Kinetics and peculiarities of thermal inactivation of volume-induced Na+/H+ exchange, Na+,K+,2Cl- cotransport and K+,Cl- cotransport in rat erythrocytes.

The kinetics of the volume-dependent activation of Na+/H+ exchange, Na+,K+,2Cl(-)-cotransport and K+,Cl(-)-cotransport in rat erythrocytes was studied. The significant increase in the rate of Na+/H+ exchange is observed within 15 min after hypertonic shrinkage while the maximum transport rate is reached by 20 min. A delay of about 5 min was found in activation of Na+,K+,2Cl(-)-cotransport, the maximum transport rate being reached 10 min after shrinkage. Activation of K+,Cl(-)-cotransport by hypotonic swelling was registered within 10 min after cell swelling, with a simultaneous achievement of the constant transport rate. Preincubation of cells at 49 degrees C has no effect on the basal Na+/H+ exchange and Na+,K+,2Cl(-)-cotransport but suppresses the activation of these systems by osmotic shrinkage. On the contrary, the rate of K+,Cl(-)-cotransport in isosmotic medium is raised 10-fold after preincubation at 49 degrees C. The thermal treatment at 49 degrees C blocks the activation of K+,Cl(-)-cotransport by swelling. On the basis of the data on thermal denaturation of spectrin at the same temperature it was suggested that the cytoskeleton of erythrocyte membrane is involved in volume regulation of the ion-transporting systems and that the molecular mechanisms which underlie the activation of Na+/H+ exchange, Na+,K+,2Cl(-)-cotransport and K+,Cl(-)-cotransport are essentially different.

The structural transitions of erythrocyte membranes induced by cyclic AMP.

The generalized structural transitions of erythrocyte membranes induced by cyclic AMP were registered by ESR, fluorescence, freeze-fracture and circular dichroism methods. Two transitions different in nature were revealed. One, which arises at 10-(11)--10-(10) M cyclic AMP, is cooperative and may be considered as a consequence of interaction of cyclic AMP with a receptor. It was calculated that a structural rearrangement in one erythrocyte ghost is induced by three cyclic AMP molecules. As a result of it the membranes are "loosened". The other transition arises at 10-(10)--10-(8) M cyclic AMP and depends on the activity of the protein kinase system. This transition was shown to be non-cooperative and due to phosphorylation of membranous proteins. During this rearrangement the membranes are "stiffened". Both transitions were demonstrated to relate to the membrane integrity.

Swelling-induced activation of Na+,K+,2Cl- cotransport in C6 glioma cells: kinetic properties and intracellular signalling mechanisms.

Swelling of C6 glioma cells in hypotonic medium (180 mOsm) results in two- to three-fold activation of K+ (86Rb+) influx suppressed by 10 microM bumetanide. Bumetanide-sensitive transport of 86Rb+ is dependent on extracellular K+, Na+ and Cl- both in iso-osmotic conditions and under hypo-osmotic shock, supporting the notion that it is mediated by Na+,K+,2Cl- cotransport. Inhibitors of protein kinase C (10 microM polymyxin B and l microM staurosporine) had no significant effect on basal cotransport but reduced its hypotonic stimulation by 70-80%. Similar results were obtained with calmodulin antagonist R24571 (10 microM), indicating Ca2+/calmodulin-dependence of the process. Influence of polymyxin B and R24571 was not additive. Swelling-activated Na+,K+,2Cl- cotransport was also suppressed by protein kinase C activator PMA (l microM). By contrast, preincubation of cells with inhibitors of protein phosphatases (100 microM vanadate, 5 mM fluoride and 0.5 microM okadaic acid) activated greatly the bumetanide-sensitive 86Rb+ uptake in isotonic conditions, while a subsequent hypotonic swelling led to smaller or no increment. These results indicate the involvement of Ca2+/calmodulin-dependent staurosporine/polymyxin B-sensitive protein kinase other than protein kinase C in swelling-induced activation of Na+,K+,2Cl- cotransport in glial cells.

[The dynamics of the domains of the IP3-binding site of the inositol-1,4,5-triphosphate-sensitive calcium channel, induced by inositol-1,4,5-triphosphate and calcium].

The dynamics of the inositol-1,4,5-triphosphate-sensitive calcium channel after binding of inositol-1,4,5-triphosphate and Ca2+ was analyzed by the Monte Carlo minimization technique. It was shown that the binding of Ca2+ with the unliganded receptor (channel) leads to a turning of the beta-sheet domain relative to the alpha-helical domain with the formation of the receptor conformation that is open for the entry of ions into the cytoplasmic channel vestibule, sterically closed for their passage through the vestibule in the part adjacent to the alpha-helical domains, and unfavourable for subsequent binding of inositol-1,4,5-triphosphate with the receptor. When both co-agonists bind to the receptor, the structure rearrangements induced eliminate both these steric obstacles for the passage of ions through the IP3-binding domain: one at the entrance of the channel cytoplasmic vestibule and the other that is placed deeper in the vestibule near the alpha-domains. The role of the dynamics of the receptor binding core in the IP3-sensitive channel gating is discussed.

[Lanthanides induce neurotransmitter release from the vesicular pool in rat brain synaptosomes].

The influence of lanthanoids on exocytosis was investigated. It was shown that gadolinium increases the spontaneous release of the glutamate nonmetabolizing analogue [3H]D-aspartate. It was established using the fluorescent dye acridine orange that gadolinium and lanthanum induce exocytosis. The effect was dose-dependent and was maximum at 300 microM Gd3+. The exocytosis induced by gadolinium was calcium-independent. It is suggested that lanthanides induce a vesicular release of neurotransmitters by the mechanisms common for all polyvalent cations.

Hypoosmotic shock activates Ca2+ channels in isolated nerve terminals.

Influence of hypotonic swelling on Ca2+ (45Ca2+) uptake in rat brain synaptosomes was studied. A decrease in medium osmolality from 310 to 260-180 mOsm led to a progressive stimulation of 45Ca2+ accumulation. The effect was blocked by verapamil (IC50 = 5 microM), CoCl2 (IC50 = 58 microM) and retained at a fixed concentration of external sodium indicating the involvement of Ca2+ channels rather than Na+/Ca2+ exchange in swelling-induced Ca2+ influx. The populations of calcium channels observed in hypoosmotic and depolarizing conditions are different in three aspects: (i) kinetics of 45Ca2+ entry; (ii) insensitivity to dihydropyridines and omega-conotoxin GVIA; (iii) insensitivity to preliminary depolarization by high potassium. The effects of swelling and depolarization on Ca2+ uptake were additive. No change in membrane potential monitored with diS-C3-(5) was recorded during synaptosome hypotonic swelling. The results suggest the existence in synaptosomal plasma membrane of volume-dependent calcium-permeable channels with properties distinct from those of the voltage-dependent calcium channels. Activation of these channels may constitute an early event in volume regulation of nerve terminals in anisoosmotic conditions.

Influence of plasma membrane depolarization on cAMP level in rat brain synaptosomes.

We studied the influence of plasma membrane depolarization on cAMP content in presynaptic nerve endings (synaptosomes) isolated from brain hemispheres (HS) and cerebellum (CS). Depolarization by elevated [K+]o decreased basal cAMP level in both types of synaptosomes; reduced cAMP content in HS and increased cAMP in CS in the presence of IBMX; and lowered forskolin-stimulated cAMP accumulation in both the HS and the CS. Similar results were obtained when depolarization was induced by veratrine or when [Ca2+]i was elevated by treatment of the synaptosomes with the ionophore A23187. In Ca2+-free media, depolarization was not able to affect the synaptosomal cAMP levels. These data suggest that in brain synaptosomes intracellular cAMP pathway is modulated by alterations in [Ca2+]i.

Osmotic regulation of sodium pump in rat brain synaptosomes: the role of cytoplasmic sodium.

The effect of hypoosmolality of incubation medium on the rat of ouabain-sensitive 86Rb+ transport in rat brain synaptosomes was studied. A decreased osmolality from 310 to 250 mOsm increased the rate of 86Rb+ uptake from 3.72 to 6.23 nmol/mg of protein min. To evaluate the involvement of cytoplasmic sodium in sodium pump stimulation inhibitors of ion channels and transport pathways able to increase [Na+]in were used. Tetrodotoxin (1 microM), amiloride (0.5 mM) and verapamil (0.1 mM) had no influence on the osmotic response of the sodium pump. The decrease of sodium concentration in incubation medium to 15 mM, leading to a practical loss of its transmembrane gradient, did not abolish stimulation of pump. No increase in 22Na+ influx or intrasynaptosomal sodium content was registered at hypotonic conditions. It is suggested that osmotic regulation of Na+,K(+)-ATPase is not connected with an increase of internal sodium through opening of sodium channels, or with activation of other membrane sodium-transporting systems.

Swelling-induced K+ influx in cultured primary astrocytes.

The effect of swelling of cultured primary astrocytes from rat brain in hypotonic medium on K+ influx has been studied. A decrease in osmolality from 310 to 180 mOsm increased the activity of sodium pump (ouabain-inhibited 86Rb+ influx) and Na+,K+,2Cl- cotransport (ouabain-insensitive bumetanide-inhibited 86Rb+ influx) by 70 and 35%, respectively. It is suggested that activation of these transport systems makes it possible to retain a high potassium concentration in the cells under regulatory volume decrease.

Fluorescent probing of the ligand-binding ability of blood plasma in the acute-phase response.

The acute-phase response alters the composition of carrier proteins in plasma, which may affect the blood deposition and transport of biomediators and drugs. The effect of the acute-phase response on the ligand binding ability of plasma was studied in leukemic children with and without systemic inflammation (sepsis and septic shock). To target different transport proteins, differentially charged fluorescent dyes were used: anionic ANS (8-anilinonaphthalene-1-sulfonate), uncharged Nile red, and cationic Quinaldine red. Human serum albumin was a principal carrier for ANS and competed for Nile red binding with lipoproteins. The synchro-scan fluorescence spectra of Nile red in plasma distinguished two species of the dye bound to serum albumin and to low-density and/or very low-density lipoproteins. The binding of Quinaldine red did not correlate with albumin and lipoprotein levels, and was probably determined by alpha(1)-acid glycoprotein. Compared with the control group, leukemia increased Quinaldine red binding by 65% and did not significantly affect the binding of other probes. Sepsis and septic shock did not change the binding of Quinaldine red, but progressively decreased ANS binding, finally by about 33%, and shifted Nile red distribution from serum albumin toward lipoproteins. These changes reflected a modified composition of the three principal transport proteins in plasma in the acute-phase response. Simple and rapid fluorescent tests developed in this study can be used to evaluate the acute-phase response and to optimize drug administration protocols in clinical practice.

Sensitivity of the brain synaptosomal membrane Mg(2+)-ATPase activity to arachidonic acid is under control of the Na+,K(+)-ATPase state.

Evidence is presented for the sensitivity of the synaptosomal plasma membrane Mg(2+)-ATPase activity to arachidonic acid being dependent on the functional state of Na+,K(+)-ATPase. An "Inversion effect" was observed at arachidonic acid concentrations exceeding 80 mumol/l when the Mg(2+)-ATPase activity (after ouabain addition) is higher than the total ATPase activity (without ouabain). The "Inversion effect" is reduced by cyclooxygenase inhibitor indomethacin or acetylsalicylic acid and restored by prostaglandin PGA2 or PGD2.

Inactivation of muscarinic acetylcholine receptors in brain synaptic membranes by free fatty acids. Evaluation of the role of lipid phase.

Arachidonic, linolenic and linoleic acids decreased the binding of the m-cholinergic antagonist [3H] QNB and did not affect the ratio of high to low affinity binding sites to the agonist carbamoylcholine in rat brain synaptic membranes. In the presence of arachidonic acid, SH-reagent N-ethylmaleimide acquired the ability to block QNB binding to receptor. Lipids in the bilayer and annular regions were probed by fluorescence of 1,6-diphenyl-1, 3, 5-hexatriene and pyrene. A microviscosity drop induced by increasing temperature from 10 to 37 degrees C did not affect the level of QNB equilibrium binding, whereas arachidonic acid strongly inhibited the binding at concentrations inducing the same drop in microviscosity as that induced by heating. For various unsaturated fatty acids an equal extent of receptor blocking was reached at quite different degrees of bilayer fluidization, the state of annular lipid being not changed under these conditions. It is suggested that the effect of unsaturated acids is reached through their direct interaction with the receptor, which undergoes a conformational change, rather than by an alteration of the physical state of the lipid phase of the membrane.

On the mechanism of shrinkage-induced potassium influx in rat and human erythrocytes.

The rates of 86Rb influx into human and rat erythrocytes were studied in media of various tonicity. At sucrose concentrations below 0.3 mol/l, the ouabain-insensitive, furosemide-inhibited component of influx increased in rat but not in human erythrocytes; this may be explained by a rise in the rate of Na+, K+, Cl-- and/or K+, Cl-cotransport. An increase in osmolarity resulted in a reduction of this as well as of the ouabain and furosemide-insensitive component in rat erythrocytes. At the same conditions a drastic inhibition of Na+, K(+)-pump occurred both in rat and human erythrocytes. We failed to observe a lag-phase in the activation of the cotransport in rat erythrocytes; i. e. the process of activation parallels the shrinkage of cells. In rat erythrocyte ghosts, the shrinkage-induced stimulation of the cotransport was lost, and the direction of their osmotic reaction (inhibition of transport pathways) was similar to that in human erythrocyte ghosts. It is suggested that the mechanism of volume regulation of ion transport in intact cells involves a step of physical amplification via a change in interactions between the protein carcass and the lipid bilayer.

[Several structural aspects of the interaction of yeast hexokinase with insulin]. / Nekotorye strukturnye aspekty vzaimodeistviia drozhzhevoi geksokinazy s insulinom

It has been noted that due to formation of the complex with insuline light and hear resistance of hexokinase from baker's yeast rises. Variation of luminescence parameters shows structural modification of enzyme upon complex formation. On the basis of comparison the photoinactivation data and hexokinase photolysis conclusion has been drawn that the tryptophanyl residues are not directly involved in the enzyme active site, although play an important role in supporting the specific structure.

[Effect of the state of the lipid phase of the membrane on the effectiveness of photochemical modification of erythrocyte acetylcholinesterase]. / Vliianie sostoianiia lipidnoi fazymembrany na éffektivnost' fotokhimicheskoi modifikatsii éritrotsitarnoi atsetilkholinésterazy.

Modification of the lipid phase structure of the erythrocyte membrane by phospholipases A2, C and D as well as the partial depletion of cholesterol was shown to be accompanied by the change of the acetylcholinesterase (AChE) UV-sensitivity. The ability of UV-light to change the catalytic properties (Km) of the membrane-bound AChE not observed for free AChE (constant value of Km) and known as the phenomenon of photochemical allotopy, is retained in the cholesterol depleted membranes and disappears after an enzymatic treatment of the membranes by phospholipases. The possible non-photochemical influence of the membrane lipid phase in response to UV-damage of membrane-bound AChE is discussed.

[Influence of UV-light on erythrocyte membrane structure and catalytic behaviour of membrane acetylcholine esterase]. / Vliianie UF-sveta na strukturu membran zritrotsitov i na kataliticheskie svoistva membrannoi atsetilkholinzsterazy.

UV-light is shown to induce the structural transitions in the erythrocyte membrane described by S-shape curves in plots of the structural response versus the irradiation dose. In contrast to the free acetylcholine esterase (AChE) UV-light acts on the membrane enzyme as a mixed inhibitor (simultaneous change in Vmax and Km). The modification of the environment structure of residual enzyme is suggested to be the main reason of this phenomenon. The effect is under the control of membrane integrity and disappears after its desintegration. Membrane AChE treated ultrasonically both prior to and after irradiation is inactivated without a Km change. The data obtained show the influence of erythrocyte membrane structure on the catalytic behaviour of membrane-bound AChE.

[Influence of heavy water (D20) on the conformation and UV-sensitivity of proteins]. / Vliianie tiazheloi vody (D20) na konformatsiiu i UF-chvstvitel'nost' belkov.

The heavy water (D2O) has been shown to induce the conformational transitions in trypsin, chymotrypsin and pepsin. The transfer of proteins from H2O into D2O results a change in their sensitivity to UV-light. An increase in sensitivity to the irradiation at 248 nm and a decrease in sensitivity to the irradiation at 280 nm were observed. The quantum yield of chromophore photolysis (for cystyne and tryptophan) is correspondingly changed. However, although the quantum yield of sensitized reduction of cystine by solvated electrons photochemically ejected from the aromatic acid residues during irradiation at 280 nm increases instead of a rise a drop in the quantum yield of protein inactivation is registered. The data obtained are discussed in terms of importance of solvated shell for conformational stability of proteins. The solvated electrons are suggested to be transfered mainly to nonessential disulfide bridges within trypsin molecule. Rupture of these bonds does not result in trypsin inactivation.

[The structural membrane control of the reduction of exogenous quinones catalysed by mitochondrial 2-methyl-1,4-naphthoquinone reductase]. / Strukturno-membrannyi kontrol' vosstanovleniia ekzogennykh khinonov, kataliziruemogo mitokhondrial'noi 2-metil-1,4-naftokhinon-reduktazoi.

The catalytic parameters of mitochondrial 2-methyl-1, 4-naphthoquinone-reductase were studied. Maximal velocity and Michaelis constant of quinone-reducing reaction were shown to depend strongly on the functional state of mitochondria (I, II and IV Chance's states). The changes in catalytic parameters were found for four quinone substrates essentially varying in their structures. For the same conditions the Michaelis constant of isolated enzyme remains unchanged. The data obtained indicate the possibility to control the activity and substrate specificity by structural rearrangements within the biological membrane.

[Interaction of bromophenol blue with serum albumin: criteria for using dyes for assessing the state and quantity of protein]. / Vzaimodeistvie bromfenolovogo sinego s syvorotochnym al'buminom: kriterii ispol'zovaniia krasiteliia dliia otsenki sostoianiia i kolichestva belka.

The optical properties of the complexes of the pH-dependent dye bromophenol blue (BPB) with human serum albumin were investigated by the spectrophotometric method. The solvatochromic longwave displacement of bound BPB-2 absorption and BPB-1/BPB-2 redistribution were shown to form the optical signal of complexes. Because of the distortion of the bound BPB-2 signal its quantity was determined as delta A630 = A630 - A660 and the use of lambda max as structural parameter was limited to low pH less than or equal to 3. The conclusion was made that BPB is inapplicable as a structural probe on account of low structural dependence of delta A630 and pH-limitation of lambda max used. The maximal absorption delta Amax = Amax - A660 and its structural independence were obtained in the region of 70-100% occupation of the dye-binding centers of the protein. It is the optimal conditions for the quantitative determination of protein. After maximal dye binding (15-16 molecules of BPB per 1 molecule of albumin) the aggregation and precipitation of the complexes occurred.

[cGMP-binding centers in photoreceptor membranes]. / cGMP-sviazyvaiushchie tsentry v fotoretseptornykh membranakh.

The binding of cGMP by structural components of bovine rod outer segments was studied. The discs and plasma membranes were shown to contain two types of the specific binding sites for cGMP which are distinct from cyclic GMP phosphodiesterase. The sites have a "high" and "low" (Kd = 0.1 divided by 0.35 and 1.5 divided by 2.0 X 10(-6) M respectively) affinity for cGMP. They belong to membraneous integral proteins presumably associated with phospholipids. Their affinity for cGMP is controlled by GTP and calmodulin.