Analysis of transcellular water movement inNitella

Summary The mechanism of transcellular osmosis was analyzed on the assumption that the driving force, which is equal to the osmotic pressure of the mannitol solution given to the exosmosis side, is divided into two parts; one causing the inward water flow on the water side, the other causing the outward water flow on the solution side, when each force drives an equal amount of water. Based on this analysis a new procedure was developed to measure the endosmotic and exosmotic water permeabilities of the membranes independently. It involved measurement of volume of water transported transcellularly, change in turgor pressure, and water permeability of the cell wall alone.Experiments following the new procedure revealed that in aNitella internode positioned across a partition wall with equal length both the endosmotic and exosmotic water permeabilities remained constant during transcellular osmosis induced with 0.4M mannitol, at least for the first minute. It was found that the permeability coefficient for endosmosis (3.9 × 10^–5 cm sec^–1 atm^–1) was very much higher than that for exosmosis (1.4× 10^–5 cm sec^–1 atm^–1). Treatment of the endosmotic cell part with 5% ethanol conspicuously decreased the water permeability of the cell on this side down to 1/2.4 the value obtained without ethanol but never affected the permeability on the other side (exosmosis side).This work was supported partly by a Research Grant from the Ministry of Education of Japan.     

Rectification of the water permeability in COS-7 cells at 22, 10 and 0°C

The osmotic and permeability parameters of a cell membrane are essential physico-chemical properties of a cell and particularly important with respect to cell volume changes and the regulation thereof. Here, we report the hydraulic conductivity, L(p), the non-osmotic volume, V(b), and the Arrhenius activation energy, E(a), of mammalian COS-7 cells. The ratio of V(b) to the isotonic cell volume, V(c iso), was 0.29. E(a), the activation energy required for the permeation of water through the cell membrane, was 10,700, and 12,000 cal/mol under hyper- and hypotonic conditions, respectively. Average values for L(p) were calculated from swell/shrink curves by using an integrated equation for L(p). The curves represented the volume changes of 358 individually measured cells, placed into solutions of nonpermeating solutes of 157 or 602 mOsm/kg (at 0, 10 or 22°C) and imaged over time. L(p) estimates for all six combinations of osmolality and temperature were calculated, resulting in values of 0.11, 0.21, and 0.10 μm/min/atm for exosmotic flow and 0.79, 1.73 and 1.87 μm/min/atm for endosmotic flow (at 0, 10 and 22°C, respectively). The unexpected finding of several fold higher L(p) values for endosmotic flow indicates highly asymmetric membrane permeability for water in COS-7. This phenomenon is known as rectification and has mainly been reported for plant cell, but only rarely for animal cells. Although the mechanism underlying the strong rectification found in COS-7 cells is yet unknown, it is a phenomenon of biological interest and has important practical consequences, for instance, in the development of optimal cryopreservation.     

Osmotic gradient-induced water permeation across the sarcolemma of rabbit ventricular myocytes

The mechanism of water permeation across the sarcolemma was characterized by examining the kinetics and temperature dependence of osmotic swelling and shrinkage of rabbit ventricular myocytes. The magnitude of swelling and the kinetics of swelling and shrinkage were temperature dependent, but the magnitude of shrinkage was very similar at 6 degrees, 22 degrees, and 37 degrees C. Membrane hydraulic conductivity, Lp, was approximately 1.2 x 10(-10) liter.N-1.s-1 at 22 degrees C, corresponding to an osmotic permeability coefficient, Pf, of 16 microns.s-1, and was independent of the direction of water flux, the magnitude of the imposed osmotic gradient (35-165 mosm/liter), and the initial cell volume. This value of Lp represents an upper limit because the membrane was assumed to be a smooth surface. Based on capacitive membrane area, Lp was 0.7 to 0.9 x 10(-10) liter.N-1.s-1. Nevertheless, estimates of Lp in ventricle are 15 to 25 times lower than those in human erythrocytes and are in the range of values reported for protein- free lipid bilayers and biological membranes without functioning water channels (aquaporin). Evaluation of the effect of unstirred layers showed that in the worst case they decrease Lp by < or = 2.3%. Analysis of the temperature dependence of Lp indicated that its apparent Arrhenius activation energy, Ea', was 11.7 +/- 0.9 kcal/mol between 6 degrees and 22 degrees C and 9.2 +/- 0.9 kcal/mol between 22 degrees and 37 degrees C. These values are significantly greater than that typically found for water flow through water-filled pores, approximately 4 kcal/mol, and are in the range reported for artificial and natural membranes without functioning water channels. Taken together, these data strongly argue that the vast majority of osmotic water flux in ventricular myocytes penetrates the lipid bilayer itself rather than passing through water-filled pores.     

Radial hydraulic conductivity along developing onion roots

Although most studies have shown that water uptake varies along the length of a developing root, there is no consistent correlation of this pattern with root anatomy. In the present study, water movement into three zones of onion roots was measured by a series of mini-potometers. Uptake was least in the youngest zone (mean hydraulic conductivity, Lpr = 1.5 x 10(-7) +/- 0.34 x 10(-7) m MPa-1 s-1; +/- SE, n = 10 roots) in which the endodermis had developed only Casparian bands and the exodermis was immature. Uptake was significantly greater in the middle zone (Lpr = 2.4 x 10(-7) +/- 0.43 x 10(-7) m MPa-1 s-1; +/- SE, n = 10 roots) which had a mature exodermis with both Casparian bands and suberin lamellae, and continued at this level in the oldest zone in which the endodermis had also developed suberin lamellae (Lpr = 2.8 x 10(-7) +/- 0.30 x 10(-7) m MPa-1 s-1; +/- SE, n = 10 roots). Measurements of the hydraulic conductivities of individual cells (Lp) in the outer cortex using a cell pressure probe indicated that this parameter was uniform in all three zones tested (Lp = 1.3 x 10(-6) +/- 0.01 x 10(-6) m MPa-1 s-1; +/- SE, n = 60 cells). Lp of the youngest zone was lowered by mercuric chloride treatment, indicating the involvement of mercury-sensitive water channels (aquaporins). Water flow in the older two root zones measured by mini-potometers was also inhibited by mercuric chloride, despite the demonstrated impermeability of their exodermal layers to this substance. Thus, water channels in the epidermis and/or exodermis of the older regions were especially significant for water flow. The results of this and previous studies are discussed in terms of two models. The first, which describes maize root with an immature exodermis, is the 'uniform resistance model' where hydraulic resistances are evenly distributed across the root cylinder. The second, which describes the onion root with a mature exodermis, is the 'non-uniform resistance model' where resistances can be variable and are concentrated in a certain layer(s) on the radial path.     

Simultaneous diffusion of inositol and mannitol in the rat brain

The diffusion of both inositol and mannitol has been determined simultaneously by the integral bolus method in rat brain. The permeability constant (Kin) of inositol averaged 0.27 +/- 0.02 ml X (100 g)-1 X min-1 or 4 X 10(-7) cm X s-1 at a cerebral capillary surface area of 100 cm2 x g-1. The permeability of mannitol was 0.08 +/- 0.01 ml X (100 g)-1. min-1 or 1 X 10(-7) cm X s-1. Neither glucose nor galactose affected the inositol permeability. Hypoglycemia increased somewhat the Km value for mannitol. The basal ganglia showed an increase Km for both substrates as compared with those obtained for cortex, temporal and parietal tissues.     

Analysis of double knockout mice lacking aquaporin-1 and urea transporter UT-B. Evidence for UT-B-facilitated water transport in erythrocytes

We reported increased water permeability and a low urea reflection coefficient in Xenopus oocytes expressing urea transporter UT-B (former name UT3), suggesting that water and urea share a common aqueous pathway (Yang, B., and Verkman, A. S. (1998) J. Biol. Chem. 273, 9369-9372). Although increased water permeability was confirmed in the Xenopus oocyte expression system, it has been argued (Sidoux-Walter, F., Lucien, N., Olives, B., Gobin, R., Rousselet, G., Kamsteeg, E. J., Ripoche, P., Deen, P. M., Cartron, J. P., and Bailly, P. (1999) J. Biol. Chem. 274, 30228-30235) that UT-B does not transport water when expressed at normal levels in mammalian cells such as erythrocytes. To quantify UT-B-mediated water transport, we generated double knockout mice lacking UT-B and the major erythrocyte water channel, aquaporin-1 (AQP1). The mice had reduced survival, retarded growth, and defective urinary concentrating ability. However, erythrocyte size and morphology were not affected. Stopped-flow light scattering measurements indicated erythrocyte osmotic water permeabilities (in cm/s x 0.01, 10 degrees C): 2.1 +/- 0.2 (wild-type mice), 2.1 +/- 0.05 (UT-B null), 0.19 +/- 0.02 (AQP1 null), and 0.045 +/- 0.009 (AQP1/UT-B null). The low water permeability found in AQP1/UT-B null erythrocytes was also seen after HgCl(2) treatment of UT-B null erythrocytes or phloretin treatment of AQP1 null erythrocytes. The apparent activation energy for UT-B-mediated water transport was low, <2 kcal/mol. Estimating 14,000 UT-B molecules per mouse erythrocyte, the UT-B-dependent P(f) of 0.15 x 10(-4) cm/s indicated a substantial single channel water permeability of UT-B of 7.5 x 10(-14) cm(3)/s, similar to that of AQP1. These results provide direct functional evidence for UT-B-facilitated water transport in erythrocytes and suggest that urea traverses an aqueous pore in the UT-B protein.     

Vanadium nitrogenase of Azotobacter chroococcum. MgATP-dependent electron transfer within the protein complex.

The kinetics of MgATP-induced electron transfer from the Fe protein (Ac2V) to the VFe protein (AclV) of the vanadium-containing nitrogenase from Azotobacter chroococcum were studied by stopped-flow spectrophotometry at 23 degrees C at pH 7.2. They are very similar to those of the molybdenum nitrogenase of Klebsiella pneumoniae [Thorneley (1975) Biochem. J. 145, 391-396]. Extrapolation of the dependence of kobs. on [MgATP] to infinite MgATP concentration gave k = 46 s-1 for the first-order electron-transfer reaction that occurs with the Ac2V MgATPAclV complex. MgATP binds with an apparent KD = 230 +/- 10 microM and MgADP acts as a competitive inhibitor with Ki = 30 +/- 5 microM. The Fe protein and VFe protein associate with k greater than or equal to 3 x 10(7) M-1.s-1. A comparison of the dependences of kobs. for electron transfer on protein concentrations for the vanadium nitrogenase from A. chroococcum with those for the molybdenum nitrogenase from K. pneumoniae [Lowe & Thorneley (1984) Biochem. J. 224, 895-901] indicates that the proteins of the vanadium nitrogenase system form a weaker electron-transfer complex.     

Vasopressin-enhanced urea transport by rat inner medullary collecting duct cells in culture

The distal inner medullary collecting duct (IMCD) is critical in the urinary concentrating process, in part because it is the site of vasopressin (AVP)-regulated permeability to urea. The purpose of these experiments was to develop a cell culture model of the IMCD on permeable structure and to characterize the responsiveness to AVP. Rat IMCD cells were grown to confluence on collagen-coated Millipore filters glued onto plastic rings. To assess the time required to achieve confluence, the transepithelial resistance was measured periodically and was found to be stable after 2 weeks, at a maximal value of 595 +/- 22 omega cm2. In separate monolayers the effect of AVP on inulin and urea permeability was determined. While inulin permeability was unchanged after AVP, urea permeability increased from 6.0 +/- 0.4 to peak values of 16.0 +/- 3.8 (10 nM), 23.1 +/- 3.9 (1 microM) and 28.1 +/- 4.9 (10 microM) x 10(-6) cm s-1 (n = 24). In 10 other monolayers, after the addition of 1 mM 8-Br-cAMP, urea permeability increased from 5.1 +/- 0.3 to 8.1 +/- 1.6 x 10(-6) cm s-1 and, after 8-Br-cAMP + 3-isobutyl-1-methylxanthine, to 12.2 +/- 0.7 x 10(-6) cm s-1. We conclude that rat IMCD cells grown in culture exhibit the characteristics of a 'tight' epithelium. Inulin and urea permeability are not different in the absence of AVP, consistent with high resistance junctional complexes. Furthermore, IMCD cells retain the capacity for AVP-regulated urea permeability, a characteristic feature of this nephron segment in vivo.     

Genetic engineering of redox donor sites: measurement of intracomplex electron transfer between ruthenium-65-cytochrome b5 and cytochrome c.

The de novo design and synthesis of ruthenium-labeled cytochrome b5 that is optimized for the measurement of intracomplex electron transfer to cytochrome c are described. A single cysteine was substituted for Thr-65 of rat liver cytochrome b5 by recombinant DNA techniques [Stayton, P. S., Fisher, M. T., & Sligar, S. G. (1988) J. Biol. Chem. 263, 13544-13548]. The single sulfhydryl group on T65C cytochrome b5 was then labeled with [4-(bromomethyl)-4'-methylbipyridine] (bisbipyridine)ruthenium2+ to form Ru-65-cyt b5. The ruthenium group at Cys-65 is only 12 A from the heme group of cytochrome b5 but is not located at the binding site for cytochrome c. Laser excitation of the complex between Ru-65-cyt b5 and cytochrome c results in electron transfer from the excited state Ru(II*) to the heme group of Ru-65-cyt b5 with a rate constant greater than 10(6) s-1. Subsequent electron transfer from the heme group of Ru-65-cyt b5 to the heme group of cytochrome c is biphasic, with a fast-phase rate constant of (4 +/- 1) x 10(5) s-1 and a slow-phase rate constant of (3 +/- 1) x 10(4) s-1. This suggests that the complex can assume two different conformations with different electron-transfer properties. The reaction becomes monophasic and the rate constant decreases as the ionic strength is increased, indicating dissociation of the complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochalasin B inhibition and temperature dependence of 3-O-methylglucose transport in fat cells

The transport of 3-O-methylglucose in white fat cells was measured under equilibrium exchange conditions at 3-O-methylglucose concentrations up to 50 mM with a previously described method (Vinten, J., Gliemann, J. and Osterlind, K. (1976) J. Biol. Chem. 251, 794--800). Under these conditions the main part of the transport was inhibitable by cytochalasin B. The inhibition was found to be of competitive type with an inhibition constant of about 2.5 . 10(-7) M, both in the absence and in the presence of insulin (1 micrometer). The cytochalasin B-insensitive part of the 3-O-methylglucose permeability was about 2 . 10(-9) cm . s-1, and was not affected by insulin. As calculated from the maximum transport capacity, the half saturation constant and the volume/surface ratio, the maximum permeability of the fat cell membrane to 3-O-methylglucose at 37 degrees C and in the presence of insulin was 4.3 . 10(-6) cm . s-1. From the temperature dependence of the maximum transport capacity in the interval 18--37 degrees C and in the presence of insulin, an Arrhenius activation energy of 14.8 +/- 0.44 kcal/mol was found. The corresponding value was 13.9 +/- 0.89 in the absence of insulin. The half saturating concentration of 3-O-methylglucose was about 6 mM in the temperature interval used, and it was not affected by insulin, although this hormone increased the maximum transport capacity about ten-fold to 1.7 mmol . s-1 per 1 intracellular water at 37 degrees C.     

Permeability of human medial collateral ligament in compression transverse to the collagen fiber direction

This study quantified the apparent and intrinsic hydraulic permeability of human medial collateral ligament (MCL) under direct permeation transverse to the collagen fiber direction. A custom permeation device was built to apply flow across cylindrical samples of ligament while monitoring the resulting pressure gradient. MCLs from 5 unpaired human knees were used (donor age 55 +/- 16 yr, 4 males, 1 female). Permeability measurements were performed at 3 levels of compressive pre-strain (10%, 20% and 30%) and 5 pressures (0.17, 0.34, 1.03, 1.72 and 2.76 MPa). Apparent permeability was determined from Darcy's law, while intrinsic permeability was determined from the zero-pressure crossing of the pressure-permeability curves at each compressive pre-strain. Resulting data were fit to a finite deformation constitutive law [Journal of Biomechanics 23 (1990) 1145-1156]. The apparent permeability of human MCL ranged from 0.40 +/- 0.05 to 8.60 +/- 0.77 x 10(-16) m(4)/Ns depending on pre-strain and pressure gradient. There was a significant decrease in apparent permeability with increasing compressive pre-strain (p=0.024) and pressure gradient (p<0.001), and there was a significant interaction between the effects of compressive pre-strain and pressure (p<0.001). Intrinsic permeability was 14.14 +/- 0.74, 6.30 +/- 2.13 and 4.29 +/- 1.71 x 10(-16) m(4)/Ns for compressive pre-strains of 10%, 20% and 30%, respectively. The intrinsic permeability showed a faster decrease with increasing compressive pre-strain than that of bovine articular cartilage. These data provide a baseline for investigating the effects of disease and chemical modification on the permeability of ligament and the data should also be useful for modeling the poroelastic material behavior of ligaments.     

Urea permeability of human red cells

The rate of unidirectional [14C]urea efflux from human red cells was determined in the self-exchange and net efflux modes with the continuous flow tube method. Self-exchange flux was saturable and followed simple Michaelis-Menten kinetics. At 38 degrees C the maximal self-exchange flux was 1.3 X 10(-7) mol cm-2 s-1, and the urea concentration for half-maximal flux, K1/2, was 396 mM. At 25 degrees C the maximal self-exchange flux decreased to 8.2 X 10(-8) mol cm-2 s-1, and K1/2 to 334 mM. The concentration-dependent urea permeability coefficient was 3 X 10(-4) cm s-1 at 1 mM and 8 X 10(-5) cm s-1 at 800 mM (25 degrees C). The latter value is consonant with previous volumetric determinations of urea permeability. Urea transport was inhibited competitively by thiourea; the half-inhibition constant, Ki, was 17 mM at 38 degrees C and 13 mM at 25 degrees C. Treatment with 1 mM p-chloromercuribenzosulfonate inhibited urea permeability by 92%. Phloretin reduced urea permeability further (greater than 97%) to a "ground" permeability of approximately 10(-6) cm s-1 (25 degrees C). This residual permeability is probably due to urea permeating the hydrophobic core of the membrane by simple diffusion. The apparent activation energy, EA, of urea transport after maximal inhibition was 59 kJ mol-1, whereas in control cells EA was 34 kJ mol-1 at 1 M and 12 kJ mol-1 at 1 mM urea. In net efflux experiments with no extracellular urea, the permeability coefficient remained constantly high, independent of a variation of intracellular urea between 1 and 500 mM, which indicates that the urea transport system is asymmetric. It is concluded that urea permeability above the ground permeability is due to facilitate diffusion and not to diffusion through nonspecific leak pathways as suggested previously.     

Diffusional water permeability of human erythrocytes and their ghosts

The diffusional water permeability of human red cells and ghosts was determined by measuring the rate of tracer efflux by means of an improved version of the continuous flow tube method, having a time resolution of 2-3 ms. At 25 degrees C, the permeability was 2.4 x 10(3) and 2.9 x 10(3) cm s-1 for red cells and ghosts, respectively. Permeability was affected by neither a change in pH from 5.5 to 9.5, nor by osmolality up to 3.3 osmol. Manganous ions at an extracellular concentration of 19 mM did not change diffusional water permeability, as recently suggested by NMR measurements. A "ground" permeability of 1 x 10(3) cm s-1 was obtained by inhibition with 1 mM of either p- chloromercuribenzoate (PCMB) or p-chloromercuribenzene sulfonate (PCMBS). Inhibition increased temperature dependence of water permeability for red cells and ghosts from 21 to 30 kJ mol-1 to 60 kJ mol-1. Although diffusional water permeability is about one order of magnitude lower than osmotic permeability, inhibition with PCMB and PCMBS, temperature dependence both before and after inhibition, and independence of osmolality showed that diffusional water permeability has qualitative features similar to those reported for osmotic permeability, which indicates that the same properties of the membrane determine both types of transport. It is suggested that the PCMB(S)- sensitive permeability above the ground permeability takes place through the intermediate phase between integral membrane proteins and their surrounding lipids.     

Water permeability of asymmetric planar lipid bilayers: leaflets of different composition offer independent and additive resistances to permeation

To understand how plasma membranes may limit water flux, we have modeled the apical membrane of MDCK type 1 cells. Previous experiments demonstrated that liposomes designed to mimic the inner and outer leaflet of this membrane exhibited 18-fold lower water permeation for outer leaflet lipids than inner leaflet lipids (Hill, W.G., and M.L. Zeidel. 2000. J. Biol. Chem. 275:30176-30185), confirming that the outer leaflet is the primary barrier to permeation. If leaflets in a bilayer resist permeation independently, the following equation estimates single leaflet permeabilities: 1/P(AB) = 1/P(A) + 1/P(B) (Eq. l), where P(AB) is the permeability of a bilayer composed of leaflets A and B, P(A) is the permeability of leaflet A, and P(B) is the permeability of leaflet B. Using for the MDCK leaflet-specific liposomes gives an estimated value for the osmotic water permeability (P(f)) of 4.6 x 10(-4) cm/s (at 25 degrees C) that correlated well with experimentally measured values in intact cells. We have now constructed both symmetric and asymmetric planar lipid bilayers that model the MDCK apical membrane. Water permeability across these bilayers was monitored in the immediate membrane vicinity using a Na+-sensitive scanning microelectrode and an osmotic gradient induced by addition of urea. The near-membrane concentration distribution of solute was used to calculate the velocity of water flow (Pohl, P., S.M. Saparov, and Y.N. Antonenko. 1997. Biophys. J. 72:1711-1718). At 36 degrees C, P(f) was 3.44 +/- 0.35 x 10(-3) cm/s for symmetrical inner leaflet membranes and 3.40 +/- 0.34 x 10(-4) cm/s for symmetrical exofacial membranes. From, the estimated permeability of an asymmetric membrane is 6.2 x 10(-4) cm/s. Water permeability measured for the asymmetric planar bilayer was 6.7 +/- 0.7 x 10(-4) cm/s, which is within 10% of the calculated value. Direct experimental measurement of P(f) for an asymmetric planar membrane confirms that leaflets in a bilayer offer independent and additive resistances to water permeation and validates the use of.     

Behavior of red king crab larvae: phototaxis, geotaxis and rheotaxis

Zoeae of Paralithodes camtschatica were positively phototactic to white light intensities above 1 x 10(13) q cm-2 s-1. Negative phototaxis occurred at low (1 x 10(12) q cm-2 s-1), but not high intensities (2.2 x 10(16) q cm-2 s-1). Phototactic response was directly related to light intensity. Zoeae also responded to red, green and blue light. Zoeae were negatively geotactic, but geotaxis was dominated by phototaxis. Horizontal swimming speed of stage 1 zoeae < 4 d old was 2.4 +/- 0.1 (SE) cm s-1 and decreased to 1.7 +/- 0.1 cm s-1 in older zoeae (P < 0.01). Horizontal swimming speed of stage 2 zoeae was not significantly different from > or = 4 d old stage 1 zoeae. Vertical swimming speed, 1.6 +/- 0.1 cm s-1, and sinking rate, 0.7 +/- 0.1 cm s-1, did not change with ontogeny. King crab zoeae were positively rheotactic and maintained position in horizontal currents less than 1.4 cm s-1. Starvation reduced swimming and sinking rates and phototactic response.     

Evidence of nitric oxide and angiotensin II regulation of circulation and cutaneous drinking in Bufo marinus

The nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (l-NAME) increased vascular resistance (VR) 10% above baseline of 3.08+/-0.08 (n=11) mmHg/mL/min at 10 mg/kg and 20% above 3.05+/-0.08 (n=9) at 50 mg/kg in anesthetized toads (Bufo marinus). Blood pressure was unaffected by either dose of L-NAME. Blood flow decreased at the higher dose of L-NAME. L-arginine (300 mg/kg) reversed the effects of L-NAME on VR and blood flow in toads treated with 10 mg/kg but not with 50 mg/kg. Injection of 50 mg/kg L-NAME into empty-bladder toads produced a 10% decrease in water uptake, J(v), resulting in a J(v) of 1,267+/-11 cm(3)/cm(2)/s x 10(-7) (n=9) compared to 1,385+/-12 (n=8) for controls. Injection of 10 microg/kg angiotensin II (ANG II) increased J(v) 15% across the pelvic patch (J(v), cm(3)/cm(2)/s x 10(-7)), resulting in a J(v) of 1,723+/-12 cm(3)/cm(2)/s x 10(-7) (n=8) compared to 1,471+/-12 (n=8) for controls. It is hypothesized that during cutaneous drinking blood flow into the capillary bed of the pelvic patch is regulated by nitric oxide and ANG II.     

Reactions of prostaglandin H synthase in the presence of the stabilizing agents diethyldithiocarbamate and glycerol

Both cyclooxygenase and peroxidase reactions of prostaglandin H synthase were studied in the presence and absence of diethyldithiocarbamate and glycerol at 4 degrees C in phosphate buffer (pH 8.0). Diethyldithiocarbamate reacts with the high oxidation state intermediates of prostaglandin H synthase; it protects the enzyme from bleaching and loss of activity by its ability to act as a reducing agent. For the reaction of diethyldithiocarbamate with compound I, the second-order rate constant k2,app, was found to fall within the range of 5.8 x 10(6) +/- 0.4 x 10(6) M-1.s-1 less than k2,app less than 1.8 x 10(7) +/- 0.1 x 10(7) M-1.s-1. The reaction of diethyldithiocarbamate with compound II showed saturation behavior suggesting enzyme-substrate complex formation, with kcat = 22 +/- 3 s-1, Km = 67 +/- 10 microM, and the second-order rate constant k3,app = 2.0 x 10(5) +/- 0.2 x 10(5) M-1.s-1. In the presence of both diethyldithiocarbamate and 30% glycerol, the parameters for compound II are kcat = 8.8 +/- 0.5 s-1, Km = 49 +/- 7 microM, and k3,app = 1.03 x 10(5) +/- 0.07 x 10(5) M-1.s-1. The spontaneous decay rate constants of compounds I and II (in the absence of diethyldithiocarbamate) are 83 +/- 5 and 0.52 +/- 0.05 s-1, respectively, in the absence of glycerol; in the presence of 30% glycerol they are 78 +/- 5 and 0.33 +/- 0.02 s-1, respectively. Neither cyclooxygenase activity nor the rate constant for compound I formation using 5-phenyl-4-pentenyl-1-hydroperoxide is altered by the presence of diethyldithiocarbamate.(ABSTRACT TRUNCATED AT 250 WORDS)     

The transverse location of the retinal chromophore in the purple membrane by diffusion-enhanced energy transfer

We have used fluorescence energy transfer in the rapid-diffusion limit (RDL) to estimate the trans-membrane depth of retinal in the purple membrane (PM). Chelates of Tb(III) are excellent energy donors for the retinal chromophore of PM, having a maximum Ro value for F?rster energy transfer of approximately 62 A (assuming a donor quantum yield of 1). Energy transfer rates were measured from the time-resolved emission kinetics of the donor. The distance of closest approach between chelates and the chromophore was estimated by simulating RDL energy-transfer rate constants according to geometric models of either PM sheets or membrane vesicles. The apparent rate constant for RDL energy transfer between Tb(III)HED3A and retinal in PM sheets is 1.5(+/- 0.1) x 10(6) M-1 s-1, corresponding to a depth of approximately 10 +/- 2 A for the retinal chromophore. Cell envelope vesicles (CEVs) from Halobacterium halobium were studied by using RDL energy transfer to assess the proximity of retinal to either the extracellular or intracellular face of the PM. The estimated depth of retinal from the extravesicular face of the PM is 10 +/- 3 A, based on the RDL energy-transfer rate constant. Energy-transfer levels to retinal in the PM were estimated by an indirect method with energy donors trapped in the inner-aqueous space of CEVs. The rate constants derived for this arrangement are too low to be consistent with the shortest depth of retinal deduced for PM sheets. Thus, the intravesticular face of CEVs, corresponding to the cytoplasmic face of cells, is the more distant surface from the chromophore of bacteriorhodopsin.     

Measurement of the water permeability of the membranes of boar, ram, and rabbit spermatozoa using concentration-dependent self-quenching of an entrapped fluorophore

Published values for sperm membrane water permeability (L(p)) obtained using a time-to-lysis methodology have produced anomalous results when used to model optimal cooling rates for cryopreservation of spermatozoa. As the lysis method is dependent on potentially questionable assumptions, we describe an alternative method for measuring sperm L(p). Spermatozoa were exposed to hypo- and hyperosmotic conditions using a stopped-flow apparatus and the time course of resulting volume changes was measured using concentration-dependent self-quenching of the entrapped fluorophore, carboxyfluorescein (CF). L(p) was measured for boar, rabbit, and ram spermatozoa using a range of osmotic stresses (+/-50-100 mOsm). Values for exosmotic and endosmotic flow showed no evidence of rectification. Mean L(p) values were 0.84 microm/min/atm (boar), 0.28 microm/min/atm (rabbit), and 2.79 microm/min/atm (ram). These values are lower than the lysis method estimates, with the ram value reduced by approximately two-thirds using the current methodology. The value for boar spermatozoa showed good agreement with published values obtained using an electronic cell-sizing technique. Substitution of the revised values for L(p) into the model for optimal cooling rates brings the calculated optimal rate closer to the lower empirically observed value but does not fully account for the previously reported discrepancies.     

Unitary permeability of gap junction channels to second messengers measured by FRET microscopy

Gap junction channels assembled from connexin protein subunits mediate intercellular transfer of ions and metabolites. Impaired channel function is implicated in several hereditary human diseases. In particular, defective permeation of cAMP or inositol-1,4,5-trisphosphate (InsP(3)) through connexin channels is associated with peripheral neuropathies and deafness, respectively. Here we present a method to estimate the permeability of single gap junction channels to second messengers. Using HeLa cells that overexpressed wild-type human connexin 26 (HCx26wt) as a model system, we combined measurements of junctional conductance and fluorescence resonance energy transfer (FRET) emission ratio of biosensors selective for cAMP and InsP(3). The unitary permeabilities to cAMP (47 x 10(-3) +/- 15 x 10(-3) microm(3)/s) and InsP(3) (60 x 10(-3) +/- 12 x 10(-3) microm(3)/s) were similar, but substantially larger than the unitary permeability to lucifer yellow (LY; 7 +/- 3 x 10(-3) microm(3)/s), an exogenous tracer. This method permits quantification of defects of metabolic coupling and can be used to investigate interdependence of intercellular diffusion and cross-talk between diverse signaling pathways.