Isolation and characterization of a molybdenum iron-sulfur protein from Desulfovibrio africanus.

A molybdenum-containing iron-sulfur protein has been isolated from the sulfate reducer $\text{Desulfovibrio}\text{africanus}$. The protein appears to be a complex protein of high molecular weight (112,000) composed of 10 subunits (mol. wt. 11,500) and containing a high amount of molybdenum (5–6 atoms/mole) with approx. 20 atoms each of iron and labile sulfide. The spectrum shows peaks at around 615, 410 and 325 nm with a protein peak at 280 nm. Its millimolar extinction coefficients at 615, 410 and 280 nm are 48.4, 64.4 and 141 respectively. The protein contains 106 amino-acid residues per subunit of mol. wt. 11,262 and the number of cysteine residues is 2 per subunit. The N-terminal sequence which has been determined up to 26 residues is characterized by its high degree of hydrophobicity.     

Flexibility of coupling and stoichiometry of ATP formation in intact chloroplasts

Since coupling between phosphorylation and electron transport cannot be measured directly in intact chloroplasts capable of high rates of photosynthesis, attempts were made to determine $\text{ATP}\text{2}\text{e}$ ratios from the quantum requirements of glycerate and phosphoglycerate reduction and from the extent of oxidation of added NADH via the malate shuttle during reduction of phosphoglycerate in light. These different approaches gave similar results. The quantum requirement of glycerate reduction, which needs 2 molecules of ATP per molecule of NADPH oxidized was found to be pH-dependent. 9–11 quanta were required at pH 7.6, and only about 6 at pH 7.0. The quantum requirement of phosphoglycerate reduction, which consumes ATP and NADPH in a $\text{1}\text{1}$ ratio, was about 4 both at pH 7.6 and at 7.0. $\text{ATP}\text{2}\text{e}$ ratios calculated from the quantum requirements and the extent of phosphoglycerate accumulation during glycerate reduction were usually between 1.2 and 1.4, occasionally higher, but they never approached 2.Although the chloroplast envelope is impermeable to pyridine nucleotides, illuminated chloroplasts reduced added NAD via the malate shuttle in the absence of electron acceptors and also during the reduction of glycerate or CO₂. When phosphoglycerate was added as the substrate, reduction of pyridine-nucleotides was replaced by oxidation and hydrogen was shuttled into the chloroplasts to be used for phosphoglycerate reduction even under light which was rate-limiting for reduction. This indicated formation of more ATP than NADPH by the electron transport chain. From the rates of oxidation of external NADH and of phosphoglycerate reduction at very low light intensities $\text{ATP}\text{2}\text{e}$ ratios were calculated to be between 1.1 and 1.4.Fully coupled chloroplasts reduced oxaloacetate in the light at rates reaching 80 and in some instances 130 μmoles · mg^?1 chlorophyll · h^?1 even though ATP is not consumed in this reaction. The energy transfer inhibitor phlorizin did not significantly suppress this reduction at concentrations which completely inhibited photosynthesis. Uncouplers stimulated oxaloacetate reduction by factors ranging from 1.5 to more than 10. Chloroplasts showing little uncoupler-induced stimulation of oxaloacetate reduction were highly active in photoreducing CO₂. Measurements of light intensity dependence of quantum requirements for oxaloacetate reduction gave no indication for the existence of uncoupled or basal electron flow in intact chloroplasts. Rather reduction is brought about by loosely coupled electron transport. It is concluded that coupling of phosphorylation to electron transport in intact chloroplasts is flexible, not tight. Calculated $\text{ATP}\text{2}\text{e}$ ratios were obtained under conditions, where coupling should be expected to be optimal, i.e. at low phosphorylation potentials $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{]}\text{[}\text{P}{}_{\mathrm{<mtext>i</mtext>}}\text{]}$. Flexible coupling implies, that $\text{ATP}\text{2}\text{e}$ ratios should decrease with increasing phosphorylation potentials inside the chloroplasts.     

Tetrahydropterin: Reduction of cytochrome c and coupled phosphorylation at mitochondrial site 3

Incubation of rat liver mitochondria with tetrahydropterin results in ATP production with a P:O ratio of 0.85, consistent with the entry of reducing equivalents into the mitochondrial electron transport chain at cytochrome $\text{c}$. No evidence for an enzymatic reduction of cytochrome $\text{c}$ was found. The reduction of either soluble or mitochondrial cytochrome $\text{c}$ was not diminished by superoxide dismutase or anaerobic conditions, indicating that the reaction is not dependent on the autoxidation of the reduced pterin and the formation of an active species of oxygen. The experiments indicate a potential pathway for the production of ATP coupled to the oxidation of NADPH through the activity of NADPH-dependent pteridine reductases.     

Complex formation by 3 H-aldosterone in rat kidney and liver

Low molecular weight polar complexes were shown to be formed $\text{in vivo}$ from ³H-aldosterone in both kidney and liver subcellular fractions, the majority being present in the cytosol fractions. Significant differences were observed between the quantities of polar complexes present in kidney subcellular fractions from intact and adrenalectomized male rats and also between the quantities of these kidney polar complexes from spironolactone treated male rats. ³H-aldosterone macro-molecule complexes were shown to exist in appreciable quantities only in the kidney cytosol fractions of adrenalectomized male rats. These gel filtration studies also showed the ³H-aldosterone labeled macromolecule complexes to consist of two protein peaks; one of high molecular weight and the other of lower molecular weight (～50,000 mol. wt.). The amount of ³H-aldosterone labeled protein complexes in kidney cytosol was greatly reduced when adrenalectomized rats were pretreated $\text{in vivo}$ with spironolactone.     

?Glutathione: Its reaction with NADP and its oxidationredutionpotenial.

The equilibrium constant for the reaction of GSH with NADP at pH 7 and 298 °K is 0.005. The standard electrode potential of the $\text{GSSG}\text{GSH}$ couple at pH 7 is ?0.25 V, based on the value of ?0.32 V for the potential of $\text{NADP}\text{NADPH}$. The discrepancy from other determinations is discussed.     

Highly purified detergent-solubilized NADPH-cytochrome P-450 reductase from phenobarbital-induced rat liver microsomes

NADPH-cytochrome P-450 reductase was highly purified from liver microsomes of phenobarbital-induced rats by column chromatography on DEAE-cellulose, DEAE-Sephadex A-50, and hydroxylapatite in the presence of deoxycholate or Renex 690, a nonionic detergent. The purified enzyme gave a single major band with a molecular weight of 79,000 daltons on SDS-polyacrylamide gel electrophoresis. FMN and FAD were present in about equal amounts. The most active reductase preparation catalyzed the reduction of 40.9 μmoles of cytochrome $\text{c}$ per min per mg of protein and, as an indirect measure of cytochrome P-450 reduction, the oxidation of 2.0 μmoles of NADPH per min per mg of protein in a reconstituted hydroxylation system containing benzphetamine as the substrate.     

The role of the membrane-bound iron-sulphur centres A and B in the Photosystem I reaction centre of spinach chloroplasts

Photosystem I particles prepared from spinach chloroplast using Triton X-100 were frozen in the dark with the bound iron-sulphur Centre A reduced. Illumination at cryogenic temperatures of such samples demonstrated the photoreduction of the second bound iron-sulphur Centre B. Due to electron spin-electron spin interaction between these two bound iron-sulphur centres, it was not possible to quantify amounts of Centre B relative to the other components of the Photosystem I reaction centre by simulating the line-shape of its EPR spectrum. However, by deleting the free radical signal I from the EPR spectra of reduced Centre A alone or both Centres A plus B reduced, it was possible to double integrate these spectra to demonstrate that Centre B is present in the Photosystem I reaction centre in amounts comparable to those of Centre A and thus also signal I ($\text{P-700}$) and X.Oxidation-reduction potential titrations confirmed that Centre A had $\text{E}{}_{\mathrm{<mtext>m</mtext>}}\text{? ?550}\text{mV}$, Centre B had $\text{E}{}_{\mathrm{<mtext>m</mtext>}}\text{? ?585}\text{mV}$. These results, and those presented for the photoreduction of Centre B, place Centre B before Centre A in the sequence of electron transport in Photosystem I particles at cryogenic temperatures. When both A and B are reduced, $\text{P-700}$ photooxidation is reversible at low temperature and coupled to the reduction of the component X. The change from irreversible to reversible $\text{P-700}$ photooxidation and the photoreduction of X showed the same potential dependence as the reduction of Centre B with $\text{E}{}_{\mathrm{<mtext>m</mtext>}}\text{? ?585}$ mV, substantiating the identification of X as the primary electron acceptor of Photosystem I.     

Heat stabilization dependence on redox state of cytochrome cd1 oxidase from Pseudomonas aeruginosa

The irreversible thermal denaturation of cytochrome cd₁ oxidase from $\text{P.}\text{aeruginosa}$ as a function of the oxidation-reduction states of its hemes was observed with a differential scanning calorimeter. Upon full reduction of the four hemes, the apparent denaturation temperature decreases by about 10° and the denaturation enthalpy decreases slightly: oxidized, 5.9 cal/gm; reduced, 5.4 cal/gm. At pH 7.5, the first order rate constants for denaturation at 90°C are: reduced, 33 × 10^?3s^?1; oxidized, 3 × 10^?3s^?1. Thus, oxidation of the hemes reuults in heat stabilization of the cytochrome oxidase. The activation energy for denaturation of fully reduced oxidase, 53 kcal/mol, is less than that for fully oxidized protein (73 kcal/mol).     

DNA-binding proteins of Xenopus laevis: synthesis during oogenesis-ovulation and early embryogenesis

DNA-binding proteins of Xenopus laevis synthesized during two periods of early development (oogenesis-ovulation and early embryogenesis) were co-chromatographed on DNA-cellulose. Proteins with an affinity for DNA were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Most of the proteins eluted from DNA-cellulose with 0.6 M NaCl had mol. wts less than 40 000; some of these proteins were synthesized to a greater extent by developing embryos than by oocytes. A DNA-binding protein or group of proteins with a mol. wt of approx. 70 000 was synthesized during oogenesis-ovulation but not during embryogenesis. Differential labeling of developing embryos with $\text{[}{}^3\text{H}\text{]}\text{tryptophan}$ and $\text{[}{}^{14}\text{C}\text{]}\text{lysine}$ indicated that some of the low mol. wt DNA-binding proteins are histones. Some of these proteins also incorporated monosodium $\text{[}{}^{32}\text{P}\text{]}\text{phosphate}$. A greater fraction of the proteins synthesized by oocytes and developing embryos were bound to DNA-histone-cellulose than to DNA-cellulose. A group of low mol. wt proteins made during oogenesis-ovulation were bound more to DNA-histone-cellulose than were proteins with similar mol. wts made by developing embryos.     

Active calcium transport in red cell ghosts resealed in dextran solutions

1.Human erythrocytes when lysed and resealed to Ca in the presence of dextran can be readily separated from the suspending medium by low-speed centrifugation. 2. Ghosts trapped Ca and EGTA at the same ratio as present in the haemolytic medium and remained tight to Ca after washing and subsequent incubation for up to 90 min at 37°C. 3. Ca extrusion could be promoted by substrates other than ATP only from ghosts that had been loaded with low free Ca concentrations (1–22 μM). The order of activation by the various substrates employed was $\text{ATP}\text{>}\text{adenine}\text{+}\text{inosine}\text{>}\text{inosine}$. 4. The kinetics of extrusion depended markedly on internal free Ca. The system showed a high affinity state ($\text{K}{}_{\mathrm{<mtext>Ca</mtext>}}\text{about}\text{3 μ}\text{M}$; $\text{V = 0.34 μ}\text{mol Ca}\text{/}\text{ml ghosts per min}$) at low concentrations (1–22 μM) and a low affinity state ($\text{K}{}_{\mathrm{<mtext>Ca</mtext>}}\text{about}\text{250 μ}\text{M}$; $\text{V = 0.17 μ}\text{mol Ca}\text{/}\text{ml ghosts per min}$) at high concentrations (0.2–4.0 mM). 5. Both at low and at high free Ca, La-sensitive ATP hydrolysis was closely correlated with La-dependent Ca efflux, in keeping with an stoichiometry of 1. 6. The rate of extrusion was maximal in the presence of 160 mM KCl and decreased to various extents when K was fully replaced by different cations, following the order $\text{K}\text{>}\text{Na = choline}\text{>}\text{Mg}$. 7. The efflux rate of high-K ghosts, resealed to alkaline cations, was stimulated by external Na, whilst Mg and choline were practically without effect. 8. The results indicate that human red cells possess a powerful Ca extrusion mechanism, the activity of which can be modulated by alkaline cations.     

Evidence for essential disulfide bonds in the β-subunit of (Na+ + K+)-ATPase

$\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ from dog kidney lost its activity when heated at 55°C in the presence of 0.3 M 2-mercaptoethanol. Either heat treatment alone or addition of reducing agent at around 25°C caused little inactivation. One disulfide bond per protomer (mol. wt. 146000) was reduced in the inactivated sample but in active samples no reduction occurred. Neither K⁺-dependent phosphatase activity nor phosphoenzyme formation in the presence of Na⁺ was detected in the inactivated sample, suggesting that the disulfide bond was essential for the catalytic cycle of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$. This essential disulfide bond belonged to the β-subunit, the glycoprotein component of the enzyme, indicating that the β-subunit may be an integral component of the $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ system.     

Alteration of the conductance of Na+ channels in the nodal membrane of frog nerve by holding potential and tetrodotoxin

(1) Na⁺ currents and Na⁺-current fluctuations were measured in myelinated frog nerve fibres at 15°C during 7.7 ms depolarizations to $\text{V = 40, 60}\text{and}\text{80}\text{mV}$. (2) The conductance γ of a single Na⁺ channel and the number $\text{N}{}_0$ of channels per node were calculated from ensemble average values of the mean Na⁺ current and the variance of Na⁺-current fluctuations. (3) For a hyperpolarizing holding potential of $\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{= ?28}\text{mV}$ the mean values of the channel conductance and number were $\text{γ = 9.8}\text{pS}$ and $\text{N}{}_0\text{= 74 000}$. (4) After changing the holding potential to the resting potential ($\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{= 0}$) the conductance γ increased by a factor of 1.37 whereas the number $\text{N}{}_0$ decreased by a factor of 0.60. (5) Addition of 8 nM tetrodotoxin at a holding potential of $\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{= ?28}\text{mV}$ increased γ by a factor of 1.55 and reduced $\text{N}{}_0$ by a factor of 0.25. (6) The increase of the channel conductance at reduced channel numbers suggests negative cooperativity between Na⁺ channels in the nodal membrane.     

Bound cytochrome C as proton donor and acceptor during enzymic oxidoreduction

When ferrocytochrome $\text{c}$ reacts with delipidated cytochrome oxidase under conditions which prevent oxidation, one proton is taken up per molecule of ferrocytochrome $\text{c}$ bound to cytochrome oxidase. When ferricytochrome $\text{c}$ reacts with delipidated Complex III, one proton is released per molecule of ferricytochrome $\text{c}$ bound to Complex III. From these data it can be concluded that the oxidation of ferrocytochrome $\text{c}$ by cytochrome oxidase leads to the release of a proton and an electron, whereas the reduction of ferricytochrome $\text{c}$ by Complex III leads to the uptake of a proton and an electron. Thus ferrocytochrome $\text{c}$ like QH₂ and NADH is both an electron and proton donor, and ferricytochrome $\text{c}$ like Q and O₂ is both an electron and proton acceptor. The pattern for the three mitochondrial electron transfer sequences NADH → Q, QH₂ → ferricytochrome $\text{c}$ and ferrocytochrome $\text{c}$ → O₂ involves separation of an electron and proton on the side of the membrane where electron transfer is initiated and recombination of an electron and a proton in the terminal acceptor on the side of the membrane where electron transfer terminates.     

Oxygen exchange associated with electron transport and photophosphorylation in spinach thylakoids

O₂ uptake in spinach thylakoids was composed of ferredoxin-dependent and -independent components. The ferredoxin-independent component was largely 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) insensitive (60%). Light-dependent O₂ uptake was stimulated 7-fold by 70 μM ferredoxin and both uptake and evolution (with O₂ as the only electron acceptor) responded almost linearly to ferredoxin up to 40 μM. NADP⁺ reduction, however, was saturated by less than 20 μM ferredoxin. The affinity of O₂ uptake for for O₂ was highly dependent on ferredoxin concentration, with $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{(O}{}_2\text{)}$ of less than 20 μM at 2 μM ferredoxin but greater than 60 μM O₂ with 25 μM ferredoxin. O₂ uptake could be suppressed up to 80% with saturating NADP⁺ and it approximated a competitive inhibitor of O₂ uptake with a K_i of 8–15 μM. Electron transport in these thylakoids supported high rates of photophosphorylation with NADP⁺ (600 μmol ATP/mg Chl per h) or O₂ (280 μmol/mg Chl per h) as electron acceptors, with $\text{ATP}\text{2}\text{e}$ ratios of 1.15–1.55. Variation in $\text{ATP}\text{2}\text{e}$ ratios with ferredoxin concentration and effects of antimycin A indicate that cyclic electron flow may also be occurring in this thylakoid system. Results are discussed with regard to photoreduction of O₂ as a potential source of ATP in vivo.     

Ammonium (methylammonium) transport by Klebsiella pneumoniae

Klebsiella pneumoniae can accumulate methylammonium up to 80-fold by means of a transport system as indicated by the energy requirement, saturation kinetics and a narrow pH profile around pH 6.8. Methylammonium transport (apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 100 μ}\text{M}$, $\text{V = 40 μ}\text{mol/min}$ per g dry weight at 15°C) is competitively inhibited by ammonium (apparent $\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 7 μ}\text{M}$). The low $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ value and the finding that methylammonium cannot serve as a nitrogen source indicate that ammonium rather than methylammonium is the natural substrate. Uphill transport is driven by a component of the protonmotive force, probably the membrane potential. The transport system is under genetic control; it is partially repressed by amino acids and completely by ammonium. Analysis of mutants suggest that the synthesis of the ammonium transport system is subject to the same ‘nitrogen control’ as nitrogenase and glutamine synthetase.     

A peptide-like inhibitor of N-methyltransferase in rabbit brain

After pretreatment with pheniprazine, rabbits were administered C-14-tryptamine i.v. and the lung was assayed for the N-methylated derivatives. Unoxidized tryptamine was present, but no N-methyl or N, N-dimethyltryptamine was found in this tissue, which contains high levels of N-methyltransferase. It appears that the indolamine-N-methyltransfer reaction is inhibited in the intact tissue. Our investigation of the possible inhibitory mechanism has led to the purification and characterization of a dialysable factor which inhibits the enzyme $\text{in}$$\text{vitro}$. The factor, which is present in most tissues, was purified from newborn rabbit brain. It is present in two forms, one having approximate mol. wt. 1,500 and one mol. wt. 1,300. Both were inactivated by crystalline trypsin. The 1,300 form was digested by carboxypeptidase A to a smaller, but still active form. It is suggested that these peptides may control $\text{in}$$\text{vivo}$ the activity of the non-specific N-methyltransferase against tryptamine and serotonin.     

The protein kinases of rat liver nuclei

Two compounds with properties of Factor F-430 were purified from $\text{Methanobacterium}\text{bryantii}$ by column chromatography. Analysis of these compounds by neutron activation and atomic absorption spectroscopy revealed the presence of nickel and the absence of other metals commonly associated with molecules of biological origin. For the two compounds, the masses are 3300 daltons per mol Ni and 1500 daltons per mol Ni. The absorbance at 430 nm of both compounds is between 2.7?2.1 × 10⁴ cm^?1 L (mol Ni)^?1. Factor F-430 appears to be a unique, nickel-containing compound of low molecular weight.     

Quantitative analysis of the binding of melittin to planar lipid bilayers allowing for the discrete-charge effect

The interaction of melittin with lecithin bilayers was studied using the resulting surface potentials at the bilayer/water interfaces to monitor the association. Melittin added to the aqueous phase binds strongly to the interface but remains localized on that side of the bilayer to which it is added. The analysis of the binding curves reveals the inadequacy of the Gouy-Chapman theory for the fixed-charge surface potential in describing the electrostatic potential experienced by the adsorbed molecules. Calculations based on the Stern equation, modified for a discrete charge distribution, give a good fit to the experimental data. The thermodynamic analysis revealed different binding energies, Δ$\text{G}$°, at 10 and 100 mM ionic strength (?7.85 and ?8.26 kcal/mol, respectively). Binding saturates at an area of 650 Ų per melittin molecule. A change in the surface dipole potential corresponding to ?1.1 debye/?_a ($\text{?}{}_{\mathrm{a}}\text{= dielectric constant of the adsorption region}$) had to be postulated. The Debye-Hückel length for a charge bound to the membrane/solution interface was found to be about one-third smaller than in bulk solution.     

Properties of carnitine transport in rat kidney cortex slices

The properties of carnitine transport were studied in rat kidney cortex slices. Tissue: medium concentration gradients of 7.9 for L-[methyl-¹⁴C]carnitine were attained after 60-min incubation at 37°C in 40 μM substrate. L- and D-carnitine uptake showed saturability. The concentration curves appeared to consist of (1) a high-affinity component, and (2) a lower affinity site. When corrected for the latter components, the estimated K_m for L-carnitine was 90 μM and $\text{V = 22}\text{nmol/min}$ per ml intracellular fluid; for D-carnitine, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 166}\text{μM}$ and $\text{V = 15}\text{nmol/min}$ per ml intracellular fluid. The system was stereospecific for L-carnitine. The uptake of L-carnitine was inhibited by (1) D-carnitine, γ-butyrobetaine, and (2) acetyl-L-carnitine. γ-Butyrobetaine and acetyl-L-carnitine were competitive inhibitors of L-carnitine uptake. Carnitine transport was not significantly reduced by choline, betaine, lysine or γ-aminobutyric acid. Carnitine uptake was inhibited by 2,4-dinitrophenol, carbonyl cyanide $\text{m-}\text{chlorophenylhydrazone}$, N₂ atmosphere, KCN, $\text{N-}\text{ethylmaleimide}$, low temperature (4°C) and ouabain. Complete replacement of Na⁺ in the medium by Li⁺ reduced L- and D-carnitine uptake by 75 and 60%, respectively. Complete replacement of K⁺ or Ca²⁺ in the medium also significantly reduces carnitine uptake. Two roles for the carnitine transport system in kidney are proposed: (1) a renal tubule reabsorption system for the steady-state maintenance of plasma carnitine; and (2) maintenance of normal carnitine levels in kidney cells, which is required for fatty acid oxidation.     

Evidence for a protonmotive Q cycle mechanism of electron transfer through the cytochrome b-c1 complex.

A protein named oxidation factor can be reversibly removed from succinate-cytochrome $\text{c}$ reductase complex and shown to be required for electron transfer between succinate and cytochrome $\text{c}$. This protein is required for reduction of cytochrome $\text{c}{}_1$ and, in the presence of antimycin, for reduction of both cytochromes $\text{b}$ and $\text{c}{}_1$. These results are consistent with a protonmotive Q cycle mechanism in which the oxidation factor catalyzes electron transfer from reduced quinone to cytochrome $\text{c}{}_1$ and thus liberates from reduced quinone one of two protons required for energy conservation during electron transfer through the cytochrome $\text{b}\text{-}\text{c}{}_1$ complex.