Generation of electric current by chromatophores of Rhodospirillum rubrum and reconstitution of electrogenic function in subchromatophore pigment-protein complexes.

Lipoprotein complexes, containing (1) bacteriochlorophyll reaction centers, (2) bacteriochlorophyll light-harvesting antenna or (3) both reaction centers and antenna, have been isolated from chromatophores of non-sulphur purple bacteria Rhodospirillum rubrum by detergent treatments. The method of reconstituting the proteoliposomes containing these complexes is described. Being associtated with planas azolectin membrane, ptoteoliposomes as well as intact chromatophores were found to generate a light-dependent transmembrane electric potential difference measured by Ag/AgC1 electrodes and voltmeter. The direction of the electric field inproteoliposomes can be regulated by the addition of antenna complexes to the reconstitution mixture. The reaction center complex proteoliposomes generate an electric field of a direction opposite to that in chromatophores, whereas proteoliposomes containing reaction center complexes and a sufficient amount of antenna complexes produce a potential difference as in chromatophores. ATP and inorganic pyrophosphate, besides light, were shown to be usable as energy sources for electric generation in chromatophores associated with planar membrane.

[Reconstitution of cytochrome oxidase with liver and hepatoma 27 mitochondrial lipids]. / Osobennosti rekonstruktsii tsitokhromoksidazy lipidami mitokhondrii pecheni i gepatomy 27.

Activity of cytochrome oxidase, incorporated into the lipoprotein vesicles /proteoliposomes/ in the course of self assembly procedure, was studied. For this purpose the lipids were used, which were isolated from mitochondria of rat liver tissue and of hepatoma 27. The enzymatic activity was distinctly increased after the reconstriction using liver mitochondrial phospholipids; the hepatoma lipids were not effective. An agent uncoupling the oxidative phosphorylation activated the oxygen utilization by cytochrome oxidase in proteoliposomes containing phospholipids of liver mitochondria, but not of hepatoma organelles. A difference in electrical potentials, generated by cytochrome oxidase reaction on proteoliposome membranes containing the phospholipids from liver mitochondria, was distinctly higher as compared with proteoliposomes containing the lipids from mitochondria of tumors.

[Characteristics of immobilization and catalytic properties of the Soviet commercial preparation of L-asparaginase in liposomes composed of soybean phospholipids]. / Issledovanie osobennostei immobilizatsii i kataliticheskikh svoistv otechestvennogo kommercheskogo preparata L-asparaginazy v liposomakh, sformirovannykh iz fosfolipidov soevykh bobov.

The commercial preparation of 1-asparaginase was incorporated into liposomes formed from soybean phospholipids (asolectin). The inside phase volume of liposomes did not exceed 1% as calculated using fluorescence dye calcein but the efficiency of the enzyme incorporation into liposomes reached approximately 60%. The enzyme was adsorbed on outside surface due to electrostatic and hydrophobic interactions. The Km value of immobilized 1-asparaginase (2.7 X 10(-5)M) did not exceed considerably the Km values of free enzyme (1.8 X 10(-5)M) when l-asparagine was used as a substrate. The incorporation of 1-asparaginase into asolectin liposomes led to considerable increase in the enzyme thermostability at 70 degrees and also to an increase in its sensitivity to proteases and, particularly, to trypsin. The half-life periods of free and immobilized enzymes were practically similar in buffer solutions. However, the half-life period of immobilized l-asparaginase in blood serum was more than 6-fold higher as compared with that of the free enzyme.

[Structural organization of membranes reconstituted from phospholipids and subchromatophore pigment-protein complexes]. / Strukturnaia organizatsiia membran, i rekonstruirovannykh iz fosfolipidov i subkhromatofornykh pigment-belkovykh kompleksov.

Pigment--protein complexes of the P870 reaction centers and complexes of the bacteriochlorophyll light-harvesting antenna were isolated from the chromatophores of the non-sulfur purple bacterium Rhodospirillum rubrum by solubilization with detergents. The proteoliposomes containing the reaction centers or reaction centers and the light-harvesting antenna as well as liposomes formed from phospholipids were obtained by a self-assembly procedure using seya bean phospholipids. The freeze-fracture study showed that the proteoliposomes contain a large amount of globular particles. The particles incorporated into the two types of the proteoliposomes were distinguished in size. The globules of the reaction center and antenna complexes were bigger in size than the reaction center globules. The globular structures were not found in the liposomal membranes. The liposomes formed in the absence of the pigment--protein complexes were predominantly the multilamellar vesicles. The proteoliposomes were mostly represented as monolamellar membrane vesicles. The spatial arrangement of the reaction center complexes in the membranes is discussed.

[Generation of the differences of the electric potentials by Rhodospirillum rubrum reaction center complexes devoid of the heavy subunit]. / Obrazovanie raznosti élektricheskikh potentsialov kompleksami reaktsionnykh tsentrov Rhodospirillum rubrum, lishennymi tiazheloi sub"edinitsy.

The electrogenic activity of Rhodospirillum rubrum P870 reaction center complexes devoid of the heavy (H) subunit and retaining the light (L) and medium (M) subunits, was studied. The proteoliposomes containing such reaction center complexes were formed by a self-assembly procedure, using soya bean phospholipids. In the presence of Ca2+ the reaction center proteoliposomes were incorporated into a phospholipid-impregnated Teflon filter separating two solutions of identical composition. After addition of N,N,N',N'-tetra-methyl-p-phenylenediamine (or cytochrome c) and qinone (menadione), illumination caused generation of an electric potential difference between the two filter-separated compartments, the proteoliposome-free compartment being negatively charged. The illuminated proteoliposomes took up penetrating tetraphenylphosphonium cations and, in a lesser degree, tetraphenylborate anions. The data obtained suggest that the reaction center complexes containing only L- and M-subunits possess the electrogenic activity. The H-subunit is not directly involved in membrane potential generation.

[Properties of reconstituted transhydrogenase from mitochondria]. / O svoistvakh rekonstruirovannoi transgidrogenazy mitokhondrii.

The membrane vesicles (proteoliposomes) have been reconstituted from soya bean phospholipids and mitochondrial transhydrogenase (EC 1.6.1.1) by a self-assembly procedure. Palmitoyl-CoA and Mg2+ inhibit the rate of NAD+ reduction by NADPH in these proteoliposomes as well as in these submitochondrial particles. After solubilization of transhydrogenase from submitochondrial particles membranes the specific activity of the enzyme decrease and then again increases (more than 4.5 times) after its incorporation into the proteoliposomal membranes. An addition of potassium cholate to the proteoliposomal suspension further increases the rate of the direct transhydrogenase reaction (by 35--40%) due to the function of the oppositely oriented molecules of transhydrogenase. The Michaelis constants for NAD+ and NADPH for transhydrogenase proteoliposomes are 27 microM and 30 microM, respectively. These data are practically coincident with the results obtained for submitochondrial particles (21 and 33 microM, respectively). Thus, the incorporation of transhydrogenase into proteoliposomal membranes results in reconstitution of both electrogenic and the most essential kinetic properties of the enzyme.

Reconstitution of biological molecular generators of electric current. Inorganic pyrophosphatase.

Proteoliposomes have been reconstituted from soy-bean phospholipids (asolectin) and inorganic pyrophosphatase isolated from Rhodospirillum rubrum chromatophores. In the presence of Mg2+ ions, pyrophosphatase proteoliposomes were incorporated into a phospholipid-impregnated Teflon filter separating two solutions of an identical electrolyte content. Addition of inorganic pyrophosphate to the same compartment as proteoliposomes was found to induce generation of an electric potential difference between the two filter-separated compartments, the proteoliposomes-containing compartment being negatively charged. An electric potential difference of 15 mV and a current of 20 pA were observed. The electrogenic effect required Mg2+ and proved to be sensitive to fluoride, an inorganic pyrophosphatase inhibitor. Treatment with 10 microM N,N'-dicyclohexylcarbodiimide for several minutes was without influence upon pyrophosphate-induced membrane potential generation. Similar results were obtained in experiments with a proteoliposome suspension and a penetrating anion, tetraphenyl borate, which is a probe for membrane potential. The obtained data are discussed in connection with the results of studies on other enzymes as molecular generators of electric current.

[Reversible electric current generation by reconstituted mitochondrial transhydrogenase]. / Obratimaia generatsiia elektricheskogo toka rekonstruirovannoi transgidrogenozoi mitokhondrii.

A direct measurement of the electrogenic activity of purified mitochondrial transhydrogenase has been carried out. For this purpose beef heart transhydrogenase was isolated and reconstituted with phospholipids to form proteoliposomes. The transhydrogenase proteoliposomes were incorporated into a membrane filter impregnated with a decane solution of phospholipids. It was shown that the addition of substrates of either forward (NADPH and NAD+) or reverse (NADH and NADP+) transhydrogenase reaction gives rise to formation of electric potential difference across the proteoliposome-treated membrane filter. The electric vector depends on the direction of the reaction. The proteoliposome-supplemented compartment charges negatively in the case of the reverse one. The addition of the reaction products after substrate equalizes the potentials. The transhydrogenase-treated membrane retains the ability to the transhydrogenase-linked electrogenesis after removal of an excess of non-incorporated proteoliposomes.

Orientation of reaction center complexes from Rhodobacter sphaeroides in proteoliposomes and the effect of o-phenanthroline on electrogenesis during primary photochemical reaction.

The orientation of Rhodobacter sphaeroides reaction center complexes (RC complexes) in proteoliposomal membranes was investigated by a direct electrometric method. Conditions were found that allow monitoring of only that RC complex fraction that is oriented with its donor side to the inner part of the proteoliposome. It is shown that o-phenanthroline, an inhibitor of electron transfer between primary (QA) and secondary (QB) quinone acceptors, can also inhibit the photoinduced QA reduction. The efficiency of this inhibition depends on the concentration of added ubiquinone. It is assumed that the laser flash-induced o-phenanthroline inhibition of primary dipole (P-870+.QA-) formation is of a competitive nature.

Subcellular distribution of the R-subunits of cAMP-dependent protein kinase in LS-174T human colon carcinoma cells.

The analysis of the particulate preparations of LS-174T human colon carcinoma cells in confluent stage of growth revealed different distribution for regulatory subunits (R) of cAMP-dependent protein kinase (PKA) in the subcellular compartments. The LS-174T cell lysates were subjected to differential and discontinuous sucrose density gradient centrifugation. The obtained fractions were assayed for marker enzymes and photoaffinity labeled with 8-N3[32P]cAMP. The whole lysates and cytoplasmic fraction exhibited the presence of both RI alpha and RII alpha--subunits of PKA. The fractions exhibiting high activity of the marker enzymes for plasma membranes, Golgi apparatus and mitochondria contained mainly RII alpha. In the fractions of lysosomes and microsomes RI alpha and RII alpha were found in nearly equal amounts.

[Reconstruction of the function of membrane potential formation by isolated pigment-protein complexes of Rhodospirillum rubrum]. / Rekonstruktsiia funktsii obrazovaniia membrannogo potentsiala izolirovannymi pigment-belkovymi kompleksami Rhodospirillum rubrum

The pigment-protein complexes, containing the photosynthetic reaction centers and chlorophyll antenna, were isolated from non-sulfur purple bacteria Rhodospirillum rubrum, by use of cholate. Absorption spectra and quantum yield of photo-oxidation of bacteriochlorophyll P870 of complexes were similar to those of chromatophores. The procedure of reconstitution of proteoliposomes from phospholipids and complexes was proposed. The proteoliposomes incubated with CoQ6 and TMPD upon illumination are generating the transmembrane electric potential difference (plus inside proteoliposomes). The electric potential difference was registered by three independent methods: by transmembrane electrophoresis of penetrating anions PCB-, by direct measurement with using voltmeter in system "proteoliposome--planar membrane" and electrochromic absorption band shifts of bacteriochlorophyll.

[Role of phospholipids in the generation of membrane potentials by proteoliposomes]. / O roli fosfolipidov v generatsii menbrannogo potentsiala proteoliposomani

Closed protein-phospholipid particles (proteoliposomes), obtained by self-assembly method, are capable to generate and to maintain the membrane potential in the case if their protein complex is represented by: a) a complex of mitochondrial ATPase; b) a complex of cytochrome oxidase and cytochrome c and c) bacteriorhodopsin from Halobacterium halobium; and their phospholipid component is represented by phosphatidylethanolamine or by a mixture of mitochondrial phospholipids. Only cytochromoxidase and bacteriorhodopsin (but not ATPase) proteoliposomes with phosphatidylserine are active. Cardiolipin also is not active in experiments with ATPase. Phosphatidylcholine produces in all the cases proteoliposomes incapable of maintaining the membrane potential. It is concluded that the inefficiency of phosphatidylcholine in the formation of proteoliposomes, generating the membrane potential, is due to the impossibility of obtaining closed membrane forms with a high electric resistance. The inefficiency of phosphatidylserine and cardiolipine, in the case of ATPase protein component of proteoliposomes, may be due to a specific requirement of this generator of the membrane potential in phosphatidylethanolamine.

Functional role of soluble mitochondrial ATPase subunits.

A preparation of soluble mitochondrial ATPase (coupling factor F1) containing no gamma and delta 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 gamma and delta subunits are necessary to form specific contacts between the F1 molecule and components of the mitochrondrial membrane.

Membrane-reversible H+-ATPase from Micrococcus lysodeikticus.

Treatment of phosphorylating fragments of bacterial membrane from Micrococcus lysodeikticus with trypsin leads to increase ATPase activity. As a result of this treatment, the membrane fragments acquire the ability to transform the ATP energy into transmembrane difference in potential. Dithiothreitol has a similar effect to that of trypsin on the membrane fragments from M. lysodeikticus. Dicyclohexylcarbodimide inhibits ATPase of the membrane fragments of M. lysodeikticus, and also the ATPase-reaction-coupled generation of membrane potential. It has been suggested that the increased ATPase activity of membranes from M. lysodeikticus during treatment with trypsin and dithiothreitol is connected with the effect of these agents on the protein inhibitor of ATPase.

Reconstitution of biological molecular generators of electric current. Transhydrogenase.

Direct measurement of the electrogenic activity of purified mitochondrial transhydrogenase has been carried out. To this end, beef-heart transhydrogenase was isolated and reconstituted with phospholipids to form proteoliposomes. The transhydrogenase proteoliposomes were incorporated into a membrane filter impregnated with a decane solution of phospholipids. It is shown that addition of substrates of either the forward (NADPH and NAD+) or the reverse (NADH and NADP+) transhydrogenase reaction gives rise to an electric potential difference across the proteoliposome-treated membrane filter. The electric vector depends upon the direction of the reaction. The proteoliposome-supplemented compartment charges negatively in the case of the forward reaction and positively in the case of the reverse one. Addition of the reaction products after substrates equalizes the potentials. The transhydrogenase-treated membrane filter retains the ability to perform transhydrogenase-linked electrogenesis after removal of excess non-incorporated proteoliposomes. The electric potential difference reaching 20 mV immediately after the transhydrogenase substrate addition, slowly decreases due to accumulation of the reaction products. Such decay is prevented when the mixture is supplemented with the substrate-regenerating and product-utilizing enzymic systems. Under these conditions, a steady continuous electric current of about 10 pA can be observed.

[Reconstitution of electrogenic function of pyrophosphatase isolated from Rhodospirillum rubrum membranes]. / Rekonstruktsiia elektrogennoi funktsii izolirovannoi membrannoi pirofosfatazy Rhodospirillum Rubrum.

The membrane vesicles (proteoliposomes) have been reconstituted from phospholipids and inorganic pyrophosphatase (EC 3.6.1.1) isolated from Rhodospirillum rubrum chromatophores. An addition of inorganic pyrophosphate (PPi) causes a Mg2+-dependent formation of a transmembrane electric potential difference and an uptake of penetrating tetraphenylborate anions by the proteoliposomes. Thus, isolated pyrophosphatase, being incorporated into the phospholipid membrane, functions as a MgPPi-dependent protein generator of the electric current.

[Electrogenic function of submitochondrial particles at the water-octane interphases]. / Elektrogennaia funktsiia submitokhondrial'nykh chastits na granitse razdela faz oktan/voda.

Studies on submitochondrial particles (SMP) preparation showed that in the sourse of the redox reactions at the octane-water interface, catalyzed by SMP enzymes, the charges are transferred from the aqueous to the octane phase. The effects were detected by a shift of the Volta potential, using the vibrating electrode method. In the presence of 2-N-methyl-amino-1,4-naphthoquinone in octane, acting as electron acceptor, the negative charges were transferred from water to octane following the oxidation of NADH, succinate and ascorbate. The charging of the octane phase was sensitive to the inhibitors of the respiratory chain, e. g. rotenone, antimycin and cyanide. In the presence of 2,4-DNP in octane, acting as a proton acceptor, the oxidation of NADH and succinate by ferricyanide, catalyzed by CMP in the presence of antimycin and cyanide correspondingly, was followed by a transfer of positive charges from water to octane. The positive charging of the octane phase, coupled with NADH oxidation, was found insensitive to rotenone, and that coupled with succinate oxidation, was completely inhibited by antimycin. The positive charging of the octane phase was also observed during the reverse transhydrogenase reaction, catalyzed by SMP at the division of the phases. The effect was inhibited by palmitoyl-CoA.

Reconstitution of biological molecular generators of electric current. Bacteriochlorophyll and plant chlorophyll complexes.

1. Electric generation by bacteriochlorophyll reaction center complexes from Rhodospirillum rubrum and by photosystem I complexes from pea chloroplasts has been studied. 2. The methods for the proteoliposome reconstitution from azolectin and bacteriochlorophyll- or plant chlorophyll-containing protein complexes have been elaborated. Light-dependent electric responses of the proteoliposomes were detected using (a) phenyldicarbaundecarborane anion (PCB-) probe and (b) direct measurement by a voltmeter in the proteoliposome-planar phospholipid membrane system. 3. Both PCB- and direct measurements demonstrated that bacteriochlorophyll proteoliposomes are competent in light-dependent electric generation (plus outside proteoliposomes). The photoelectric effect was shown to increase on addition of tetramethyl-p-phenylenediamine (TMPD), CoQ6, and vitamin K3, and to decrease on addition of ferricyanide, o-phenanthroline and a protonophorous uncoupler. Estimation of the photoelectromotive force of the bacteriochlorophyll proteoliposome-planar membrane system gave a value of about 0.2 V. The action spectrum of the photoelectric effect was similar to the absorption spectrum of the bacteriochlorophyll complex. 4. Reconstitution of proteoliposomes containing bacteriochlorophyll centers and bacteriorhodopsin resulted in the system generating an electric field whose direction can be changed by varying the spectral composition of the light: the red light, exciting bacteriochlorophyll, induces negative, and the green light, exciting bacteriorhodopsin, induces positive charging of the proteoliposome interior. 5. Association of isolated R. rubrum chromatophores with planar phospholipid membrane was found to give a system demonstrating light-induced electric generation as high as 215 mV in the presence of napthoquinone, TMPD (or phenazine methosulfate, PMS), and ascorbate. Under the same conditions, addition of inorganic pyrophosphate or ATP results in formation of an electric field of the same direction as that induced by light. 6. Proteoliposomes with plant chlorophyll complexes of photosystem I demonstrated light-induced PCB- responses indicating formation of the electric field with plus inside vesicles. The effect required PMS addition. A protonophorous uncoupler and o-phenanthroline were inhibitory. Electric responses in the chlorophyll proteoliposome-planar membrane system were very small (not higher than 10 mV).