Lack of binding of glyceraldehyde-3-phosphate dehydrogenase to erythrocyte membranes under in vivo conditions.

A filtration method is described for separating membrane-free cytoplasm from concentrated erythrocyte haemolysates. The method has been used to assess glyceraldehyde-3-phosphate dehydrogenase binding to erythrocyte membranes. The relative amounts of glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase in the cytoplasm (either oxygenated or deoxygenated) indicate there is no detectable binding of glyceraldehyde-3-phosphate dehydrogenase to the membranes under physiological conditions.

The effects of the membrane-penetrating polypeptide segment of the human erythrocyte MN-glycoprotein on the permeability of model lipid membranes.

The insoluble peptide, T(is), prepared by trypsin hydrolysis of the MN-glycoprotein (glycophorin) of the human erythrocyte has been incorporated into phospholipid membranes in the form of liposomes and black lipid membranes. The permeability of liposome membranes to 42K+ and of black lipid membranes to water and ions is increased significantly by the presence of the T(is) peptide. Electrophoresis measurements indicate that these effects are not due to the T(is) peptide carrying a net charge. The results suggest that the peptide causes local disordering of the bilayer membrane structures. This is considered in the light of findings published elsewhere: that the MN-glycoprotein penetrates through the cell membrane via a non-polar segment of its polypeptide chain, which is contained intact within the T(is) peptide; that the T(is) peptide is partially helical when associated with phospholipid and forms multimeric 8.0 nm structures within the hydrophobic plane of phospholipid bilayers.

Effect of osmolality on the hydraulic permeability coefficient of red cells.

The osmotic water permeability coefficient, L(p), for human and dog red cells has been measured as a function of medium osmolality, and found to depend on the osmolality of the bathing medium. In the case of human red cells L(p) falls from 1.87 x 10(-11) cm(3)/dyne sec at 199 mOSM to 0.76 x 10(-11) cm(3)/dyne sec at 516 mOSM. A similar decrease was observed for dog red cells. Moreover, L(p) was independent of the direction of water movement and the nature of the solute used to provide the osmotic pressure gradient; it depended only on the final osmolality of the medium. Furthermore, L(p) was not affected by pH in the range of 6 to 8 nor by the presence of drugs such as valinomycin (1 x 10(-6)M) and tetrodotoxin (3.2 x 10(-6)M). The instantaneous nature of the response to changes in external osmolality suggests that the hydraulic conductivity of the membrane is controlled by a thin layer at the outer face of the membrane.

Determination of urea permeability in red cells by minimum method. A test of the phenomenological equations.

A new method has been developed for measuring the permeability coefficient, omega, of small nonelectrolytes. The method depends upon a mathematical analysis of the time course of cell volume changes in the neighborhood of the minimum volume following addition of a permeating solute to an isosmolal buffer. Coefficients determined by the minimum volume method agree with those obtained using radioactive tracers. omega for urea in human red cells was found to decrease as the volume flow, J(v), into the cell increased. Such behavior is entirely unexpected for a single uniform rate-limiting barrier on the basis of the linear phenomenological equations derived from irreversible thermodynamics. However, the present findings are consonant with a complex membrane system consisting of a tight barrier on the outer face of the human red cell membrane and a somewhat less restrictive barrier behind it closer to the inner membrane face. A theoretical analysis of such a series model has been made which makes predictions consistent with the experimental findings.

Permeability studies on red cell membranes of dog, cat, and beef.

Water permeability coefficients of dog, cat, and beef red cell membranes have been measured under an osmotic pressure gradient. The measurements employed a rapid reaction stop flow apparatus with which cell shrinking was measured under a relative osmotic pressure gradient of 1.25 to 1.64 times the isosmolar concentration. For the dog red cell the osmotic permeability coefficient is 0.36 cm(4)/(sec, osmol). The water permeability coefficient for the dog red cell under a diffusion gradient was also measured (rate constant = 0.10/msec). The ratio between the two permeabilities was used to calculate an equivalent pore radius of 5.9 A. This value agrees welt with an equivalent pore radius of 6.2 A obtained from reflection coefficients of nonelectrolyte water-soluble molecules, and is consistent with data on the permeability of the dog red cell membrane to glucose. These data provide evidence supporting the existence of equivalent pores in single biological membranes.

The effect of the unstirred layer on human red cell water permeability.

A study has been made of water entry into human red blood cells under an osmotic pressure gradient. The measurements were made using a rapid reaction stop flow apparatus, whose construction, calibration, and performance are described in detail. Red cell volume changes were determined from 90 degrees scattered light. The permeability coefficient for water entry under a relative isosmolar concentration of 1 to 1.5 was found to be 0.22 +/- 0.01 cm(4)/sec osmol, which agrees well with our previously published value. The experiments were also designed to measure the thickness of the unstirred layer around the6 red cells. This was found to be 5.5 +/- 0.8 micro under the present experimental conditions. It is concluded that our previously measured permeability coefficient for water entrance under a diffusion gradient does not require correction on account of the unstirred layer.

Low pH enhances the transfer of carotene from carrot juice to olive oil.

A current model for carotenoid transport and absorption in the gut proposes an initial solubilization in the oil phase of dietary emulsions followed by incorporation of the carotenoids in mixed bile salt micelles. To assess the relevance of the first stage of this model to what is observed in vivo we have examined the transfer of carotene from carrot juice to olive oil. Increased acidity enhanced the transfer from both whole juice and carotene crystals isolated from carrot chromoplasts. The transfer was significantly slower from whole juice. By using exogenous beta-carotene and measuring its transfer to oil in the presence and absence of carrot juice we have demonstrated that the inhibition of the transfer in juice arises, at least in part, from soluble juice factors. The inhibition is relieved by a fall in pH, which leads to lowering of the electric potential at the oil/aqueous phase interface and aggregation of carrot tissue including crystalline carotene. Under conditions of low pH, oil droplets adhere to the tissue aggregates, allowing carotene to pass into the oil. Our results provide an explanation for why carotene absorption in vivo is depressed by conditions of low gastric acidity.

Glyceraldehyde-3-phosphate dehydrogenase release from erythrocytes during haemolysis. No evidence for substantial binding of the enzyme to the membrane in the intact cell.

Glyceraldehyde-3-phosphate dehydrogenase (G3PDH, EC 1.2.1.12) release from haemolysing erythrocytes, and its redistribution between free and membrane-bound states, were studied with a new type of rapid-mixing filtration apparatus. The apparatus is described. The results indicate that the rate of G3PDH appearance in filtrates is determined not only by the enzyme redistribution but also by the kinetics of haemolysis. We have quantified the extent of haemolysis as a function of time, by measuring the amounts of filterable K+ and lactate dehydrogenase. These are cytoplasmic components that are not membrane-bound. When we correct for incomplete haemolysis, extrapolation to zero times indicates that very little G3PDH is membrane-bound in the intact cell.

High-pressure treatment of milk: effects on casein micelle structure and on enzymic coagulation.

High isostatic pressures up to 600 MPa were applied to samples of skim milk before addition of rennet and preparation of cheese curds. Electron microscopy revealed the structure of rennet gels produced from pressure-treated milks. These contained dense networks of fine strands, which were continuous over much bigger distances than in gels produced from untreated milk, where the strands were coarser with large interstitial spaces. Alterations in gel network structure gave rise to differences in rheology with much higher values for the storage moduli in the pressure-treated milk gels. The rate of gel formation and the water retention within the gel matrix were also affected by the processing of the milk. Casein micelles were disrupted by pressure and disruption appeared to be complete at treatments of 400 MPa and above. Whey proteins, particularly beta-lactoglobulin, were progressively denatured as increasing pressure was applied, and the denatured beta-lactoglobulin was incorporated into the rennet gels. Pressure-treated micelles were coagulated rapidly by rennet, but the presence of denatured beta-lactoglobulin interfered with the secondary aggregation phase and reduced the overall rate of coagulation. Syneresis from the curds was significantly reduced following treatment of the milk at 600 MPa, probably owing to the effects of a finer gel network and increased inclusion of whey protein. Levels of syneresis were more similar to control samples when the milk was treated at 400 MPa or less.

Estimation of the free [Ca2+] gradient across endoplasmic reticulum membranes by a null-point method.

The rate of unidirectional efflux of 45Ca from rat liver microsomal vesicles loaded with 45Ca and then treated with thapsigargin is not inhibited by increased [Ca2+] in the external medium, although the net efflux rate is substantially inhibited. We have used this property to measure the electrochemical gradient of Ca2+ from the inside to the outside of the vesicles at a series of Ca2+ loadings, by measuring the external [Ca2+]free at which there is zero net efflux. At a loading of 7.9 +/- 0.6 nmol/mg of microsomal protein, the apparent internal [Ca2+]free is 21 +/- 1.6 microM. As the loading is increased, the internal [Ca2+]free increases linearly up to a value of 47 +/- 3.6 microM at a loading of 21.6 +/- 1.6 nmol/mg. Using a similar technique, the value for [Ca2+]free in the endoplasmic reticulum of permeabilized L1210 cells was found to be 12.5 microM.

Effects of CoA and acyl-CoA on Ca(2+)-permeability of endoplasmic-reticulum membranes from rat liver.

We have studied the effects of CoA and palmitoyl-CoA on Ca2+ movements and GTP-dependent vesicle fusion in rat liver microsomes. (1) Inhibition of membrane fusion by CoA depends on esterification of CoA to long-chain acyl-CoA using endogenous non-esterified fatty acids. (2) Binding of long-chain acyl-CoA to microsomal membranes is inhibited by BSA, which also relieves inhibition of membrane fusion. (3) Under conditions where acyl-CoA binding is inhibited, CoA causes increased Ca2+ accumulation, apparently by decreasing the Ca2+ leak rate. (4) Conversely, palmitoyl-CoA, in the presence of BSA, causes Ca2+ efflux. (5) The decrease in Ca(2+)-permeability caused by CoA does not depend on the presence of ATP or GTP, and is irreversible in the short term. (6) Using 14C-labelled CoA we show that CoA derivatives can be formed from endogenous components of microsomal membranes in the absence of ATP. (7) The results are interpreted in terms of a Ca(2+)-permeability which is controlled by CoA and/or long-chain acyl-CoA esters.

Thiopentone and propofol: a compatible mixture?

The physical compatibility of thiopentone and propofol mixtures was investigated. The investigations used were macroscopic and microscopic observations, zeta potential and oil droplet size measurements. There was no evidence of instability in the mixtures. The thiopentone-propofol mixture has the potential advantage of reducing the pain on injection, provides synergistic interaction, does not prolong recovery when used for induction of anaesthesia, may reduce the incidence of convulsions and is cost-effective.