Water transporting properties of hepatocyte basolateral and canalicular plasma membrane domains

Previous work from our laboratory supports an important role for aquaporins (AQPs), a family of water channel proteins, in bile secretion by hepatocytes. To further define the pathways and molecular mechanisms for water movement across hepatocytes, we directly assessed osmotic water permeability (Pf) and activation energy (Ea) in highly purified, rat hepatocytes basolateral membrane vesicles (BLMV) and canalicular membrane (CMV) vesicles by measuring scattered light intensity using stopped-flow spectrophotometry. The time course of scattered light for BLMV and CMV fit well to a single-exponential function. In BLMV, Pf was 108 +/- 4 mum.s-1 (25 degrees C) with an Ea of 7.7 kcal/mol; in CMV, Pf was 86 +/- 5 mum.s-1 (25 degrees C) with an Ea of 8.0 kcal/mol. The AQP blocker, dimethyl sulfoxide, significantly inhibited the Pf of both basolateral (81 +/- 4 mum.s-1; -25%) and canalicular (59 +/- 4 mum.s-1; -30%) membrane vesicles. When CMV were isolated from hepatocytes treated with dibutyryl cAMP, a double-exponential fit was needed, implying two functionally different vesicle populations; one population had Pf and Ea values similar to those of CMV from untreated hepatocytes, but the other population had a very high Pf (655 +/- 135 mum.s-1, 25 degrees C) and very low Ea (2.8 kcal/mol). Dimethyl sulfoxide completely inhibited the high Pf value in this second vesicle population. In contrast, Pf and Ea of BLMV were unaltered by cAMP treatment of hepatocytes. Our results are consistent with the presence of both lipid- and AQP-mediated pathways for basolateral and canalicular water movement across the hepatocyte plasma membrane barrier. Our data also suggest that the hepatocyte canalicular membrane domain is rate-limiting for transcellular water transport and that this domain becomes more permeable to water when hepatocytes are exposed to a choleretic agonist, presumably by insertion of AQP molecules. These data suggest a molecular mechanism for the efficient coupling of osmotically active solutes and water transport during canalicular bile formation.     

A study of protoplasmic streaming in Nitella by laser Doppler spectroscopy.

Laser light scattered from particles in the streaming protoplasm of a living cell is shifted in frequency by the Doppler effect. The spectrum of the scattered light can be measured and interpreted to infer details of the velocity distribution in the protoplasm. We have developed this approach to study the protoplasmic streaming in the fresh-water alga Nitella. Our results indicate a characteristic flow pattern to which diffusion makes a negligible contribution. No difference in the velocity of particles of different size is indicated. The streaming velocity linearly with temperature with a supraoptimal temperature of 34 degrees C, and the velocity distribution becomes narrower at high temperatures. The protoplasmic streaming can be inhibited by laser light, and this effect has been used to study the photoresponse of the algae. Using beam diameters of about 50 mum, we have shown that the inhibition is very local, becoming minimal at a displacement of about 200 mum in the upstream direction and 400 mum in the downstream direction. Prolonged exposure produces a bleached area free of chloroplasts, which is three orders of magnitude less sensitive to photoinhibition.     

Translational and Rotational Diffusion Constants of Tobacco Mosaic Virus from Rayleigh Linewidths

The translational and rotational diffusion constants of tobacco mosaic virus (TMV) have been determined from homodyne and heterodyne measurements of the spectrum of laser light scattered from dilute aqueous solutions of TMV. Our results for the translational and rotational constants respectively, reduced to 20 degrees C, are: D(T) = 0.280 +/- 0.006 x 10(-7) cm(2)/sec, and D(R) = 320 +/- 18 sec(-1). We include a theoretical derivation of the spectrum of light scattered from rod-shaped molecules which reproduces results obtained previously by Pecora, but which is specialized at the outset to the problem of dilute solutions so that simple single-particle correlation functions may be utilized. An analysis of the photocurrent spectrum for both the homodyne and heterodyne detection schemes is given. Various data reduction schemes utilized in the analysis of our spectra are described in some detail, and our results are compared with values of the diffusion constants obtained from other experiments.     

Effects of MgATP and MgADP on the cross-bridge kinetics of rabbit soleus slow-twitch muscle fibers.

The elementary steps surrounding the nucleotide binding step in the cross-bridge cycle were investigated with sinusoidal analysis in rabbit soleus slow-twitch muscle fibers. The single-fiber preparations were activated at pCa 4.40, ionic strength 180 mM, 20 degrees C, and the effects of MgATP (S) and MgADP (D) concentrations on three exponential processes B, C, and D were studied. Our results demonstrate that all apparent (measured) rate constants increased and saturated hyperbolically as the MgATP concentration was increased. These results are consistent with the following cross-bridge scheme: [cross-bridge scheme: see text] where A = actin, M = myosin, S = MgATP, and D = MgADP. AM+S is a collision complex, and AM*S is its isomerized form. From our studies, we obtained K0 = 18 +/- 4 mM-1 (MgADP association constant, N = 7, average +/- sem), K1a = 1.2 +/- 0.3 mM-1 (MgATP association constant, N = 8 hereafter), k1b = 90 +/- 20 s-1 (rate constant of ATP isomerization), k-1b = 100 +/- 9 s-1 (rate constant of reverse isomerization), K1b = 1.0 +/- 0.2 (equilibrium constant of isomerization), k2 = 21 +/- 3 s-1 (rate constant of cross-bridge detachment), k-2 = 14.1 +/- 1.0 s-1 (rate constant of reversal of detachment), and K2 = 1.6 +/- 0.3 (equilibrium constant of detachment). K0 is 8 times and K1a is 2.2 times those in rabbit psoas, indicating that nucleotides bind to cross-bridges more tightly in soleus slow-twitch muscle fibers than in psoas fast-twitch muscle fibers. These results indicate that cross-bridges of slow-twitch fibers are more resistant to ATP depletion than those of fast-twitch fibers. The rate constants of ATP isomerization and cross-bridge detachment steps are, in general, one-tenth to one-thirtieth of those in psoas.     

A kinetic study of the kinesin ATPase.

The mechanism of kinesin ATPase has been investigated by transient state kinetic analysis. The results satisfy the scheme [formula: see text] where T, D, and P(i) refer to nucleotide tri- and diphosphate and inorganic phosphate, respectively. The nucleotide-binding steps were measured by the fluorescence enhancement of mant (2'-(3')-O-(N-methylanthraniloyl)-ATP and mant-ADP. The initial rapid equilibrium binding steps (1) and (6) are followed by isomerizations (k2 = 170 +/- 30 s-1 at 20 degrees C, k-5 greater than 100 s-1). The increase in fluorescence is 20-25% larger for K.T** than K.D*. The rate constant of the hydrolysis step k3 is 6-7 s-1. The fluorescence decreases after formation of K.T** at a rate of 7-10 s-1. This change could occur in step 3 or in step 4 if k4 much greater than k3. The value of k4 is larger than 0.1 s-1. The steady state rate is 0.003 s-1 which agrees with the rate of ADP dissociation (k5). Step 5 is rate limiting in the scheme in agreement with the conclusion of Hackney (Hackney, D. D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6314-6318) that ADP dissociation is the rate-limiting step.     

Effects of exogenous proteins on cytoplasmic streaming in perfused Chara cells

Cytoplasmic streaming in characean algae is thought to be generated by interaction between subcortical actin bundles and endoplasmic myosin. Most of the existing evidence supporting this hypothesis is of a structural rather than functional nature. To obtain evidence bearing on the possible function of actin and myosin in streaming, we used perfusion techniques to introduce a number of contractile and related proteins into the cytoplasm of streaming Chara cells. Exogenous actin added at concentrations as low as 0.1 mg/ml is a potent inhibitor of streaming. Deoxyribonuclease I (DNase I), an inhibitor of amoeboid movement and fast axonal transport, does not inhibit streaming in Chara. Fluorescein-DNase I stains stress cables and microfilaments in mammalian cells but does not bind to Chara actin bundles, thus suggesting that the lack of effect on streaming is due to a surprising lack of DNase I affinity for Chara actin bundles. Heavy meromyosin (HMM) does not inhibit streaming, but fluorescein-HMM (FL-HMM), having a partially disabled EDTA ATPase, does. Quantitative fluorescence micrography provides evidence that inhibition of streaming by FL-HMM may be due to a tendency for FL-HMM to remain bound to Chara actin bundles even in the presence of MgATP. Perfusion with various control proteins, including tubulin, ovalbumin, bovine serum albumin, and irrelevant antibodies, does not inhibit streaming. These results support the hypothesis that actin and myosin function to generate cytoplasmic streaming in Chara.     

Characterization of a chimeric plasminogen activator consisting of a single-chain Fv fragment derived from a fibrin fragment D-dimer-specific antibody and a truncated single-chain urokinase

An Mr 57,000 single-chain chimeric plasminogen activator, K12G0S32, consisting of a variable region fragment (Fv) derived from the fibrin fragment D-dimer-specific monoclonal antibody MA-15C5 and of a 33-kDa (amino acids Ala132 to Leu411) recombinant single-chain urokinase-type plasminogen activator (rscu-PA-33k) was studied. K12G0S32, secreted by infected Spodoptera frugiperda insect cells at a rate of 1.5 micrograms/10(6) cells/48 h, was purified to homogeneity by ion-exchange chromatography and gel filtration. It was obtained essentially as a single-chain molecule with a Ka = 5.5 x 10(9) M-1 for immobilized fragment D-dimer, similar to that of MA-15C5. The specific activity of both its single-chain and two-chain forms on fibrin plates was 100,000 IU/mg of urokinase-type plasminogen activator (u-PA) equivalent. Activation of plasminogen by two-chain K12G0S32 obeyed Michaelis-Menten kinetics with Km = 2.9 +/- 0.6 microM and a k2 = 3.7 +/- 0.6 s-1 (mean +/- S.D.; n = 3), as compared to Km = 12 microM and k2 = 4.8 s-1 for rtcu-PA-32k (recombinant low Mr two-chain u-PA consisting of amino acids Leu144 to Leu411). Single-chain K12G0S32 induced a dose- and time-dependent lysis of a 125I-fibrin-labeled human plasma clot immersed in citrated human plasma; 50% lysis in 2 h was obtained with 0.70 +/- 0.07 micrograms/ml (mean +/- S.D.; n = 5), as compared with 8.8 +/- 0.1 micrograms/ml for rscu-PA-32k (recombinant low Mr single-chain u-PA consisting of amino acids Leu144 to Leu411) (mean +/- S.D.; n = 3). With two-chain K12G0S32, 50% clot lysis in 2 h required 0.25 +/- 0.03 micrograms/ml (mean +/- S.D.; n = 3), as compared with only 0.62 +/- 0.04 micrograms/ml (mean +/- S.D.; n = 2) for rtcu-PA-32k. These results indicate that low Mr single-chain u-PA can be targeted to a fibrin clot with a single-chain Fv fragment of a fibrin-specific antibody, resulting in a 13-fold increase of the fibrinolytic potency of the single-chain form and a 2.5-fold increase of the potency of the two-chain form.     

Rapid relaxation of single frog skeletal muscle fibres following laser flash photolysis of the caged calcium chelator, diazo-2

Diazo-2 is a calcium chelator based on BAPTA [(1989) J. Biol. Chem., in press], whose electron withdrawing diazoacetyl group may be rapidly (2000 s-1) converted photochemically to an electron donating carboxymethyl group by exposure to near ultraviolet light, producing an increase in its calcium affinity (Kd changes from 2.2 microM to 0.073 microM) without steric modification of the metal binding site. Photolysis of a 2 mM solution of this compound with a brief flash of light from a frequency-doubled ruby laser (347 nm) caused single skinned muscle fibres from the semitendinosus muscle of the frog Rana temporaria to relax with a mean half-time of 60.4 +/- 5 ms (range 30-100 ms, n = 15) at 12 degrees C, which is faster than the relaxation observed in intact muscles (half-time 133 ms at 14 degrees C [(1986) J. Mol. Biol. 188, 325-342]) and similar to the rate of the fast phase of tension decay in intact single fibres (20 s-1 at 10 degrees C [(1982) J. Physiol. 329, 1-20]).     

The kinetics of glucose transport in human red blood cells

A quenched-flow apparatus and a newly developed automated syringe system have been used to measure initial rates of D-[14C]glucose transport into human red blood cells at temperatures ranging from 0 degrees to 53 degrees C. The Haldane relationship is found to be obeyed satisfactorily at both 0 and 20 degrees C, but Arrhenius plots of maximum D-[14C]glucose transport rates are non-linear under conditions of both equilibrium exchange and zero trans influx. Fitting of the data by non-linear regression to the conventional model for glucose transport gives values at 0 degrees C of 0.726 +/- 0.0498 s-1 and 12.1 +/- 0.98 s-1 for the rate constants governing outward and inward movements of the unloaded carrier molecule and 90.3 +/- 3.47 s-1 and 1113 +/- 494 s-1 for outward and inward movements of the carrier-glucose complex. Activation energies for these four rate constants are respectively 173 +/- 3.10, 127 +/- 4.78, 88.0 +/- 6.17 and 31.7 +/- 5.11 kJ X mol-1. These parameters indicate that at low temperatures the marked asymmetry of the transport mechanism arises mainly from the high proportion of inward-facing carriers and carrier-glucose complexes, and that there is a relatively small difference between the affinities of the carrier for glucose in the inward and outward-facing conformations. At high (physiological) temperatures the carrier is fairly evenly distributed between outward- and inward-facing conformations and the affinity for glucose is about 2.5-times greater outside than inside.     

Fluorescence studies on modes of cytochalasin B and phallotoxin action on cytoplasmic streaming in Chara

Various investigations have suggested that cytoplasmic streaming in characean algae is driven by interaction between subcortical actin bundles and endoplasmic myosin. To further test this hypothesis, we have perfused cytotoxic actin-binding drugs and fluorescent actin labels into the cytoplasm of streaming Chara cells. Confirming earlier work, we find that cytochalasin B (CB) reversibly inhibits streaming. In direct contrast to earlier investigators, who have found phalloidin to be a potent inhibitor of movement in amoeba, slime mold, and fibroblastic cells, we find that phalloidin does not inhibit streaming in Chara but does modify the inhibitory effect of CB. Use of two fluorescent actin probes, fluorescein, isothiocyanate-heavy meromyosin (FITC-HMM) and nitrobenzoxadiazole-phallacidin (NBD-Ph), has permitted visualization of the effects of CB and phalloidin on the actin bundles. FITC-HMM labeling in perfused but nonstreaming cells has revealed a previously unobserved alteration of the actin bundles by CB. Phalloidin alone does not perceptibly alter the actin bundles but does block the alteration by CB if applied as a pretreatment, NBD-Ph perfused into the cytoplasm of streaming cells stains actin bundles without inhibiting streaming. NBD-Ph staining of actin bundles is not initially observed in cells inhibited by CB but does appear simultaneously with the recovery of streaming as CB leaks from the cells. The observations reported here are consistent with the established effects of phallotoxins and CB on actin in vitro and support the hypothesis that streaming is generated by actin-myosin interactions.     

Kinetics of carboxymethylation of histidine hydantoin.

The reaction of the imidazole group of histidine hydantoin with bromoacetate was studied as a model for carboxymethylation of histidine residues in proteins. pK values of 6.4 and 9.1 (25 degrees C) and apparent heats of ionization of 7.8 and 8.7 kcal/mol were determined for the imidazole and hydantoin rings, respectively. At pH values corresponding to the isoelectric points for histidine hydantoin, the rates of carboxymethylation at 12, 25, 37, and 50 degrees C were determined; the modified hydantoins were hydrolyzed to the corresponding histidine derivatives for quantitative amino acid analysis. At pH 7.72 and 25 degrees C, the imidazole tele-N was alkylated (k = 3.9 X 10(-5) M-1 s-1) twice as fast as the pros-N. The monocarboxymethyl derivatives were carboxymethylated at the same rate at the pros-N (k = 2.1 X 10(-5) M-1 s-1) but 3 times faster at the tele-N (k = 11 X 10(-5) M-1 s-1). The enthalpies of activation determined for carboxymethylation of the imidazole ring and its monocarboxymethyl derivatives were similar (15.9 +/- 0.7 kcal/mol). delta S for the four carboxymethylations was -25 +/- 2 eu. The electrostatic component of delta S (delta S es) was calculated from the influence of the dielectric constant on the reaction rate at 25 degrees C. delta S es was slightly negative (-4 +/- 1 eu) for mono- or dicarboxymethylations, indicating some charge separation in the transition state. The nonelectrostatic entropy of activation was -21 +/- 2 eu for all four carboxymethylations.     

Pulsed nuclear magnetic resonance study of 39K in frog striated muscle.

Samples of 1 M KCl solution and 10 samples of intact frog striated muscle were studied at 4-7 degrees C and/or at 21-22 degrees C. Field inhomogeneity was minimized by using small sample volumes and by using a superconducting magnet designed specifically to provide highly homogeneous fields. In the present experiments, magnetic field inhomogeneity was measured to contribute less than 15% to the free induction decay observed for intracellular 39K. The signal-to-noise ratio of the measurements was enhanced by means of extensive time-averaging. The rates of nuclear relaxation for 39K in aqueous solution were 22 +/- 3 (mean +/- 95% confidence limits) s-1 at 4-7 degrees C and 15 +/- 2 s-1 at 21-22 degrees C. For intracellular 39K, (1/T2) was measured to be 327 +/- 22 s-1 and 229 +/- 10 s-1 at the lower and higher temperatures, respectively. The corresponding values for (1/T1) in the same muscle samples were 198 +/- 31 s-1 and 79 +/- 15 s-1 at 4-7 degrees C and at 21-22 degrees C, respectively. These results for 39K are similar to those previously obtained for intracellular 23Na. Since less than 1% of the intracellular 23Na has been estimated to be immobilized, fractional immobilization of intracellular 39K is also likely to be insubstantial.     

Studies on the equilibria and kinetics of the reactions of ferrous catalase with ligands

The optical absorption spectrum of bovine liver catalase was found to change on light irradiation in the presence of proflavin and EDTA in a deaerated solution. Upon addition of CO to the photolyzed product, the spectrum changed to an another form, suggesting that the photolyzed product is the ferrous form of the enzyme and CO is bound to the ferrous enzyme. When O2 was introduced into the ferrous enzyme, the absorption spectrum returned to its original ferric state. An intermediate spectrum was obtained in this reaction at -20 degrees C in 33% v/v ethylene glycol. Judged from the spectral characteristics of this compound, it is probably an oxyferrous enzyme. It was converted into ferric enzyme gradually when the sample was left at room temperature. The ferrous enzyme, which was generated by flash photolysis of the CO complex of the enzyme in an air-saturated buffer, reacted with O2 to form the oxyferrous enzyme with a second order rate constant of 9.2 x 10(3) M-1.s-1 at pH 8.6 and 20 degrees C. The oxyferrous enzyme thus obtained autodecomposed into the ferric form with a rate constant of 0.1 s-1.     

Spin probe mobility in the whole blood of white rats]

By means of ESR-method the rotary mobility of a tanol spin probe is studied in the whole blood of white rats at the temperatures 5.20 and 37 degrees C. It is shown that at all the temperatures the spin probe is localized in the blood plasma and has a value HFS a = (17.1 +/- 0.1) G. By means of linear anamorphism method it is shown on the example of the spectrum central line that the contour is lorenz, i. e. the superposition of the spectra of different sample regions is absent. The spin probe rotation frequency v is a stable blood parameter, the same for 11 rats investigated and dependent only on the blood temperature. For T = 5.20 and 37 degrees C the values have been received v = (86 +/- 2) x 10(8) s-1, (98 +/- 2) x 10(8) s-1 and (107 +/- 3) x 10(8) s-1, subsequently, which compared to v value in water-glycerin system (1:1) (WGS) allow one to calculate the blood microviscosity values (7.2 +/- 0.4), (6.3 +/- 0.4) and (5.8 +/- 0.4) mPds, subsequently. For the mentioned temperatures the non-sphericity parameter epsilon of the spin probe rotation has the values 0.19 +/- 0.03, 0.22 +/- 0.04 and 0.21 +/- 0.05, subsequently that is close to this parameter value for WCS (epsilon = 0.21 +/- 0.02; v = (6 divided by 20) x 10(9) s-1).     

Macromolecular diffusion of biological polymers measured by confocal fluorescence recovery after photobleaching.

Fluorescence recovery after photobleaching with an unmodified confocal laser scanning microscope (confocal FRAP) was used to determine the diffusion properties of network forming biological macromolecules such as aggrecan. The technique was validated using fluorescein isothiocyanate (FITC)-labeled dextrans and proteins (molecular mass 4-2000 kDa) at 25 degrees C and with fluorescent microspheres (207 nm diameter) over a temperature range of 5-50 degrees C. Lateral diffusion coefficients (D) were independent of the focus position, and the degree and extent of bleach. The free diffusion coefficient (Do) of FITC-aggrecan determined by confocal FRAP was 4.25 +/- 0.6 x 10(-8) cm2 s-1, which is compatible with dynamic laser light scattering measurements. It appeared to be independent of concentration below 2.0 mg/ml, but at higher concentrations (2-20 mg/ml) the self-diffusion coefficient followed the function D = Do(e)(-Bc). The concentration at which the self-diffusion coefficient began to fall corresponded to the concentration predicted for domain overlap. Multimolecular aggregates of aggrecan ( approximately 30 monomers) had a much lower free diffusion coefficient (Do = 6.6 +/- 1.0 x 10(-9) cm2 s-1) but showed a decrease in mobility with concentration of a form similar to that of the monomer. The method provides a technique for investigating the macromolecular organization in glycan-rich networks at concentrations close to those found physiologically.     

On measuring the diffusional water permeability of human red blood cells and ghosts by nuclear magnetic resonance

The characteristics of water diffusional permeability (P) of human red blood cells were studied on isolated erythrocytes and ghosts by a doping nuclear magnetic resonance technique. In contrast to all previous investigations, systematic measurements were performed on blood samples obtained from a large group of donors. The mean values of P ranged from 2.2 X 10(-3) cm.s-1 at 5 degrees C to 8.1 X 10(-3) cm.s-1 at 42 degrees C. The reasons for some of the discrepancies in the permeability coefficients reported by various authors were found. In order to estimate the basal permeability, the maximal inhibition of water diffusion was induced by exposure of red blood cells to p-chloromercuribenzenesulfonate (PCMBS) under various conditions (concentration, duration, temperature). The lowest values of P were around 1.3 X 10(-3) cm.s-1 at 20 degrees C, 1.6 X 10(-3) cm.s-1 at 25 degrees C, 1.9 X 10(-3) cm.s-1 at 30 degrees C and 3.2 X 10(-3) cm.s-1 at 37 degrees C. The results reported here represent the largest series of determinations of water diffusional permeability of human red blood cells (without or with exposure to mercurials) available in the literature, and consequently the best estimates of the characteristics of this transport process. The values of P can be taken as references for the studies of water permeability in various cells or in pathological conditions.     

Temperature jump relaxation kinetics of the P-450cam spin equilibrium

The ferric spin-state equilibrium and relaxation rate of cytochrome P-450 has been examined with temperature jump spectroscopy using a number of camphor analogues known to induce different mixed spin states in the substrate-bound complexes [Gould, P., Gelb, M., & Sligar, S. G. (1981) J. Biol. Chem. 256, 6686]. All temperature-induced spectral changes were monophasic, and the spin-state relaxation rate reached a limiting value at high substrate concentrations. The ferric spin equilibrium constant, Kspin, is defined in terms of the rate constants k1 and k-1 via Kspin = k1/k-1 = [P-450(HS)]/[P-450(LS)] where HS and LS represent high-spin (S = 5/2) and low-spin (S = 1/2) ferric iron, respectively, and the spectrally observed spin-state relaxation rate by kobsd = k1 + k-1. A strong correlation between the fraction of high-spin species and the rate constant, k-1, is observed. For a 3 degrees C temperature jump (from 10 to 13 degrees C), the 23% high-spin tetramethylcyclohexanone complex (Kd = 45 +/- 20 microM) is characterized by a ferric spin relaxation rate of kobsd = 1990 s-1, while the rates for the d-fenchone (41% high spin, Kd = 42 +/- 10 microM) and kobsd = 1990 s-1, while the rates for the d-fenchone (41% high spin, Kd = 42 +/- 10 microM) and camphoroquinone (75% high spin, Kd = 15 +/- 5 microM) complexes are 1430 and 346 s-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

An assay for inhibitors of nucleoside transport based upon the use of 5-[125I]iodo-2'-deoxyuridine as permeant

5-[125I]Iodo-2'-deoxyuridine (IdUrd) has been shown to serve as a permeant for the nucleoside transport system of human erythrocytes and to be matabolically inert in these cells. Linear initial velocities were obtained at 20 degrees C for 125IdUrd transport, yielding a Km of 73 +/- 18 microM (n = 6). Low-affinity inhibitors of 125IdUrd transport, such as adenosine (Ki = 32 +/- 2 microM, n = 2), could be characterized by Michaelis-Menten kinetics. However, high-affinity inhibitors, such as 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, caused nonlinear initial velocities when added to the cells simultaneously with 125IdUrd. Conditions were defined (viz., 20-min pretreatment of cells with test compound followed by 5.0-min incubation with 1.0 microM 125IdUrd, all at 20 degrees C) whereby high-affinity inhibitors of IdUrd transport can be identified and evaluated according to their 50% inhibitory concentrations. The use of 125IdUrd as permeant greatly expedites the testing of compounds as inhibitors of nucleoside transport by allowing the cell pellets generated in these assays to be monitored directly in a gamma spectrometer, thereby circumventing the solubilization and decolorization of cell pellets required by assays that use 3H- or 14C-labeled nucleoside permeants.     

Binding of chara Myosin globular tail domain to phospholipid vesicles

Binding of Chara myosin globular tail domain to phospholipid vesicles was investigated quantitatively. It was found that the globular tail domain binds to vesicles made from acidic phospholipids but not to those made from neutral phospholipids. This binding was weakened at high KCl concentration, suggesting that the binding is electrostatic by nature. The dissociation constant for the binding of the globular tail domain to 20% phosphatidylserine vesicles (similar to endoplasmic reticulum in acidic phospholipid contents) at 150 mM KCl was 273 nM. The free energy change due to this binding calculated from the dissociation constant was -37.3 kJ mol(-1). Thus the bond between the globular tail domain and membrane phospholipids would not be broken when the motor domain of Chara myosin moves along the actin filament using the energy of ATP hydrolysis (DeltaG degrees ' = -30.5 kJ mol(-1)). Our results suggested that direct binding of Chara myosin to the endoplasmic reticulum membrane through the globular tail domain could work satisfactorily in Chara cytoplasmic streaming. We also suggest a possible regulatory mechanism of cytoplasmic streaming including phosphorylation-dependent dissociation of the globular tail domain from the endoplasmic reticulum membrane.     

Formation of super-reduced Chromatium high-potential iron--sulphur protein in aqueous solution by pulse radiolysis.

Both the oxidized and reduced forms of Hipip (high-potential iron--sulphur protein) are reduced (approx. 30% yields) by eaq.- in a single-stage process, rate constants 1.7 x 10(10) and 1.8 x 10(10) M-1 . s-1 respectively, at 25 degrees C, pH 7.0 (5 mM-phosphate). Super-reduced Hipip, which is formed in the latter case, has a spectrum which closely resembles that of reduced ferredoxin, i.e. Fe4S4 (SR)4(3-) clusters. The spectrum is stable over 2 s periods investigated. Super-reduced Hipip is reoxidized with O2, rate constant 4.8 x 10(6) M-1 . s-1 at 25 degrees C.