Arrangement of the electric potential-generating redox chain in the mitochondrial membrane

The intramembrane arrangement of the respiratory chain generating electric potential difference across the mitochondrial membrane has been studied. The accessibility of various respiratory carriers to the non-penetrating electron donors and acceptors, such as ferri-and ferrocyanide, cytochrome c. fumarate and nicotinamide nuclcotides has been used as a test for surface localization of the carrier in the membrane of mitochondria and inside-out (sonicated) submitochondrial particles. Membrane potential formation was detected by measuring the transmembrane flows of the penetrating anion, phenyl dicarbaundecaborne (PCB^–).It is shown that ferricyanide reduction can support PCB^– movement if this electron acceptor interacts with intact mitochondria in the region localized on the oxygen site of the antimycin-sensitive point. The same region is accessible for ferrocyanide whose oxidation by O₂ can be also coupled with PCB^– translocation. Added nicotinamide nuclcotides cannot be utilized by mitochondria for supporting PCB^– movement.PCB^– movement in the inside-out submitochondrial particles can be supported by reduction of ferricyanide or fumarate by NADH, and of NAD⁺ by NADPH, the former process being sensitive to rotenone but not to antimycin. Antimycin-insensitive reduction of feericyanide or of CoQ₆ by succinate is not coupled with PCB^– transport. Neither ferrocyanide nor ferrocytochromec can be used as electron donors in the particles.Penetrating electron donors (TMPDH₂, succinate) and acceptors (menadione) are effective both in mitochondria and particles.It is coucluded that flavin and transhydrogenase regions of the potential-generating redox chain are localized near the inner surface, cytochromec region-near the outers surface of the internal membrane of intact mitochondria. It means that the redox chain includes at least one act of the transmembrane transfer of reducing equivalents between flavins and cytochromec.     

Functional role of soluble mitochondrial ATPase subunits

A preparation of soluble mitochondrial ATPase (coupling factor F₁) containing no and minor subunits has been isolated. The minor-subunits-deficient F₁ was found to be competent in ATP hydrolysis. However, it did not demonstrate a coupling effect in EDTA-submitochondrial particles. A portion of the ATPase activity of EDTA particles, stimulated by the minor-subunits-deficient F₁, was insensitive to oligomycin. ATPase activity of Na⁺-particles was changed only slightly by this F₁. It is suggested that and subunits are necessary to form specific contacts between the F₁ molecule and components of the mitochrondrial membrane.Abbreviations SMP submitochondrial particles - F₁ coupling factor (soluble mitochondrial ATPase) - PCB^– phenyl dicarbaundecaborane anions     

Energetic basis of cadmium toxicity in Staphylococcus aureus

In washed cells of cadmium-sensitive Staphylococcus aureus 17810S oxidizing glutamate, initial Cd²⁺⁺⁺ influx via the Mn²⁺ porter down membrane potential () was fast due to involvement of energy generated by two proton pumps—the respiratory chain and the ATP synthetase complex working in the hydrolytic direction. Such an unusual energy drain for rapid initial Cd²⁺ influx is suggested to be due to a series of toxic events elicited by Cd²⁺ accumulation down generated via the redox proton pump: (i) strong inhibition of glutamate oxidation accompanied by a decrease of electrochemical proton gradient ( _H ⁺) formation via the respiratory chain, (ii) automatic reversal of ATP synthetase from biosynthetic to hydrolytic mode, which was monitored by a decrease of _H ⁺-dependent ATP synthesis, (iii) acceleration of the initial Cd²⁺ influx down generated the reversed ATP synthetase, the alternative proton pump hydrolyzing endogenous ATP. The primary, cadmium-sensitive targets in strain 17810S seem to be dithiols located in the cytoplasmic glutamate oxidizing system, prior to the membrane-embedded NADH oxidation system. Inhibition by Cd²⁺ of _H ⁺-dependent ATP synthesis and of pH gradient (pH)-linked [¹⁴C]glutamate transport is a secondary effect due to cadmium-mediated inhibition of _H ⁺ generation at the cytoplasmic level. In washed cells of cadmium-resistant S. aureus 17810R oxidizing glutamate, Cd²⁺ accumulation was prevented due to activity of the plasmid-coded Cd²⁺ efflux system. Consequently, _H ⁺-producing and -requiring processes were not affected by Cd²⁺.     

Anilinonaphthalene sulfonate fluorescence and amino acid transport in yeast

Summary Fluorescence of 1-anilinonaphthalene-8-sulfonate in yeast membranes appears to be caused predominantly by binding to lipids (ANS_proteinANS_lipid120) as indicated by the fluorescence lifetime, degree of polarization, and excitation spectra. It was insensitive to short-circuiting the membrane potential. Fluorescence intensity increased as cells (especially after pretreatment with energy donors such as glucose) were exposed to some amino acids, in particular, aspartic and glutamic acids. The character of fluorescence shifted to that of protein-bound ANS, suggesting an exposure of new protein sites accessible to the probe. This shift could be prevented by inhibitors of energy transduction as well as of transport. TheK _1/2 of the shift was at 2.5mm aspartic acid.     

Effect of chemical modifiers of amino acid residues on proton conduction by the H+-ATPase of mitochondria

The effect of chemical modifiers of amino acid residues on the proton conductivity of H⁺-ATPase in inside out submitochondrial particles has been studied. Treatment of submitochondrial particles prepared in the presence of EDTA (ESMP) with the arginine modifiers, phenylglyoxal or butanedione, or the tyrosine modifier, tetranitromethane, caused inhibition of the ATPase activity. Phenylglyoxal and tetranitromethane also caused inhibition of the anaerobic release of respiratory H⁺ in ESMP as well as in particles deprived of F₁ (USMP). Butanedione treatment caused, on the contrary, acceleration of anaerobic proton release in both particles. The inhibition of proton release caused by phenylglyoxal and tetranitromethane exhibited in USMP a sigmoidal titration curve. The same inhibitory pattern was observed with oligomycin and withN,N-dicyclohexylcarbodiimide. In ESMP, relaxation of H⁺ exhibited two first-order phases, both an expression of the H⁺ conductivity of the ATPase complex. The rapid phase results from transient enhancement of H⁺ conduction caused by respiratory H⁺ itself. Oligomycin,N,N-dicyclohexylcarbodiimide, and tetranitromethane inhibited both phases of H⁺ release, and butanedione accelerated both. Phenylglyoxal inhibited principally the slow phase of H⁺ conduction. In USMP, H⁺ release followed simple first-order kinetics. Oligomycin depressed H⁺ release, enhanced respiratory H⁺, and restored the biphasicity of H⁺ release. Phenylglyoxal and tetranitromethane inhibited H⁺ release in USMP without modifying its first-order kinetics. Butanedione treatment caused biphasicity of H⁺ release from USMP, introducing a very rapid phase of H⁺ release. Addition of soluble F₁ to USMP also restored biphasicity of H⁺ release. A mechanism of proton conduction by F _o is discussed based on involvement of tyrosine or other hydroxyl residues, in series with the DCCD-reactive acid residue. There are apparently two functionally different species of arginine or other basic residues: those modified by phenylglyoxal, which facilitate H⁺ conduction, and those modified by butanedione, which retard H⁺ diffusion.     

Passive ionic properties of frog retinal pigment epithelium

Summary The isolated pigment epithelium and choroid of frog was mounted in a chamber so that the apical surfaces of the epithelial cells and the choroid were exposed to separate solutions. The apical membrane of these cells was penetrated with microelectrodes and the mean apical membrane potential was –88 mV. The basal membrane potential was depolarized by the amount of the transepithelial potential (8–20mV). Changes in apical and basal cell membrane voltage were produced by changing ion concentrations on one or both sides of the tissue. Although these voltage changes were altered by shunting and changes in membrane resistance, it was possible to estimate apical and basal cell membrane and shunt resistance, and the relative ionic conductanceT _i of each membrane. For the apical membrane:T _K0.52,T _{HCO 3}=0.39 andT _Na=0.05, and its specific resistance was estimated to be 6000–7000 cm². From the basalT _K=0.90 and its specific resistance was estimated to be 400–1200 cm². From the basal potassium voltage responses the intracellular potassium concentration was estimated at 110mm. The shunt resistance consisted of two pathways: a paracellular one, due to the junctional complexes and another, around the edge of the tissue, due to the imperfect nature of the mechanical seal. In well-sealed tissues, the specific resistance of the shunt was about ten times the apical plus basal membrane specific resistances. This epithelium, therefore, should be considered tight. The shunt pathway did not distinguish between anions (HCO₃ ^–, Cl^–, methylsulfate, isethionate) but did distinguish between Na⁺ and K⁺.     

Orientation of the porphyrin ring in artificial chlorophyll membranes

Summary A sensitive photometric method is described by which the dichroism of lipid bilayer membranes in aqueous phase can be measured. The method is applied to black films with incorporated chlorophylla andb. With chlorophylla a relatively large dichroism is found in the Soret band and a much weaker dichroism in the red band. From the experimental data, the angles _B and _R between the blue and red transition moments and the membrane can be obtained. _B and _R are then used to calculate the angle of the porphyrin ring with respect to the membrane surface. For chlorophylla and three different lipids, values of between 44 and 49° are found.     

The Use of the [13C]/[12C] Ratio for the Assay of the Microbial Oxidation of Hydrocarbons

The study deals with a comparative analysis of the relative abundances of the carbon isotopes ¹²C and ¹³C in the metabolites and biomass of the Burkholderia sp. BS3702 and Pseudomonas putida BS202-p strains capable of utilizing aliphatic (n-hexadecane) and aromatic (naphthalene) hydrocarbons as sources of carbon and energy. The isotope compositions of the carbon dioxide, biomass, and exometabolites produced during the growth of Burkholderia sp. BS3702 on n-hexadecane (¹³C = –44.6 ± 0.2) were characterized by the values of ¹³C_{CO 2} = –50.2 ± 0.4, ¹³C_biom = –46.6 ± 0.4, and ¹³C_exo = –41.5 ± 0.4, respectively. The isotope compositions of the carbon dioxide, biomass, and exometabolites produced during the growth of the same bacterial strain on naphthalene (¹³C = –21 ± 0.4) were characterized by the isotope effects ¹³C_{CO 2} = –24.1 ± 0.4, ¹³C_biom = –19.2 ± 0.4, and ¹³C_exo = –19.1 ± 0.4, respectively. The possibility of using the isotope composition of metabolic carbon dioxide for the rapid monitoring of the microbial degradation of petroleum hydrocarbons in the environment is discussed.     

Characterization of the MHC class II region in cattle. The number of DQ genes varies between haplotypes

The organization of the major histocompatibility complex (MHC) class II region in cattle was investigated by Southern blot analysis using human probes corresponding to DO, DP, DQ, and DR genes. Exon-specific probes were also employed to facilitate the assessment of the number of different bovine class II genes. The results indicated the presence of single DO and DR genes, at least three DR genes, while the number of DQ genes was found to vary between MHC haplotypes. Four DQ haplotypes, DQ ^{1 1} to DQ ^{2 4}, possessed a single DQ and a single DQ gene whereas both these genes were duplicated in eight other haplotypes, DQ ^{3 5} to DQ ^{9 12}. No firm evidence for the presence of bovine DP genes was obtained. The same human probes were also used to investigate the genetic polymorphism of bovine class II genes. DQ DQ , DR DR , and DO restriction fragment length polymorphisms (RFLPs) were resolved and in particular the DQ restriction fragment patterns were highly polymorphic. Comparison of the present result with the current knowledge of the class II region in other mammalian species suggested that the DO, DP, DQ, DR, and DZ subdivision of the class II region was established already in the ancestor of mammals. The DP genes appear to be the least conserved class II genes among mammalian species and may have been lost in cattle. The degree of polymorphism of different class II genes, as revealed by RFLP analyses, shows striking similarities between species.     

Modular organization of the AIDA autotransporter translocator: The N-terminal β1-domain is surface-exposed and stabilizes the transmembrane β2-domain

The adhesin involved in diffuse adherence (AIDA-I) of the diarrhoeagenic Escherichia coli strain 2787 (O126:H27) is synthesized as a precursor molecule. This pre-pro-protein is N- and C-terminally processed to generate three distinct domains, which are characteristic for autotransporter secretion systems in Gram-negative bacteria: the N-terminal pre-peptide, the -domain and the C-terminal -domain. The outer membrane-integrated -domain (AIDA^C) is responsible for the surface-presentation of the -domain (AIDA-I) and is thus termed `translocator'. Characterization of extracted N-terminally truncated forms and of in vitro refolded proteins revealed a core structure at the C-terminus of the translocator which was found to be very stable even in the presence of SDS. Denaturation occurs only after additional incubation at temperatures above 80 °C. Reporter-epitope insertions were used to analyze the location of regions of membrane-integrated AIDA^C relative to the membrane. The modified topological model developed for the AIDA translocator suggests the N-terminal domain (₁) encompasses approximately 10 kDa to represent a completely surface-exposed segment while the C-terminal compact core domain (₂) remains integrated in the membrane as a -barrel-like structure. Though the ₂-core structure alone harbours all the information for the outer membrane integration of AIDA^C it is additionally stabilized by the ₁-domain. Access to large amounts of complete as well as truncated AIDA^C proteins facilitated the study of protein folding by CD and fluorescence spectroscopy. A potential pore forming activity of the translocator using the completely refolded AIDA^C or the ₂-core in black-lipid membranes could not be demonstrated.     

Cell-wall tension of the inner tissues of the maize coleoptile and its potential contribution to auxin-mediated organ growth

Plant organs such as maize (Zea mays L.) coleoptiles are characterized by longitudinal tissue tension, i.e. bulk turgor pressure produces unequal amounts of cell-wall tension in the epidermis (essentially the outer epidermal wall) and in the inner tissues. The fractional amount of turgor borne by the epidermal wall of turgid maize coleoptile segments was indirectly estimated by determining the water potential ^* of an external medium which is needed to replace quantitatively the compressive force of the epidermal wall on the inner tissues. The fractional amount of turgor borne by the walls of the inner tissues was estimated from the difference between -^* and the osmotic pressure of the cell sap (_i) which was assumed to represent the turgor of the fully turgid tissue. In segments incubated in water for 1 h, -^* was 6.1–6.5 bar at a _i of 6.7 bar. Both -^* and _i decreased during auxin-induced growth because of water uptake, but did not deviate significantly from each other. It is concluded that the turgor fraction utilized for the elastic extension of the inner tissue walls is less than 1 bar, i.e. less than 15% of bulk turgor, and that more than 85% of bulk turgor is utilized for counteracting the high compressive force of the outer epidermal wall which, in this way, is enabled to mechanically control elongation growth of the organ. This situation is maintained during auxin-induced growth.Abbreviations and Symbols _i osmotic pressure of the tissue - ₀ external water potential - ^* water potential at which segment length does not change - IAA indole-3-acetic acid - ITW longitudinal inner tissue walls - OEW outer epidermal wall - P turgor Supported by Deutsche Forschungsgemeinschaft (SFB 206).     

Light and GTP dependence of transducin solubility in retinal rods

The physical origin and functional significance of the near infra-red light scattering changes observable upon flash illumination of diluted suspensions of magnetically oriented, permeabilised frog retinal rods has been reinvestigated with particular attention paid to the degree with which transducin remains attached to the membrane. In the absence of GTP, the so called binding signal is shown to include two components of distinctive origins, widely different kinetics, and whose relative amplitudes depend on the dilution of the suspension and resulting detachment of transducin from the disc membrane. The fast component is a consequence of the fast interaction between photoexcited rhodopsin (R^*) and the transducin remaining on the membrane. Its kinetics monitors a structural modification of the discs caused by a change in electrostatic interaction between closely packed membranes upon the formation of R^*-T complexes. The slow component monitors the slow rebinding to the membrane and possible subsequent interaction with excess R^* of T-GDP which, in spite of its low solubility, had eluted into solution given the high dilution of the permeated rods. In the presence of GTP, the so called dissociation signal includes a fast, anisotropic release component that specifically monitors the release into the interdiscal space of T -GTP formed from the membrane-bound pool, and a slower isotropic loss component monitoring the leakage from the permeated rod of the excess T -GTP which did not interact with the cGMP phosphodiesterase. The amplitudes of both components depend exclusively on the membrane bound T-GDP pool. The kinetics of the loss component is limited by the size and degree of permeation of the rod fragments, rather than by the dissociation rate of T -GTP from the membrane.Abbreviations ROS rod outer segment - R rhodopsin - R^* photoactivated rhodopsin - T, T-GDP, T -GDP, T -GTP, T transducin and its various forms - T _mb, T _sol: T bound to membrane or soluble - PDE cGMP-phosphodiesterase - GTP guanosine 5-triphosphate - GDP guanosine 5-diphosphate - GDP S guanosine 5-O-(2-thiodiphosphate) - cGMP guanosine-3-5 cyclic-monophosphate - DTT dithiothreitol - HEPES 4-(2-hydroxyethyl)-1-piperazine-ethane sulfonic acid - TRIS Tris (hydroxymethyl)aminomethane - SDS sodium dodecyl sulfate     

The problem of nonstationary ion fluxes in excitable membranes

Time-dependent electrodiffusion through a membrane is analysed within a simple model treating the boundary-layers in a consistent manner. It is shown that time-independent reversal potentials for the ion fluxes exist only under steady-state conditions. We argue that this result holds very generally. Therefore nonstationary effects like ion storage and depletion inside the membrane should not contribute to the phenomena of excitability.Glossary of Symbols A _mv [V] functional cf. Equation (3) - C membrane capacitance - d one half the thickness of the membrane - F[V] functional cf. Equation (1) - g _i electrochemical potential inside membrane - g _i electrochemical potentials outside membrane at x ±d, respectively - i (index) refers to i-th ionic species - J electric current across membrane - j = j ^} = j ^< current density measured by external electrodes - j _i (x) current density inside membrane in x-direction - j _i ^inst(x) instantaneous current density - J _i ^stat steady-state current density - k Boltzmann constant - m (index) is used in Sec. 2 to denote the independent diffusion currents - n ^< ionic strength of electrolyte at x = - - n _i density of ions inside membrane - n _i density of ions outside membrane at x = ±, respectively - Q charge per unit area of boundary layers at x ± d, respectively - Q ₀ fixed charge per unit area of membrane - q elementary charge - q _i ionic charges - T temperature - it time - V membrane potential (= (-)-()) - V _i Nernst potential - V potential drops inside boundary layers (can be neglected, see Appendix II) - V ^± potential steps at x = ± d, cf. Equation (29) - V ₀ = V ^–-V ⁺ - w _i activation energy inside membrane - x spatial coordinate perpendicular to membrane - y, z spatial coordinates parallel to membrane - dielecric constant - ₀ dielectric constant of electrolyte solution ( 80) - _m dielectric constant of membrane ( 5) - (x) electrostatic potential - charge density of boundary layers - ⁰ fixed charge density inside membrane - spatial average, cf. Equation (12)     

The partially reduced species present in purified cytochrome oxidase from baker's yeast is cytochromea

Cytochrome oxidase purified from baker's yeast submitochondrial particles is found to exist in a partially reduced state in the resting enzyme. Studies utilizing optical and EPR spectroscopy indicate that the inactive fraction contains a reduced low-spin heme, cytochromea, possibly indicating a block of electron transfer from cytochromea to cytochromea ₃. There is no apparent reduction of either the EPR-detectable copper or the species associated with the 830 nm band. Oxidative titrations of the resting-state yeast cytochrome oxidase indicate that the reduction potential of the species titrating is higher than that of ferricyanide. This inactive cytochrome oxidase is not the result of the isolation procedure, but seems to represent a species which is present in the intact yeast.     

The tryptic peptide composition of the beta chains of hemoglobins A and B of the domestic cat (Felis catus)

Summary The tryptic peptides from the ^A and ^B chains of cat hemoglobins A and B have been isolated and the amino acid compositions determined. Differences between the two chains were found in two peptides,T-1 (GlySer) andT-14 (AsnSer and LysArg). The GlySer and LysArg substitutions are placed at-1 and-144 respectively from earlier work, and the third substitution, AsnSer at-139 is suggested from this work. In addition, the presence of a blocked amino terminus in ^B has been confirmed. Tentative sequences constructed by homology with known-chain structures suggest the occurrence of substitutions at _{1 1} contacts in ^A and ^B that may be functionally significant. There are at least 18 differences in amino acid composition between cat ^A and dog-chains and 22 differences between cat ^A and normal adult human-chains.     

The effect of membrane potential on the redox state of cytochromeb 561 in antimycin-inhibited submitochondrial particles

The oxidation of cytochromeb ₅₆₁ by ATP was measured in submitochondrial particles inhibited by antimycin. The redox potential of the bulk (M phase) was controlled by the ratio of fumarate:succinate, and the oxidation of cytochromeb was calculated and expressed as a change in redox potential (E _h) measured in millivolts. The oxidation of cytochromeb ₅₆₁ is an energy-driven reaction affected only by the component of the proton motive force. The oxidation (measured in millivolts) is a function of the phosphate potential, reaching a maximal value of 40 mV at G_ATP<–12 kcal/mole. The maximal measured value of ATP-dependent was 100 mV. Thus only a fraction of the membrane potential effects the redox state of cytochromeb ₅₆₁. In contrast to the ATP-induced oxidation of cytochromeb ₅₆₁, cytochromeb ₅₆₆ is in redox equilibrium with fumarate succinate either in the presence or in the absence of ATP. The selective oxidation ofb ₅₆₁ is explained within the term of theQ cycle as a reflection of on the electron electrochemical potential. The positive electric potential of theC phase causes cytochromeb ₅₆₆ to act as oxidant with respect to cytochromeb ₅₆₁. In the presence of antimycin cytochromeb ₅₆₁ cannot equilibrate with the quinone and undergoes oxidation, while cytochromeb ₅₆₆ reequilibrates with the quinone and thus regains redox equilibrium with the fumarate succinate redox buffer.Abbreviations used: ETP_H, phosphorylating submitochondrial particles; TMPD,N ₁ N ₁ NN-tetramethyl-p-phenylenediamine; FCCP, carbonylcyanidep-trifluoromethoxyphenylhydrazone; Mes, 2-(N-morpholino) ethanesulfonic acid.     

Some characteristics of anion transport at the tonoplast of oat roots,determined from the effects of anions on pyrophosphatedependent proton transport

The effects of anions on inorganicpyrophosphate-dependent H⁺-transport in isolated tonoplast vesicles from oat (Avena sativa L.) roots were determined. Both fluorescent and radioactive probes were used to measure formation of pH gradients and membrane potential in the vesicles. Pyrophosphate hydrolysis by the H⁺-translocating pyrophosphatase was unaffected by anions. Nonetheless, some anions (Cl^-, Br^- and NO₃-) stimulated H⁺-transport while others (malate, and iminodiacetate) did not. These differential effects were abolished when the membrane potential was clamped at zero mV using potassium and valinomycin. Stimulation of H⁺-transport by Cl^- showed saturation kinetics whereas that by NO₃- consisted of both a saturable component and a linear phase. For Cl^- and NO₃-, the saturable phase had a K _m of about 2 mol·m^-3. The anions that stimulated H⁺-transport also dissipated the membrane potential (.) generated by the pyrophosphatase. It is suggested that the stimulatory anions cross the tonoplast in response to the positive generated by the pyrophosphatase, causing dissipation of and stimulation of pH, as expected by the chemiosmotic hypothesis. The work is discussed in relation to recent studies of the effects of anions on ATP-dependent H⁺-transport at the tonoplast, and its relevance to anion accumulation in the vacuole in vivo is considered.Abbreviations and symools BTP 1,3-bis[tris(hydroxymethyl)-methylamino]-propane - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid - IDA iminodiacetate - membrane potential - pH pH gradient - PPase inorganic pyrophosphatase - PPi morganic pyrophosphate     

Determination of the apparent K mfor oxygen of Beneckea natriegens using the respirograph technique

The affinity for oxygen of the marine bacterium Beneckea natriegens was mesured using the Respirograph technique of Degn and Wohlrab. Values between 0.15 and 0.25 M oxygen were obtained for the apparent K _mfor oxygen regardless of the nature of the respiratory substrate. These values are an order of magnitude lower than those previously reported for B. natriegens using the conventional closed oxygen electrode system.     

L and D presequence peptides derived from the precursor of F1β subunit of the ATP synthase inhibit mitochondrial protein import by interaction with import machinery

We investigated the effect of L and D enantiomers of a 25-residue peptide derived from the N-terminal region of the presequence of Nicotiana plumbaginifolia F₁ subunit of the ATP synthase, pF₁(1, 25), on import into spinach leaf mitochondria. Three in vitro synthesized precursor proteins using different import pathways were used. Import of the precursor proteins of F₁ subunit of the ATP synthase, pre-F₁, and the alternative oxidase, pre-AOX, required addition of external ATP, whereas the chimeric precursor containing the N-terminal 84 amino acids of the cytochrome b ₂ precursor protein linked to dihydrofolate reductase, pre-b ₂(1, 84)-DHFR was not dependent on ATP. Import of pre-F₁, and pre-AOX was inhibited already at 1 M and 3 M concentration of the L and D enantiomers, whereas inhibition of import of pre-b ₂(1, 84)-DHFR, occurred at concentrations >10 M of both enantiomers. Binding efficiency of the precursor proteins was not affected by addition of the L and D enantiomers. There was no correlation between inhibition of import of pre-F₁ and pre-AOX and dissipation of membrane potential measured as a decrease of Rhodamine 123 fluorescence quenching. The inhibitory effect of the L and D presequence enantiomers on import of pre-F₁ and pre-AOX was concluded to occur within the outer membrane translocase machinery beyond the initial precursor receptor interaction. Furthermore, the fact that the D enantiomer had the same effect as the natural peptide showed that interaction of the presequence with the import machinery was not dependent on chiral properties of the presequence.     

The α2 cDNA sequence of human haptoglobin carries a bacterial promoter functionalin vivo

Various constructions of human haptoglobin (Hp) cDNA coding either for the complete ^2FS precursor protein or only for the subunit have been placed under the control of the P_R promoter in the bacterial expression vector pCQV2 (Queen, 1983). In addition to the expected 45,000 dalton polypeptide synthesized after induction of the P_R promoter, the complete ^2FS constructions constitutively express a smaller polypeptide of 30,000 dalton corresponding to a truncated Hp protein. Computer analysis of the HpcDNA revealed the presence of two strong potential bacterial promoters (²P_F and ²P_S) located in the duplicated ^2FS sequence. Both Hp promoter signals are followed by potential mRNA start sites and ribosome binding sites at a compatible distance from initiation codons. In addition, the Hp² cDNA sequence, when fused upstream to the cDNA coding for ¹-antitrypsin, constitutively promotesin vivo the efficient expression of an hybrid protein specifically recognized by antibodies raised against ¹-antitrypsin or haptoglobin.