Robert I. Macey

MEMBRANE ENZYME SYSTEMS. MOLECULAR SIZE DETERMINATIONS BY RADIATION INACTIVATION.

Abstract Classical radiation target theory is applied to radiation inactivation data on lyophilized membranes to obtain values for the molecular weights of membrane-bound enzymes. A molecular weight of 250 000 is estimated for the ( Na + + K + -activated ATPase (ATP phosphohydrolase, EC 3.6.1.3) of human erythrocyte ghosts, guinea pig kidney cortex microsomes or plasma membrane preparations, and crayfish nerve cord. Other results suggest that the Mg2+-dependent ATPase and the ( Na + + K + )-ATPase have the same molecular weight. Studies with a K+-stimulated microsomal alkaline phosphatase (orthophosphoric acid monoester phosphohydrolase, EC 3.1.3.1) indicate a molecular weight of 140 000 for this enzyme which is thought to be responsible for the dephosphorylation step in the hydrolysis of ATP via the ATPase system. The volume fraction of the red cell membrane devoted to active cation transport is estimated at 0.002–0.04%. Microsomal and plasma membrane preparations apparently contain the same Na + + K + )-ATPase judge by their similar responses to ouabain inhibition, cation stimulation, and radiation inactivation. Radiation inactivation estimates of the molecular weight of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) and of mitochondrial ATPase are also presented. Studies with intact red cells and ghosts, irradiated while frozen, suggest that it might be possible to estimate the molecular weight of enzymes in intact cells.

Erythrocyte membrane potentials determined by hydrogen ion distribution

If the extracellular fluid is left unbuffered, dynamic membrane potential changes in the red blood cell may be determined from external pH readings. For some types of experiments it is necessary to accelerate H+ equilibration by adding minute amounts of hydrogen carriers. The method is independent of hematocrit over a wide range of membrane potential changes. Membrane potential jumps produced by permeability changes or by changes in ionic composition may be measured. The method provides a convenient means of measuring parameters of both the conductive and non-conductive anion pathways in the red cell.

Perturbation of red cell volume: Rectification of osmotic flow

Abstract A method is described which allows determination of the concentration dependence of the filtration coefficient L p for membranes of intact cells. After a small, sudden perturbation of solution osmolality the cell volume exponentially approaches a new equilibrium value. The time constant of this osmotic process is measured by turbidity changes and is used to calculate L p for each set of experimental conditions. Application of this method to beef and human erythrocytes has shown: (a) Values of L p are in good agreement with those found by other investigators using other techniques. (b) L p is independent of cell size and osmolality of the suspending medium. (c) An apparent rectification of water flow occurs, inward flow being 40–50% greater than outward flow for the same osmotic gradient. The two latter results contradict the recent conclusions of Rich et al. ( J. Gen. Physiol. , 52 (1968) 941) but are consistent, nevertheless, with the experimental data reported by these investigators.

Thermodynamics of all-or-none water channel closure in red cells.

SummaryThe relation of osmotic to diffusional water permeability of human red blood cells was compared after treating the cells with different concentrations of PCMBS (p-chloromercuribenzene sulfonate). After subtracting the PCMBS-insensitive permeability (presumably the water permeability of the lipid bilayer) from each, the ratio of osmotic to diffusional permeability remains invariant (≈11) as more and more water channels are inhibited by increasing concentrations of PCMBS. This result implies that the channels close in an all-or-none way and suggests a two-state model. Analysis of the dependence of osmotic water permeability on PCMBS concentration in terms of the model reveals a 1∶1 stoichiometry and a dissociation constant for the PCMBS/membrane receptor complex of about 0.019mm at 37°C. Temperature dependence studies show that the reaction is entropically driven (ΔHo≈25 kcal/mol, ΔSo≈100 cal/moldeg) and suggest the involvement of hydrophobic interactions.

Pressure flow patterns in a cylinder with reabsorbing walls

The problem of fluid motion in renal tubules, in contrast to ordinary flow through cylinders with impermeable walls, is complicated by the existence of radial velocities generated by reabsorption processes. As a first approach to this problem, the Navier Stokes equations for axially symmetric, slow flow in an infinite cylinder whose walls reabsorb fluid are integrated. If the rate of reabsorption is constant, the solutions resemble the conventional Poiseuille flow, i.e., the longitudinal velocity profile is parabolic. In addition the drop in mean pressure is proportional to the mean axial flow, the length of tube between reference points, and inversely proportional to the fourth power of the radius. If the rate of reabsorption is a linear function of the distance from the origin, the presence of an additive term alters these relations. If, for example, the gradient in reabsorption is positive, the axial velocity profile tends to flatten and when the gradient is sufficiently large, the maximum velocity moves from the center of the stream toward the periphery, leaving a relative minimum at the center. In passing from the center of the tube to the walls, the radial velocity passes through a miximum, regardless of the reabsorption properties of the wall.

Effects of calcium on potassium and water transport in human erythrocyte ghosts

Abstract Bulk water transport in reconstituted ghosts is statistically comparable to that in the parent red cells, and is unaffected by incorporation of Ca 2+ over the range of 0.01 to 1 mM. Brief exposure of ghosts to p -chloromercuribenzene sulfonate results in a supression of osmotic water flow but leaves K + permeability unchanged. Incorporation of p -chloromercuribenzene sulfonate provokes extremely rapid K + loss which can be counteracted by simultaneous inclusion of Ca 2+ . Erythrocyte ghosts, when prepared with a small amount of Ca 2+ , demonstrate recovery of normal impermeability to choline, sucrose, Na + and inulin and have an improved K + retention over Ca 2+ -free preparations. The rate of passive transport of K + from unwashed erythrocyte ghosts was measured during the initial few minutes of efflux. The initial rates vary in a bimodal fashion with the concentration of Ca 2+ incorporated at the time of hemolysis. In low concentrations (0.01–0.1 mM), Ca 2+ protects the K + barrier while at higher concentrations (0.1–1.0 mM) it provokes a K + leakage ranging from 7 to 50 times the normal rate of passive K + loss. The Ca 2+ -induced K + leak is thus a graded response rather than a discrete membrane transport state. The transition from a Ca 2+ -protected to a Ca 2+ -damaged membrane occurs upon an increase in Ca 2+ concentration of less than 50 μmoles/l.

Diffusional water permeability of red cells. Independence on osmolality.

The osmotic permeability coefficient (Lp) for human red cells has been reported to depend on the osmolality of the suspending solution. These results are also consistent with the view that the value of Lp depends on flow rectification. In this report an NMR method is used to measure the dependence of water exchange times at constant cell volume on osmolality. Our results indicate that the diffusion water permeability is constant over a large range of osmolality (300-1000 mosM) produced by the permeable solutes urea, methanol, ethanol, and glycerol. The results support the view that the apparent dependence of Lp on osmolality is due to flow rectification.

Estimate of the number of urea transport sites in erythrocyte ghosts using a hydrophobic mercurial

SummaryIn this paper a variety of mercurials, including a pCMB-nitroxide analogue, were used to study urea transport in human red cell ghosts. It was determined that the rate of inhibition for pCMBS, pCMB, pCMB-nitroxide, and chlormerodrin extended over four orders of magnitude consistent with their measured oil/water partition coefficients. From these results, we concluded that a significant hydrophobic barrier limits access to the urea inhibition site, suggesting that the urea site is buried in the bilayer or in a hydrophobic region of the transporter. In contrast, the rate of water inhibition by the mercurials ranged by only a factor of four and did not correlate with their hydrophobicities. Thus, the water inhibition site may be more directly accessible via the aqueous phase. Under conditions that leave water transport unaffected, we determined that ≤32,000 labeled sites per cell corresponded to complete inhibition of urea transport. This rules out major transmembrane proteins such as band 3, the glucose carrier, and CHIP28 as candidates for the urea transporter. In contrast, this result is consistent with the Kidd (Jk) antigen being the urea transporter with an estimated 14,000 copies per cell. From the experimental number of urea sites, a turnover number between 2–6×106 sec−1 at 22°C is calculated suggesting a channel mechanism.

Modification of the Erythrocyte Membrane Dielectric Constant by Alcohols

SummaryAliphatic alcohols are found to stimulate the transmembrane fluxes of a hydrophobic cation (tetraphenylarsonium, TPA) and anion (AN-12) 5–20 times in red blood cells. The results are analyzed using the Born-Parsegian equation (Parsegian, A., 1969,Nature (London)221:844–846), together with the Clausius-Mossotti equation to calculate membrane dielectric energy barriers. Using established literature values of membrane thickness, native membrane dielectric constant, TPA ionic radius, and alcohol properties (partition coefficient, molar volume, dielectric constant), the TPA permeability data is predicted remarkably well by theory. If the radius of AN-12 is taken as 1.9 Å, its permeability in the presence of butanol is also described by our analysis. Further, the theory quantitatively accounts for the data of Gutknecht and Tosteson (Gutknecht, J., Tosteson, D.C., 1970,J. Gen. Physiol.55:359–374) covering alcohol-induced conductivity changes of 3 orders of magnitude in artificial bilayers. Other explanations including perturbations of membrane fluidity, surface charge, membrane thickness, and dipole potential are discussed. However, the large magnitude of the stimulation, the more pronounced effect on smaller ions, and the acceleration of both anions and cations suggest membrane dielectric constant change as the primary basis of alcohol effects.

Anion exchange in human erythrocytes has a large activation volume

Sulfate equilibrium exchange in human red cells has an activation volume of +150 +/- 20 cm3/mol over the pressure range 0.1 to 83 MPa (15 to 12000 lb/in2) at 30 degrees C. This value greatly exceeds the expected contribution from sulfate binding to the anion exchanger. We suggest that the activation volume reflects conformational changes during the transport cycle.