[The phosphorylation state of transducin beta-subunits].

The supposition that nucleoside diphosphate kinase is the enzyme that phosphorylates transducin beta-subunits on one of the histidine residues (His-266) has been analyzed. It stands the reason that 1) this enzyme is multifunctional and plays in particular the role of protein histidine kinase; and 2) the phosphorylated beta-subunit of transducin may activate transducin via the mechanism of transphosphorylation. Nevertheless, in our experiments, in which different forms of transducin preparations were incubated with α- and ß-isoforms of recombinant rat NDP kinase in the presence of [γ32P]ATP or [γ32P]GTP (specific activity of about 1 Ci/mmol) followed by separation of proteins by electrophoresis and-gel radio-autography, the phosphorylation of the transducin beta-subunit wasn't succeeded to be found. The negative result of our experiments most likely implies that the major part of transducin beta-subunits in the preparations has already been phosphorylated via a process that takes place in vivo.

[Activation of bovine retinal rod outer segment cGMP-specific phosphodiesterase by the transducin-GTP complex in a physiologically significant range of free calcium ion concentrations].

The kinetic behavior of cGMP-specific phosphodiesterase in a totally bleached bovine retinal rod outer segment suspension was studied by the pH-metric method at high and low concentrations of free calcium ions (≈ 100 µM and 10 nM, respectively). The phosphodiesterase was activated by low GTP concentrations (about 1-2 µM) that were comparable with the concentration of G-protein transducin, its GTP-binding alpha-subunit was the intrinsic activator of photoreceptor phosphodiesterase. The results allow the suggestion that besides the earlier described system of RGS proteins, participating in the acceleration of GTP hydrolysis, rod outer segments also contain an additional Ca(2+)-dependent mechanism to inactivate so called "free transducin", i.e. active transducin that has not managed to interact with phosphodiesterase during the time, restricted by duration of photoreceptor response.

[Purification of transducin betagamma-subunit complex from isotonic extracts of bovine retinal rod outer segments].

A method for obtaining a free complex of transducin betagamma-subunits from bovine retinal rod outer segments in a highly purified state has been suggested.

[Nucleoside diphosphate kinase and GTP-binding proteins. Possible mechanisms of coupling].

The results of numerous investigations during the last 20 years have shown that nucleoside diphosphate kinase (NDP kinase) is a multifunctional protein. In this paper, the current data are analyzed indicating that one of the possible mechanisms by which NDP kinase manifests its multifunctional role is its participation in the activation (or regulation) of heterotrimeric GTP-binding proteins (G proteins). We demonstrate that one of the NDP kinase isoforms dynamically interacts with the retinal rod G protein transducin (Gt) and phosphorylates its beta-subunit at histidine residue (His 266). It is also shown that it leads to the consecutive transfer of the phosphate group to the GDP in the active center of G protein alpha-subunit and G protein activation. The advantages of this mechanism are considered as compared to the classic G protein activation mechanism, GDP/GTP exchange.

Investigation of chimerical and tagged forms of recombinant rat nucleoside diphosphate kinases alpha and beta. Interaction with rhodopsin-transducin complex and thermal stability.

To elucidate the physicochemical basis of differences between the isoforms of mammalian multifunctional nucleoside diphosphate kinase (NDP), we investigated the recombinant rat homohexameric NDP kinases alpha and beta, consisting of highly homologous alpha or beta subunits of 152 residues each and differing only in variable regions V1 and V2, and their chimerical forms (NDP kinase alpha(1-130)beta(131-152) and NDP kinase beta(1-130)alpha(131-152)) and tagged derivatives (NDP kinase HA-alpha(1-130)beta(131-152), NDP kinase HA-beta(1-130)alpha(131-152), and NDP kinase HA-beta). The thermal stability of these proteins and the ability of some of them to interact with the rhodopsin-transducin (R*Gt) complex have been studied. It was found that NDP kinase alpha, NDP kinase alpha(1-130)beta(131-152), and NDP kinase HA-alpha(1-130)beta(131-152) were similar in their thermal stability (T(1/2) = 61-63 degrees C). NDP kinase beta, NDP kinase beta(1-130)alpha(131-152), NDP kinase HA-beta(1-130)alpha(131-152), and NDP kinase HA-beta were inactivated at a lower temperature (T(1/2) = 51-54 degrees C). NDP kinase HA-alpha(1-130)beta(131-152) interacted with the R*Gt complex in the same manner as NDP kinase alpha, whereas the interaction of NDP kinase HA-beta(1-130)alpha(131-152) and NDP kinase beta with the photoreceptor membranes under the same conditions was very weak. It is suggested that the variability of the region V1 is a structural basis for the multifunctionality of NDP kinase hexamers in the cell.

[Thermostable extracellular cyclic nucleotide phosphodiesterase from Physarum polycephalum plasmodium].

The cyclic nucleotide phosphodiesterase secreted by Physarum polycephalum plasmodium into extracellular medium has been partially purified by DEAE cellulose chromatography, ultrafiltration, and HPLC. The results obtained by gel filtration, HPLC, electrophoresis, and isoelectric focusing suggest that, the native enzyme in solution is a monomer with a molecular mass of about 90 kDa and pI in the range 3.6 - 4.0. The Km values were estimated to be about 0.9 mM and 7.7 mM, respectively, and Vm for both substrates were similar (up to several thousand micromoles of cAMP hydrolyzed/hour per mg of enzyme). The partially purified enzyme was shown to be extremely stable. It did not lose the activity after heat treatment at 100 degrees C during 30 min. The enzyme was active in the presence of 1% SDS, but it was fully inactivated under the same conditions in the presence of beta-mercaptoethanol. The properties of the phosphodiesterase from Physarum polycephalum are discussed.

[Calcium-dependent interaction of transducin with calmodulin-sepharose].

It has been shown by affinity chromatography on calmodulin-sepharose that transducin, a G protein of bovine retinal rod outer segments interacts with Ca(2+)-calmodulin. This result assumes that the main part of calmodulin in dark retinal rod outer segments is associated with transducin. It has been suggested that photoactivation of retinal rods induces changes in intracellular calmodulin concentration, which may be one of the steps involved in the light adaptation of photoreceptor.

[Isolation of lipofuscin from the bovine myocardium]. / Vydelenie lipofustsina iz miokarda krupnogo rogatogo skota.

The paper describes the procedure of lipofuscin isolation from bovine myocardium with the use of 15-45% (weight/volume) linear gradient of sucrose density and differential centrifugation. Two coloured populations of lipofuscin granules with different density have been distinguished. Electron microscopic studies of the isolated lipofuscin have revealed the homogeneity of fractions and good maintenance of the granules. The electron microscopic, absorptive and luminescent spectral properties of lipofuscin preparations correspond to its properties in situ. The isolation of lipofuscin preparations suggests the possibility of studying the chemical composition and intramolecular structure of the granules.

[Adenylate kinase and GDP-kinase activity of rod outer segments in the frog retina. Possible functional role of the T beta subunit of transducin]. / Adenilatkinaznaia i GDP-kinaznaia aktivnost' preparatov naruzhnykh segmentov palochek setchatki liagushki. Vozmozhnaia funktsional'naia rol' sub''edinitsy T beta transdutsina.

Pure frog retina rod outer segments (ROS) preparations (A280/A500 = 2,1-2,3) catalyze the synthesis of ATP from ADP in the presence of Mg2+. Adenylate kinase (AK) (ATP:AMP phosphotransferase, EC 2.7.4.3) specific activities for ROS preparations are within the range 2-4 mumole per hour for mg protein. The enzymatic activity of investigated preparations is due to intact, but not destroyed ROS. The component which possesses AK is found in water-soluble, but not in membranous ROS fractions and seems to be a part of the predominant ROS plasma protein--GTP-binding complex of transducin. It has been shown, that this component is the T beta subunit of transducin and its enzymatic activity is controlled by other subunits of the transducin complex. The obtained results indicate that GDP kinase (ATP:GDP phosphotransferase, EC 2.7.4.6) activity of transducin depends on the work of both of T beta and T alpha subunits. It has been shown that in the ROS preparations synthesis of the ATP from ADP and GDP phosphorylation are stimulated by a lowering of Ca2+ concentration (less than 10(-5)-10(-7) M). T beta component is suggested to play the role of phosphotransferase which phosphorylates GDP associated with the T alpha subunits and it leads to formation of a complex T alpha X GTP which is an activator of vertebrate retina ROS phosphodiesterase.

[Various characteristics of erythrocyte membrane proteins in rats with spontaneous hypertension]. / Nekotorye osobennosti membrannykh belkov éritrotsitov krys so spontannoi gipertenziei.

Differential scanning microcalorimetry demonstrated an increase in specific heat capacity of erythrocyte membranes of spontaneously-hypertensive rats around 63 degrees C (C-transition). Disc electrophoresis with sodium dodecyl sulphate showed the percentage of band 3 peptides to be increased in relation to all membrane peptides in these membranes. It is suggested that the elevated content of band 3 peptides, which are known to participate in the formation of anion channels in mammalian erythrocyte membranes, may be one of the causes of increased specific heat of C-transition.

[Guanosine triphosphate complex in rod outer segments of the frog retina]. / Guanizin-trifosfatnyi kompleks v naruzhnykh segmentakh palochek setchatki liagushki.

It is shown that nearly 70% water--soluble protein of the frog retina outer segments (ROS) consist of three polypeptides with molecular weights 39 000, 36 000 and less than 15 000 daltons. These proteins are present in equal proportions and are, apparently, the subunits of a tightly bound protein complex. The subunit of 39 000 daltons is responsible for guanyl nucleotides binding. Parameters of the investigated GTP-binding complex are similar to transducyn which transmits excitation from bleached rhodopsin to PDE molecules in the bovine retina ROS. The thermodynamic state of GTP-binding protein in frog retina ROS depends on the functional state of the photoreceptor membrane, as shown by microcalorimetric method.

[Molecular mechanisms of photoreception. IV. Photoregeneration of rhodopsin from metarhodopsin II using the artificial lipid membrane method for detection of intermediate steps of this reaction]. / Molekuliarnye mekhanizmy fotoretseptsii. VI. Fotoregeneratsiia rodopsina iz metarodopsina II ispol'zovanie metoda iskusstvennykh lipiknykh membran dlia obnaruzheniia promezhutochnykh étapov étoi reaktsii.

It was shown that photoregeneration of rhodopsin from metarhodopsin II takes place in bovine retinal rod outer segment disc membrane fragments. Photoregenerated rhodopsin is identical to unbleached rhodopsin in spectra and in stability to NH2OH. Moreover, as an unbleached pigment, photoregenerated rhodopsin can induce the transient increase of the artificial lipid membrane (ALM) conductivity in response to visible light. It was shown, that the UV-light converts metarhodopsin II to rhodopsin through the new long-lived (tau approximately 5 min) intermediate product (X500). X500 is indistinguishable from rhodopsin spectrophotometrically, but it can not induce the transient increase of ALM conductivity in response to visible light. It was shown that P467 (this product is obtained usually by photolysis of metarhodopsin II) has a retinal chromophore in all-trans configuration. This result indicates that P467 is produced from cis-metarhodopsin II and X500 (these products are the intermediates of the back reaction metarhodopsin II--rhodopsin) but not from metarhodopsin II as suggested earlier. A modified scheme of the back reaction metarhodopsin II--rhodopsin is given.

[Molecular mechanisms of photoreception. V. Reconstitution of a sodium channel on an artificial lipid membrane modified by bovine rod outer segment components]. / Molikuliarnye mekhanizmy fotoretseptsii. V. Popytka modelirovaniia tsitoplazmaticheskoi membrany fotoretseptora na iskusstvennoi lipidnoi membrane, modifitsirovannoi komponentami naruzhnykh segmentov palochek setchatki byka.

An attempt is made to reconstitute the Na+-conductivity elements of rod outer segment (ROS) plasma membrane on the artificial lipid membrane (ALM). ALM were modified by preparation with bovine ROS plasma membrane fragments. Discrete fluctuations of the ALM conductivity were observed at addition of this preparation to the ALM bathing solutions to a final concentration of 0.1--1.0 microgram/ml. The magnitude of these fluctuations was about 25 pS at 140 mM NaCl. The modified ALM possessed preferentially the Na+-conductivity, which was at least five times as great as that for K+ or Li+. The modified ALM showed practically no conductivity for Cl-. The Na+-channels were voltage-dependent. The Na+-channels were "sensitive to visible light" at some experimental conditions. The optimal conditions were obtained for reconstitution of Na+-channel on the ALM. The temporal and termal stabilities of the modified preparation were investigated. It was shown that the frequency of the modified ALM conductivity fluctuations are dependent on temperature and on lipids composition of ALM. The data obtained in our work are in a good agreement with the results of electrophysiological studies of photoreceptor cells. It may be indicated, that the investigated ALM contains the Na+-channel of the ROS plasma membranes. It is suggested that the reconstituted system will be useful for studying the principles of regulation of ROS plasma membrane sodium conductivity and the nature of a mediator in a photoreceptor transduction mechanism.

[Phosphorylation of endogenous proteins of bovine retina rod outer segments]. / Fosforilirovanie édogennykh belkov naruzhnykh segmen tov palochek setchatki byka.

The components of bovine rod outer segments (ROS) and water-soluble extracts of ROS were separated by SDS-electrophoresis after incubation with [gamma-32P]ATP or [gamma-32P]GTP at different experimental conditions. After that gels were autoradiographed to reveal the phosphorylated intermediates. Our results suggest, that ROS contains the following protein kinase systems: 1) water-soluble cAMP-dependent protein kinases, that uses ATP, but not GTP, and phosphorylates the water-soluble 30 000 molecular weight protein; 2) protein kinase that uses GTP (probably, ATP also) and phosphorylates the 20 000 molecular weight protein in light-adapted ROS; 3) water-soluble cyclic nucleotide- and Ca2+-independent protein kinase that uses ATP rather than GTP and phosphorylates the water-soluble 70 000 molecular weight protein. The concentrations of phosphorylated intermediates in bovine ROS are estimated.

[Protein kinase activity of bovine retina rod outer segments]. / Proteinkinaznaia aktivnost' naruzhnykh segmentov palochek setchatki byka.

Dark-adapted pure bovine rod outer segments (ROS) (A280/A500--2.1) can be phosphorylated in the presence of [gamma-32P]ATP and [gamma-32P]GTP. The constant levels of phosphorylation, reached within 10--15 min, are 100 +/- 30 pmol 32P/nmol of rhodopsin for [gamma-32P]ATP and 2--4 pmol 32P/nmol of rhodopsin for [gamma-32P]GTP. These processes are not controlled by 10(-4)--10(-8) cAMP, cGMP or Ca2+, but are inhibited at higher concentrations of these agents. In the presence of histone the constant level of phosphorylation is increased up to 200 +/- 30 pmol 32P/nmol of rhodopsin for [gamma-32P]ATP, but is not changed when [gamma-32P]GTP is used. 10(-5) M cAMP is found to activate the phosphorylation in the presence of histone and [gamma-32P]ATP by 5--6 times. All this evidences that ROS contains cAMP-dependent protein kinase, which utilizes ATP, but not GTP. Moreover, ROS contains cyclic nucleotides- and Ca2+-independent protein kinase. These protein kinases are the ROS endogenous enzymes. This is shown in experiments on separation of pure ROS in a sucrose density gradient.

[Fast photo-induced changes in the light scattering of vertebrate photoreceptor membrane preparations. Study of suspensions of vesicles obtained by ultrasonic treatment of the outer segments of retinal rods]. / Bystrye fotoindutsirovannye izmeneniia svetorasseianiia preparatov fotoretseptornykh membran pozvonochnykh. Izuchenie suspenzii vezikul, poluchennykh pri ul'trazvukovoi obrasbotke naruzhnykh segmentov palochek setchatki.

By light scattering method it was shown, that vesicles, obtained by sonication of rod outer segment membranes in different ionic media rapidly and irreversibly increased their volume under bleaching of rhodopsin. Apparently, this volume change was caused by the increase in the amount of free ions in vesicles under illumination. The photoinduced volume change was practically the same in all cases studied (equal ionic constitutions inside and outside of vesicles; in the presence of penetrating anion SCN-; under ionic gradients on vesicle membranes; in the presence of ionophores which induce the permeability for K+ and H+) and corresponds to the appearance of no more than one ion inside the vesicle under bleaching of one rhodopsin molecule. The obtained results testify against the hypothesis, that bleaching of rhodopsin induce essential increase in ionic permeability of rod outer segment disk membranes.