Pump and displacement currents of reconstituted ATP synthase on black lipid membranes

Summary Purified ATP synthase (F ₀ F ₁) fromRhodospirillum rubrum was reconstituted into asolectin liposomes which were than adsorbed to a planar lipid bilayer. After the addition of an inactive photolabile ATP derivative (caged ATP), ATP was released after illumination with UV light, which led to a transient current in the system. The transient photocurrent indicates that the vesicles and the planar membrane are capacitatively coupled. Stationary pump currents were obtained after addition of protonophores. These currents are specifically inhibited by oligomycin and stimulated threefold by inorganic phosphate (P _i ). In analogy oligomycin-sensitive pump currents in the reverse direction coupled to net ATP synthesis were induced by a light-induced concentration jump of ADP out of caged ADP, demonstrating the reversibility of the pump. For this, a preformed proton motive force and P _i were necessary.In a second series of experiments, proteoliposomes containing both ATP synthase and bacteriorhodopsin were adsorbed to a planar bilayer. The system was excited by a laser flash. The resulting photocurrents were measured with a time resolution of 2 sec. In the presence of ADP, the signal was modulated by the electrical activity of ATP synthase. ADP-induced charge displacements in ATP synthase, with time constants of 11 and 160 sec were obtained. The kinetics of the charge movements were slowed down byF ₀ specific inhibitors (DCCD or oligomycin) and were totally absent if ADP binding toF ₁ is prevented by the catalytic site-blocking agent NBD-Cl. The charge displacement of ATP synthase is coupled only to the membrane potential induced by the electrical activity of bacteriorhodopsin. The charge movements are interpreted as conformational transitions during early steps of the reaction cycle of ATP synthase.     

Ion channel formation by synthetic transmembrane segments of the inhibitory glycine receptor--a model study.

The inhibitory glycine receptor (GlyR) of rat spinal cord contains an intrinsic transmembrane channel mediating agonist-gated anion flux. Here, synthetic peptides modelled after the predicted transmembrane domains M2 and M4 of its ligand-binding subunit were incorporated into lipid vesicle membranes and black lipid bilayers to analyze their channel forming capabilities. Both types of peptides prohibited the establishment of, or dissipated, preexisting transmembrane potentials in the vesicle system. Incorporation of peptide M2 into the black lipid bilayer elicited randomly gated single channel events with various conductance states and life-times. Peptide M4 increased the conductance of the bilayer without producing single channels. Exchange of the terminal arginine residues of peptide M2 by glutamate resulted in a significant shift towards cation selectivity of the respective channels as compared to peptide M2. In conclusion, the peptide channels observed differed significantly from native GlyR in both conductivity and ion-selectivity indicating that individual synthetic transmembrane segments are not sufficient to mimic a channel protein composed of subunits with multiple transmembrane segments.     

Nucleotide sequence analysis of the lemur beta-globin gene family: evidence for major rate fluctuations in globin polypeptide evolution

Lemur beta-related globin genes have been isolated and sequenced. Orthology of prosimian and human epsilon-, gamma-, and beta-related globin genes was established by dot-matrix analysis. All of these lemur globin genes potentially encode functional beta-related globin polypeptides, though precisely when the gamma-globin gene is expressed remains unknown. The organization of the 18-kb brown lemur beta-globin gene cluster (5' epsilon-gamma-[psi eta-delta]-beta 3') is consistent with its evolution by contraction via unequal crossing-over from the putative ancestral mammalian beta-globin gene cluster (5' epsilon-gamma- eta-delta-beta 3'). The dwarf lemur nonadult globin genes are arranged as in the brown lemur. Similar levels of synonymous (silent) nucleotide substitutions and noncoding DNA sequence differences have accumulated between species in all of these genes, suggesting a uniform rate of noncoding DNA divergence throughout primate beta-globin gene clusters. These differences are comparable with those observed in the nonfunctional psi eta pseudogene and have therefore accumulated at the presumably maximal neutral rate. In contrast, nonsynonymous (replacement) nucleotide substitutions show a significant heterogeneity in distribution for both the same gene in different lineages and different genes in the same lineage. These major fluctuations in replacement but not silent substitution rates cannot be attributed to changes in mutation rate, suggesting that changes in the rate of globin polypeptide evolution in primates is not governed solely by variable mutation rates.      

The dose effect ofma-l+ andry+ on xanthine dehydrogenase activity inDrosophila melanogaster

Summary Two loci,ma-l ⁺ andry ⁺, necessary for xanthine dehydrogenase activity inDrosophila melanogaster have been studied for dosage effects utilizing deficiencies and duplications induced for this purpose. Comparisons of one, two and three doses ofma-l ⁺ in the female or one and two doses in the male indicate that there is no increase in specific enzyme activity with dose. On the other hand, comparisons of one, two and three doses ofry ⁺ in the male and female reveal an increase in enzyme activity that is roughly proportional to dose. Since dosage ofry ⁺ is limiting, whereas that ofma-l ⁺ is not, the final concentration of xanthine dehydrogenase is shown to depend on the number of doses ofry ⁺.The implications of these findings with respect to the hypothesis of dosage compensation and to the mechanism of control of enzyme and protein concentration are discussed.Operated by Union Carbide Corporation for the U.S. Atomic Energy Commission.     

Kinetics and mechanism of anionic ligand binding to carbonic anhydrase

The kinetics of complex formation between Co(II)-carbonic anhydrase B and the anions cyanate, thiocyanate and cyanide has been studied at different pH values employing temperature-jump relaxation spectrometry. Formation of the 1:1 complex occurs via binding of the deprotonated state of the anion to an acidic state of the enzyme. The determined formation rate constants range from 10(8) to 3 X 10(9) M-1 s-1 and are two to three orders of magnitude higher than the value estimated for a ligand coordination to the central Co2+, based on a solvate substitution mechanism. These kinetic results strongly indicate that the deprotonated anion binds to an unoccupied coordination position of the protein-bound heavy metal ion in the form of an addition reaction. Upon binding of the anion, the coordination number of the Co2+ in the acidic state of the enzyme is increased from four to five. In the case of cyanide, a 2:1 anion complex is also formed. The formation rate constant is 5 X 10(5) M-1 s-1 which provides good evidence that this binding process is controlled by a solvate substitution mechanism.     

Electrical pump currents generated by the Ca-ATPase of sarcoplasmic reticulum vesicles adsorbed on black lipid membranes

Sarcoplasmic reticulum vesicles adsorbed on a black lipid membrane generate an electrical current after a fast increment of the concentration of ATP. This demonstrates directly that the sarcoplasmic Ca²⁺-ATPase from skeletal muscle acts as an electrogenic ion pump. The increment of the concentration of ATP is achieved by the photolysis of caged ATP (P³-1-(2-nitro)phenylethyl adenosine 5′-triphosphate) a protected analogue of ATP (Kaplan, J.H. et al. (1978) Biochemistry 17, 1929–1935), which is split into ATP and 2-nitroso acetophenone. The release of ATP leads to a transient current flow across the lipid membrane indicating that the vesicles are capacitatively coupled to the underlying lipid membrane. In addition to this transient signal, a stationary current flow is obtained in the presence of ionophores which increase the conductance of the bilayer system and prevent the accumulation of Ca²⁺ in the lumen of the vesicles. The direction of the transient and the stationary current is in accordance with the concept that Ca²⁺ is pumped into the lumen of the vesicles. The transient current depends on the concentration of ATP, Ca²⁺ and Mg²⁺ as would be the case for a current generated by the sarcoplasmic Ca²⁺-ATPase. Its amplitude is half-maximal at 10 μM ATP and 1 μM Ca²⁺. At Ca²⁺ concentrations above 0.1 mM the amplitude of the current signal declines again. The Mg²⁺ concentration dependence of the current amplitude at a constant ATP concentration indicates that the MgATP complex is the substrate for the activation of the current. The pump current is inhibited by vanadate and ADP. No current signal is observed if caged ATP is replaced by caged ADP. However, the release of ADP from caged ADP generates a pump current in the presence of an ATP generating system such as creatine phosphate and creatine kinase.     

Mechanism of suppression inDrosophila: Regulation of tryptophan oxygenase by thesu(s) + allele

The suppressor gene,su(s)², inDrosophila melanogaster restores the production of red and brown eye pigments for some purple and vermilion mutant alleles, respectively. We showed previously that the product of thesu(s)⁺ allele caused inhibition of the sepiapterin synthase A produced by the purple mutant but did not affect the wild-type enzyme. Suppression was accomplished by removingsu(s)⁺ from the genome. We now report that the tryptophan oxygenase, produced by suppressible vermilion alleles, is also inhibited by extracts fromsu(s)⁺ flies. The inhibition of the vermilion enzyme can be reduced or eliminated, respectively, by prior storage of the extract at 4 or –20°C or by boiling, whereas the wild-type enzyme is not affected by extracts ofsu(s)⁺ flies. Also, when the suppressible vermilion strain is raised on certain diets, brown eye pigment production occurs. This epigenetic suppression was reduced by the presence of an extra copy ofsu(s)⁺ in the genome. These data support a posttranslational mechanism for regulation of enzyme activity in which the activity of the mutant enzyme is reduced by the product of thesu(s)⁺ allele. How thesu(s)⁺ gene product can distinguish between the normal and the mutant forms of these two enzymes is discussed, along with other mechanisms for suppression that are currently under investigation.This work was supported in part by a grant from the KOSEF, Korea Science and Engineering Foundation, and the National Science Foundation under the U.S.-East Asia Cooperative Science Program as well as the Office of Health and Environmental Research, U.S. Department of Energy, under Contract DE-AC05-840R21400 with the Martin Marietta Energy Systems, Inc.