Ion channel enzyme in an oscillating electric field.

To explain the electrical activation of several membrane ATPases, an electroconformational coupling (ECC) model has previously been proposed. The model explained many features of experimental data but failed to reproduce a window of the field intensity for the stimulated activity. It is shown here that if the affinities of the ion for the two conformational states of the transporter (one with binding site on the left side and the other on the right side of the membrane) are dependent on the electric field, the field-dependent transport can exhibit the observed window. The transporter may be described as a channel enzyme which opens to one side of the membrane at a time. It retains the energy-transducing ability of the earlier ECC models. Analysis of the channel enzyme in terms of the Michaelis-Menten kinetics has been done. The model reproduced the amplitude window for the electric field-induced cation pumping by (Na,K)-ATPase.

Mammalian phosphorylating ion-motive ATPases.

The pumps discussed in this review are three members of the phosphorylating class of ion transport ATPases. They are the Na(+)-K(+)-, Ca(2+)- and H(+)-K(+)-ATPases. Recent work on their topology, possible transport mechanisms, ion-binding sites and role of the different subunits found for the Na(+)-K(+)- and H(+)-K(+)-ATPases is presented, with a suggestion of a unifying 10-membrane segment model for the catalytic subunit of this class of enzyme.

Reconstitution of a chloride-translocating ATPase from Aplysia californica gut.

Basolateral membranes of Aplysia foregut epithelia contain both a Cl(-)-stimulated ATPase activity and an ATP-dependent Cl- transport. The protein responsible for both of these biochemical activities (Cl- pump) can be solubilized and reconstituted into liposomes with the aid of the detergent digitonin. Proteoliposomal Cl- pump activity was inhibited by vanadate.

Effects of microgravity on liposome-reconstituted cardiac gap junction channeling activity.

Effects of microgravity on cardiac gap junction channeling activity were investigated aboard NASA zero-gravity aircraft. Liposome-reconstituted gap junctions were assayed for channel function during free-fall, and the data were compared with channeling at 1 g. Control experiments tested for 0 g effects on the structural stability of liposomes, and on the enzyme-substrate signalling system of the assay. The results demonstrate that short periods of microgravity do not perturb reconstituted cardiac gap junction channeling activity.

Highly cooperative feedback control of retinal rod guanylate cyclase by calcium ions.

Visual excitation in retinal rod cells is mediated by a cascade that leads to the amplified hydrolysis of cyclic GMP (cGMP) and the consequent closure of cGMP-activated cation-specific channels in the plasma membrane. Recovery of the dark state requires the resynthesis of cGMP, which is catalysed by guanylate cyclase, an axoneme-associated enzyme. The lowering of the cytosolic calcium concentration (Cai) following illumination is thought to be important in stimulating cyclase activity. This hypothesis is supported by the finding that the cGMP content of rod outer segments increases several-fold when Cai is lowered to less than 10 nM. It is evident that cGMP and Cai levels are reciprocally controlled by negative feedback. Guanylate cyclase from toad ROS is strongly stimulated when the calcium level is lowered from 10 microM to 10 nM, but only if they are excited by light. We show here that the guanylate cyclase activity of unilluminated bovine rod outer segments increases markedly (5 to 20-fold) when the calcium level is lowered from 200 nM to 50 nM. This steep dependence of guanylate cyclase activity on the calcium level in the physiological range has a Hill coefficient of 3.9. Stimulation at low calcium levels is mediated by a protein that can be released from the outer segment membranes by washing with a low salt buffer. Calcium sensitivity is partially restored by adding the soluble extract back to the washed membranes. The highly cooperative activation of guanylate cyclase by the light-induced lowering of Cai is likely to be a key event in restoring the dark current after excitation.

[The channel function of the hydrophobic fragment of Ca ATPase in the sarcoplasmic reticulum of rabbit skeletal muscles in the early period after x-ray radiation exposure]. / Kanal'naia funktsiia gidrofobnogo fragmenta Ca-ATFazy sarkoplazmaticheskogo retikuluma skeletnykh myshts krolika v rannii period vozdeistviia rentgenovskim izlucheniem.

At early times (1 and 24 h) following local single exposure of rabbit hindlimbs to 0.21 C/kg X-radiation Ca ATPase was isolated from sarcoplasmic reticulum of skeletal muscles and inserted in liposomes the treatment of which with trypsin resulted in the formation of proteoliposomes with a Ca ATPase hydrophobic fragment (Mr 45-50 kDa). It was shown that X-radiation increased the permeability of univalent and bivalent cations through a channel formed by the hydrophobic fragment of Ca ATPase and diminished ion selectivity of this channel.

Comparative effects of vinpocetine and 8-Br-cyclic GMP on the contraction and 45Ca-fluxes in the rabbit aorta.

Vinpocetine is a highly specific inhibitor of calmodulin-dependent phosphodiesterase (CaM-PDE) with an IC50 of 19 microM and produces a significant accumulation of cyclic GMP but not cyclic AMP in rabbit aorta. In isolated rabbit aortic strips, vinpocetine (0.01 and 0.1 mM) inhibited the contraction and 45Ca uptake due to both phenylephrine (1 microM) and KCl (40 mM), whereas 8-Br-cyclic GMP (0.1-1mM) selectively impaired phenylephrine-induced responses. Furthermore, the KCl-stimulated 45Ca efflux in normal Ca2+ buffer, which reflects elevated cytosolic Ca2+, was greatly diminished by vinpocetine but not by 8-Br-cyclic GMP. However, phenylephrine-induced 45Ca efflux and contraction in Ca2+-free buffer, which reflect Ca2+ release from intracellular sites, were similarly inhibited by both vinpocetine and 8-Br-cyclic GMP. The results suggest that vinpocetine may effect vasodilatation through blockade of the slow channel and selective inhibition of CaM-PDE in the vascular smooth muscle.

Phorbol ester translocation of protein kinase C in guinea-pig synaptosomes and the potentiation of calcium-dependent glutamate release.

The distribution of protein kinase C activity and specific phorbol ester binding sites between soluble and particulate fractions of isolated guinea-pig cerebral cortical synaptosomes is examined following preincubation with phorbol esters. Half-maximal decrease in cytosolic activity requires 10 nM 4 beta-phorbol myristoyl acetate. Specific [3H]phorbol dibutyrate binding sites are translocated from cytoplasmic to particulate fractions in parallel with protein kinase C activity. Depolarization of the plasma membrane by 30 mM KCl does not cause translocation of protein kinase C. 1 microM 4 beta-phorbol myristoyl acetate and 1 microM 4 beta-phorbol didecanoate (but not 1 microM 4 alpha-phorbol didecanoate) enhance the release of glutamate from synaptosomes partially depolarized by 10 mM KCl; however, 4 beta-phorbol myristoyl acetate is ineffective at 20 nM. 1 microM 4 beta-phorbol myristoyl acetate slightly increases the cytosolic free Ca2+ concentration of polarized synaptosomes, but not that following partial depolarization. 4 beta-Phorbol myristoyl acetate causes a concentration-dependent increase in the Ca2+-dependent glutamate release induced by sub-optimal ionomycin concentrations, but is without effect on the release induced by maximal ionomycin. It is concluded that phorbol esters stereospecifically enhance the Ca2+-sensitivity of glutamate release, but that higher concentrations may be required than for protein kinase C translocation in the same preparation. Instead the enhancement may be related to the rapid inactivation of protein kinase C which occurs with phorbol esters.

Structural properties of the proton translocating complex of the clathrin-coated vesicle.

The clathrin-coated vesicle proton pump is a representative member of the new class of endomembrane proton ATPases that share an inhibitor profile which distinguishes them from classic F1F0 and E1E2-type proton pumps. The coated vesicle proton pump is a large (530 kDa) heteroligomer composed of eight polypeptides with molecular masses of 116, 70, 58, 40, 38, 34, 33 and 17 kDa. The 200-fold purified enzyme catalyses ATP-generated proton pumping when reconstituted in liposomes composed of pure lipids. Subunit function has been determined by partial reaction analysis of subunit and subcomplex activities. The isolated 17 kDa subunit, when co-reconstituted with bacteriorhodopsin, forms a dicyclohexylcarbodiimide-inhibitable proton channel. Selective removal of the 116 kDa subunit transforms the proton ATPase from a Mg2+-activatable to a Ca2+-activatable ATPase. Subsequent dissociation and reconstitution of subunits reveals that the 70, 58, 40 and 33 kDa components are required, in composite, to form a functional ATP-hydrolytic core, and that no single subunit or subcomplex deficient in these subunits can catalyse ATP hydrolysis.

Regulation of the cardiac calcium channel by protein phosphatases.

The calcium current (ICa) through the L-type channel in cardiac ventricular cells is enhanced by phosphorylation of a channel protein [Kameyama, M., Hofmann, F. & Trautwein, W. (1985) Pflügers Arch. Eur. J. Physiol. 405, 285-293]. We investigated the possible contribution of the 'catalytic subunits' of protein phosphatase 1 and 2A in the down-regulation of the cardiac calcium channel. Single guinea-pig ventricular myocytes were voltage clamped and the following results were obtained. (1) Intracellular perfusion of the myocyte with the catalytic subunits of protein phosphatase 1 (2 microM) as well as 2A (2.3 microM) completely abolished the increase of ICa induced by isoprenaline (0.05 microM) but did not decrease the basal level of ICa. Alkaline and acid phosphatases were without detectable effect. (2) Cell dialysis with the modulator of protein phosphatase 1 (inhibitor-2) under control conditions (without addition of isoprenaline) caused a slow significant increase of ICa. (3) The time course for the wash-out of the isoprenaline effect was considerably prolonged in the presence of high concentrations of inhibitor-2. (4) Perfusion of the myocyte under basal conditions with adenosine 5'-[gamma-thio]triphosphate led to a slow increase of ICa. Additional superfusion of the cell with a threshold concentration of isoprenaline (0.01 microM) resulted in a rapid increase of ICa which could not be washed out during at least 10 min. From these results we make the following conclusions. (1) The calcium channel from guinea-pig myocytes is regulated by phosphorylation-dephosphorylation. (2) The catalytic subunits of the protein phosphatases 1 as well as 2A, purified from rabbit skeletal muscle, catalyse the down-regulation of the channel. (3) Indirect evidence suggests that endogenous protein phosphatase 1 contributes only partially to the dephosphorylation of the calcium channel in the intact myocyte.

Partial characterization of two (Ca+2 + Mg+2)-dependent ATPase activities from bovine brain synaptic membrane homogenates.

Synaptic plasma membranes isolated from bovine brain exhibited a low and high affinity (Ca+2 + Mg+2)-dependent ATPase as evidenced by kinetic constants for ATP. One activity which hydrolyzed ATP maximally at pH 7.4 and 7.8 exhibited an 8-fold higher affinity when compared to the second or lower affinity activity which hydrolyzed ATP maximally at pH 7.0. Both activities exhibited submicromolar kinetic constants for Ca+2 (Km = 0.24 micromolar). Km values for magnesium differed significantly; the lower affinity activity being approximately 6.5 times higher (120 microM) than that observed for the high affinity activity (18 microM). Vmax values obtained under optimal assay conditions (low and high) were 110-135 and 43-55 nmol/min/mg protein, respectively. Both activities were KCN, NaN3 and ruthenium red insensitive. Only slight inhibition was observed in the presence of rotenone and oligomycin. Although both activities were observed to be trifluoperazine sensitive, they differed significantly with regard to other parameters. Na+1 and NH4+1 ions preferentially inhibited the low affinity activity greater than 90%. Cs+1 ions completely inhibited the high affinity activity while reducing the low affinity only 22%. Li+1, Al+3 and Mn+2 significantly inhibited the high affinity activity while reducing the low activity only moderately. Both the low and high activity were inhibited by vanadate with half maximum inhibition occurring at 2 and 5 microM, respectively indicating the plasma membrane origin of these activities. Thermal denaturation studies indicated the high affinity activity to be stable for 2 minutes at 45 degrees C after which 50% of the activity is lost at 2.5 minutes.(ABSTRACT TRUNCATED AT 250 WORDS)

[Detection by thermal denaturation of damages to the Na pump in the myocardial sarcolemma in stress and the role of lipid peroxidation in this process]. / Vyiavlenie s pomoshch'iu termodenaturatsii povrezhdenii Na-nasosa sarkolemmy miokarda pri stresse i rol' perekisnogo okisleniia lipidov v étom protsesse.

The determination of ATP-hydrolytic activity of Na pump does not always reveal the enzyme damage in vivo. The method assessing Na, K-ATPase molecular conformational stability in the rat heart sarcolemma based on thermal denaturation is suggested. After a prolonged emotional-painful stress (EPS) the activity of Na, K-ATPase dropped by 20%, as the rate of its thermal denaturation in the range of 50-60 degrees C increased 2-3-fold. Thermodynamic calculations have demonstrated a decrease in Ea, delta H and delta S* of Na, K-ATPase thermal denaturation process after EPS. An analogous enzyme damage was found after the activation of lipid peroxidation in sarcolemma membrane suspension. These results imply that essential changes in intra- and supra-molecular properties of Na, K-ATPase under EPS may be detected by thermal denaturation. Lipid peroxidation is a most likely reason for EPS-induced Na pump damage.

Evidence for a high and specific concentration of (Na+,K+)ATPase in the plasma membrane of the osteoclast.

During bone resorption, the osteoclast actively acidifies a limited extracellular compartment. We hypothesized that, like other cells engaged in ion transport and proton translocation, the osteoclast's membrane might be highly enriched in sodium pumps. Using monoclonal antibodies to both the alpha and the beta subunits, immunoblot analysis, and [3H]ouabain binding, we have demonstrated that the osteoclast plasma membrane is both highly and specifically enriched in (Na+,K+)ATPase, compared with other bone cells, monocytes, macrophages, and other blood and bone marrow cells. The density of binding sites on the osteoclast is equivalent to that of kidney tubule cells. This observation is consistent with the hypothesis that the (Na+,K+)ATPase plays a role in the mechanism of bone resorption, possibly coupled with secondary active calcium and/or proton transport. Monoclonal antibodies against the (Na+,K+)ATPase can therefore be used as specific markers for the osteoclast in bone and bone marrow preparations.

Sodium pathway markers in normal and kindled frog brains.

The present report evaluates Na,K-ATPase activity as well as Na channel levels in the frog telencephalon after kindling, i.e. the acquisition of an epileptic focus through localized low-voltage electrical stimulation of one hemisphere. K-dependent phosphatase activity and binding of tritiated ouabain were measured, revealing no change in Na,K-ATPase activity 14 h after the last seizure. Na channels were measured by binding assays using a tritiated ethylenediamine tetrodotoxin derivative. Na channels were reduced in kindled brain as compared to controls.

Is there sufficient experimental evidence to consider the mitochondrial cytochrome bc1 complex a proton pump? Probably no.

The electron flow through the cytochrome bc1 complex of the mitochondrial respiratory chain is accompanied by vectorial proton translocation, though the mechanism of the latter phenomenon has not yet been clarified. Several proposed hypotheses are briefly presented and discussed here. Recently, a number of papers have appeared claiming the existence of a proton pump in the enzyme mainly on the basis of the interaction of the complex with N,N'-dicyclohexylcarbodiimide. These data are reviewed here with the aim of showing their ability to fit multiple interpretations. This together with some other arguments leads to the conclusion that a proton pump in the mitochondrial bc1 complex has not yet been demonstrated.

Effects of digitalis on cell biochemistry: sodium pump inhibition.

It is now generally agreed that Na+-K+ adenosine triphosphatase (ATPase), a transport enzyme derived from the sarcolemmal sodium pump, is the primary site at which digitalis exerts its effects on the myocardial cell. Inhibition of the ability of this ion transport enzyme to catalyze Na+ efflux from the cell in exchange for K+ leads to both the therapeutic and toxic effects of the cardiac glycosides. The mechanism by which digitalis inhibits the sodium pump has been established in studies of Na+-K+ ATPase which show that the ability of cardiac glycosides to inhibit adenosine triphosphate (ATP)-supported transport of Na+ is reduced in the presence of elevated levels of K+. These studies explain the ability of hypokalemia to potentiate the effects of cardiac glycosides on the heart, and of high K+ concentrations to overcome the inhibition of sodium pump activity by the cardiac glycosides. Recent demonstrations that the positive inotropic effect of the cardiac glycosides is correlated with an increased intracellular Na+ provide strong evidence that these effects of digitalis to impair sodium efflux are responsible for the increased myocardial contractility caused by digitalis.

The relationship between channel size and the number of C9 molecules in the C5b-9 complex.

We have recently shown by dose-response analyses with resealed erythrocyte ghosts that the channel formed by complement is a monomer of C5b-9 of the composition C5b61C71C81C9n, in which n = 1 for channels permitting passage of sucrose (0.9 nm molecular diameter) and n = 2 for channels allowing transit of inulin (3 nm molecular diameter) (1). We have now continued these experiments and expanded them by including ribonuclease A (molecular diameter, 3.8 nm) as a marker to assess whether additional C9 molecules enlarge the functional C5b-9 channel. Our results show that formation of C5b-9 channels displays one-hit characteristics with respect to C5b6 when tested by transmembrane passage of inulin or ribonuclease A. By contrast, analysis of dose-response curves of C9 indicate that n = 2-3 for channels allowing transit of inulin and n = 4 for channels allowing transit of ribonuclease A. We have also performed sieving experiments with ghosts carrying C5b-7 and containing two small markers, inositol and sucrose. Dose-response curves for C8 were performed in the presence of excess C9 to ensure conversion of all C5b-8 to C5b-9 channels. The results indicate that small channels (approximately 0.8 nm effective diameter) are not formed at high C9 multiplicity, thus confirming the results obtained with the larger markers, i.e., increase of C9 input leads to formation of larger channels.