magnetic resonance spectroscopy

Seven microbial and one mammalian species of cytochrome c have been reacted with O-methylisourea to convert lysine residues to homoarginines containing enriched ¹³C. This set of guanidinated cytochromes has been assayed for electron transport reactivity and the nuclear magnetic resonance spectra of incorporated label have been obtained. The set consisted of c-type cytochromes from horse, Saccharomyces cerevisiae, Candida krusei, Paracoccus denitrificans, Pseudomonas aeruginosa, Rhodospirillum rubrum, Rhodopseudomonas capsulata, and Rhodopseudomanas spheroides. All derivatives demonstrated high electron transport reactivity with cytochrome oxidases; at some concentrations this rate was 100% or higher compared to corresponding native rates. All labeled ferricytochrome spectra followed a common pattern giving about five resolved or partially resolved resonance peaks. Two of these, at approximately 158.1 and 157.3 parts per million, correspond to single carbon sites. They have been assigned to labeled lysine 27 and lysine 79 (horse numbering), respectively, on the basis of sequence comparisons and an approximate chemical shift calculation. Labeled ferrocytochrome spectra were obtained and shown to be more diverse than the set of ferric spectra. Poly-[¹³C]homoarginine was prepared and shown to be an inhibitor of the horse cytochrome c-cytochrome oxidase reaction but an activator for the reactions of Paracoccus cytochrome c550. Relaxation measurements indicated that polyhomoarginine forms a complex with both cytochromes c.     

A study of the interaction of Escherichia coli elongation factor-Tu with aminoacyl-tRNAs by partial digestion with cobra venom ribonuclease

The hydrolysis of several aminoacylated transfer RNAs, by double-strand-specific ribonuclease from Naja oxiana was studied. The sensitivity to this enzyme of Phe-tRNA^Phe, Glu-tRNA^Glu and Met-tRNA_m^Met from Escherichia coli and Phe-tRNA^Phe from yeast was examined, both in the free state and complexed to E. coli elongation factor Tu. The hydrolysis patterns in the isolated state were similar for all aminoacylated tRNAs except Glu-tRNA₂^Glu, which exhibited striking differences probably arising from the existence of several subpopulations of tRNA₂^Glu. When engaged in a ternary complex with EF-Tu and GTP, the aminoacyl-tRNAs were efficiently protected in the amino acid acceptor and TΨC helices, showing that the interaction with EF-Tu primarily takes place at the -C-C-A end and at the amino acid acceptor and TΨC helices. In all cases an increased reactivity of the anticodon stem was observed in the complexed tRNA, possibly resulting from a conformational change in this region of the tRNAs.