Oxygenation-linked subunit interactions in human hemoglobin: experimental studies on the concentration dependence of oxygenation curves

An experimental study on the concentration dependence of oxygenation curves for human hemoglobin has been carried out between 4 X 10(-8) M heme and 5 X 10(-4) M heme in 0.1 M tris(hydroxymehtyl)aminomethane hydrochloride, 0.1 M NaCl, 1 mM disodium ethylenediaminetetraacetic acid, pH 7.4, 21.5 degrees C. With decreasing hemoglobin concentration the curves show pronounced shifts in position and shape, consistent with dissociation of tetrameric hemoglobin into dimeric species of high affinity and low cooperativity. Combination of these data with independently determined values of dissociation constants for unliganded and fully liganded hemoglobin permits a resolution of the seven parameters necessary to define the linked binding and subunit association processes. The total oxygenation-linked subunit dissociation energy (6.34 kcal) was resolved into intersubunit contact energy changes between alphabeta dimers in tetrameric hemoglobin which accompany binding of the first, middle two, and last oxygen molecules. The resolution is accurate to within approximately +/-0.3 kcal. To within this limit the isolated dimers are found to bind oxygen noncooperatively and with the same affinity as isolated alpha and beta chains. Equally good fits to the data are obtained when dimers are slightly anticooperative. At least three major energetic states are apparently assumed by hemoglobin tetramers, with respect to the alpha1beta2 contact region, corresponding to (a) unliganded, (b) singly liganded, (c) triply and quadruply liganded species. The results do not establish whether these states may be assumed by a single molecule, or whether they arise as averages over a distrubution of conformational states. They do provide unequivocal evidence against a concerted transition at any particular binding step in a system with only two energetic states of tetramer (i.e., an all or none switchover between T and R states at a particular binding step).     

A study of the relationship between inhibition of anion exchange and binding to the red blood cell membrane of 4,4'-diisothiocyano stilbene-2,2'-disulfonic acid (DIDS) and its dihydro derivative (H2DIDS)

DIDS (4,4'-diisothiocyano stilbene-2,2'-disulfonic acid) and H2DIDS (4,4'-diisothiocyano-1,2-diphenyl ethane-2,2'-disulfonic acid) binding to the human red cell membrane proteins were studied as a function of concentration, temperature and time. Most binding sites were common to both. The common sites were in band 3 of SDS polyacrylamide gel electropherograms (Steck, 1974. J. Cell Biol. 62:1), an unidentified adjacent band, and glycophorin. Reversible and irreversible binding occurred; both inhibited sulfate equilibrium exchange. The time courses of irreversible binding to band 3 and total binding to the membrane as a whole were biphasic. About 20% of H2DIDS and greater 60% of DIDS binding were rapid, independent of temperature. Slow H2-DIDS binding was monoexponential, activation enthalpy 23 kcal/mole. The stoichiometry of irreversible H2DIDS binding to band 3 was 1.1-1.2, concentration-dependent. Under the conditions studied (0-50 muM, hematocrit 10%, 5-37 degrees C) binding to band 3 was a constant fraction of total binding, 0.7 for H2DIDS and 0.8 for DIDS. Inhibition was a linear function of total binding, binding to band 3, and therefore also to nonband 3 sites, with either inhibitor during both phases, H2DIDS inhibition was complete at 1.9 X 10(6) or 1.2 X 10(6) molecules/cell total and band 3 binding respectively. For DIDS the corresponding figures were 1.3 X 10(6) and 1.1 X 10(6). It is shown how reagents of mixed function can react with biphasic kinetics. Binding to multiple contiguous sites may exhibit concentration-dependent stoichiometry. Under such conditions a linear inhibition-binding relationship is neither a necessary nor a sufficient condition for the identification of transport sites.     

Role of membrane-bound Ca in ghost permeability to Na and K

The permeability of red cell ghosts to K is determined by the amount of membrane-bound Mg which, in turn, depends on internal Mg. Contrasting with such effect, an increase in cellular Ca raises K permeability. To test whether this action is due to a competitive displacement of membrane Mg, the free Ca content of human red cell ghosts was altered by means of Ca-EGTA buffers. Net Na and K movements as well as Ca and Mg bindings were assessed after incubation in a Na-medium at 37 degrees C. Raising Ca from 3 X 10(-7) to 1 X 10(-2) M caused a large K efflux with very little Na gain. Under similar conditions, Ca binding was increased without affecting membrane-bound Mg. Both Ca binding and K loss were markedly diminished by either adding ATP to the hemolytic medium or increasing internal Mg at a fixed Ca concentration. A Scatchard analysis showed three Ca binding sites, two of them having high affinity. It is concluded that Ca action does not arise from a displacement of membrane-bound Mg but from binding to different sites in the membrane. Presumably, high affinity sites are involved in the control of K permeability.     

Biotransformation of 4-dimethylaminophenol: reaction with glutathione, and some properties of the reaction products

4-Dimethylaminophenol (DMAP) forms ferrihemoglobin by catalytic transfer of electrons from ferrohemoglobin to oxygen. In solutions of purified human hemoglobin, quick binding of oxidized DMAP to the globin moiety of hemoglobin terminates this reaction. Reduced glutathione in high concentrations, as in the red cell, substantially diminished binding of oxidized DMAP to hemoglobin by formation of S,S,S-(2-dimethylamino-5-hydroxy-1,3,4-phenylene)-tris-glutathione (tris-(GS)-DMAP), which does not form ferrihemoglobin. In the presence of reduced glutathione, DMAP disappeared more rapidly from hemoglobin solutions than in its absence. The formation of tris(GS)-DMAP in red cells was found to be of importance for the termination of catalytic ferrihemoglobin formation by DMAP in vivo. With low concentrations of GSH, DMAP in hemoglobin solutions formed another conjugate, (GS)-DMAP, S,S(2-dimethylamino-5-hydroxy-1,3-phenylene)-bis-glutathione. Similar to DMAP, bis(GS)-DMAP catalyzed the formation of ferrihemoglobin. As the oxidized bis(GS)-DMAP was bound to hemoglobin more slowly and to a lesser extent, it produced more ferrihemoglobin than DMAP. In contrast to the reactions of DMAP with hemoglobin, hydrogen peroxide and superoxide radicals are involved in the ferrihemoglobin formation by bis(GS)-DMAP. The radicals accelerate the oxidation of bis(GS)-DMAP and thereby the ferrihemoglobin formation.     

Effect of calcium and magnesium on binding of beta, gamma-methylene ATP to sarcoplasmic reticulum

Isolated sarcotubular membranes (SR) from skeletal muscle bound 3.7 nmol of beta, gamma-methylene [8-3H]ATP (AMP-PCP) per mg of membrane protein. Only one class of binding site was identified and the dissociation constant (K) for this site was 1.5 X 10(-5) M. Addition of 0.05% Triton X-100 increased the number of binding sites to 5.7 nmol/mg. ATP and ADP competitively inhibited AMP-PCP binding. The dissociation constants for ATP and ADP were 3.5 X 10(-5) M and 3.3 X 10(-6) M, respectively. Since this data was obtained in the presence of 5 mM EDTA, it was established that the sarcoplasmic reticulum has a high affinity for the metal free forms of ATP, ADP, and AMP-PCP. Magnesium concentrations in excess of 1 X 10(-4) M inhibited AMP-PCP binding. Lower concentrations of magnesium had little effect on AMP-PCP binding. The effect of calcium on AMP-PCP binding was biphasic. Calcium concentration between 1 X 10(-6) and 1 X 10(-4) M inhibited AMP-PCP binding. Inhibition was maximal at 1 X 10(-5) M. Calcium concentration above 1 X 10(-4) M facilitated analogue binding. Possible sites of magnesium and calcium actions are discussed.     

Binding of insulin analogs to partially purified insulin receptor from rat liver membrane (author's transl)

The insulin binding fraction was solubilized from rat liver membrane vesicles by triton and partially purified up to the specific binding activity of 2.1 pmol/mg. A further characteristic of the partially purified soluble receptor is a decrease in irreversible binding to 0.36% (+/- 0.28) with regard to total iodine insulin and to (+/- 1.8) in comparison to reversible binding. From Scatchard plots a high affinity binding site (KD = 5 X 10(-10)M) with low capacity (5 pmol/mg) and a low affinity binding site (KD = 3 X 10(-8) M) with high capacity (30 pmol/mg) can be seen. With carefully prepared liver membrane vesicles, the dissociation constant of the high affinity binding site from Scatchard plot is only 2 X 10(-9)M. With liver membrane vesicles, isolated for preparative purification procedure, the high affinity binding site could not be demonstrated. Displacement studies with insulin analogs were performed with [A1-D-alanine] insulin, [A1-L-alanine] insulin, [des-Gly-A1, NB1, NB29-(Msc)2]-insulin, proinsulin and [desoctapeptid B23-B30]-insulin. Results of binding measurements are presented in half-maximal iodo-insulin binding, in determination of inhibitor- and dissociation constants from Dixon-, Scatchard- and Lineweaver-Burk plots. There are equal relative binding potencies of analogs, observed with crude membrane vesicles and partially purified soluble receptor, although there is a 50-fold difference in specific binding activity. Biologically active insulins are characterized by strong binding to the high affinity binding site. The binding to the low affinity binding site is not correlated to the biological activity of the insulin analog. With insulin and biologically responsive analogs a non-linear curve in the double-reciprocal Lineweaver-Burk plot can be observed. Analogs with low biological activity show a linear dependency. Functional interactions of insulin with the receptor can be demonstrated in a high affinity binding with the first binding site of the Scatchard plot and in a non-linear hyperbolic Lineweaver-Burk plot.     

Distribution and role of aspartate in the nervous system of the chaetognath Sagitta

Forty-nine female workers in the shoemaking industry, exposed to a solvent mixture containing benzene and twenty-seven non-exposed controls, were investigated. Concentrations of benzene and toluene in the working atmosphere, as well as benzene and toluene in blood and phenols in pre- and post-shift urine as parameters of biological monitoring, were determined. In order to assess hematotoxic risk, a complete blood cell count with differential, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, reticulocytes, serum iron, alkaline phosphatase in neutrophils and red blood cell glycerol lysis time were determined in all subjects. Benzene concentrations in the workplace atmosphere at the shoemaking factory ranged from 1.9 to 14.8 ppm (median = 5.9). Significant difference in benzene in blood (p = 0.005) and phenol in post-shift urine (p = 0.003) between exposed workers and controls confirmed exposure to benzene. Hemoglobin level (p = 0.02) and mean corpuscular hemoglobin concentration (p = 0.0002) in the shoe workers were lower, and band neutrophils (p = 0.005) and mean corpuscular volume (p = 0.03) higher, than in controls. Red blood cell glycerol lysis time was significantly higher (p = 0.000001) in shoe workers (X +/- SD = 41.6 +/- 8.9) than in controls (X +/- SD = 31.1 +/- 6.5) and showed a significant correlation with exposure biomarkers. The results confirm that benzene exposure below 15 ppm may produce qualitative abnormalities, particularly macroerythrocytosis and increased red cell glycerol resistance, in the absence of an overt quantitative decrease in circulating blood cells. Increased resistance to the hemolytic action of glycerol is a potentially useful biological monitoring procedure in medical surveillance of benzene exposed workers. The results of this study suggest that potential threshold concentration for hematologic effects of benzene is lower than 15 ppm.     

Glucose transport in isolated perfused proximal tubules of snake kidney

Glucose transport was studied in isolated, perfused snake (Thamnophis spp.) renal tubules. When 14C-labeled and unlabeled glucose concentrations for bath and perfusate were identical, net transepithelial glucose transport occurred from lumen to bath. Maximum rates of transport were 1.24 X 10-12 and 2.17 X 10-12 mol min-1 mm-1 in proximal-proximal and distal-proximal segments, respecitvely. Glucose concentration in cells of perfused tubules of both segments was less than that of bath and lumen when tubules spontaneously stopped transporting glucose. Transepithelial glucose permeability ath leads to lumen) was about 0.25 X 10-5 cm sec-1 for both segments. Peritubular membrane permeability (bath leads to cell) was about 0.50 X 10-5 cm sec-1 for both segments. Luminal membrane permeabilities (cell leads to lumen) were 0.29 X 10-5 and 0.65 X 10-5 cm sec-1 for proximal-proximal and distal-proximal segments, respectively. Luminal membrane permeability in opposite direction (lumen leads to cell) was about 10.0 X 10-5 cm sec-1 for both segments. These results indicate that, during maximum glucose absorption, glucose enters cells down concentration gradient across luminal membrane by a mediated process and is transported out of the cells against concentration gradient at peritubular membrane.     

Moxisylyte: a review of its pharmacodynamic and pharmacokinetic properties, and its therapeutic use in impotence

A technique is described for opening the membrane of a red blood cell by electroporation in a manner which permits free exchange of the native hemoglobin with exogenous hemoglobin in the surrounding medium. After resealing the RBC's demonstrate near normal size and hemoglobin content and retain an effective methemoglobin reduction system. This method can be used to introduce natural or genetically engineered hemoglobins with altered oxygen binding characteristics. Allosteric effectors and other non-diffusible small molecules can be encapsulated during the same procedure. A fish Root effect hemoglobin exchanged into rat RBC's produced oxygen transport characteristics, unloading at high pressure at acidic pH, which should be useful to treat tissue hypoxia from a variety of causes.     

The adaptation of the fetal red cells of newborn lambs to extrauterine life: the role of 2,3-diphosphoglycerate and and adult hemoglobin

The purpose of this study was to determine the interrelationship of the rise and fall of 2,3-diphosphoglycerate (DPG) with the increase in adult hemoglobin and the decrease in red cell oxygen hemoglobin affinity after birth in normal lambs. It was found that the mean maximum DPG level was 26.71 +/- 4.98 mol/g Hb at 7.5 +/- 1.1 days. At the same time the mean P50 and adult hemoglobin level was 27.0 +/- 1.4 mm Hg and 31.1 +/- 11.i%, respectively. In the individual lambs, the level of their maximum DPG correlated inversely with the amount of adult hemoglobin (r-0.77, P less than 0.05). Once the DPG began to decrease, there was an inverse correlation between the DPG and the adult hemoglobin present in the red cell (r = 0.68, P less than 0.001). It appeared that the rise in DPG postanatally is only a compensatory mechanism until an adequate amount of adult hemoglobin is present. This fact was borne out by the second part of the study in which exchange transfusions with adult red cells were performed on five newborn lambs during the first 24 hr after birth and aborted the rise in DPG.     

Kinetics of bicarbonate-chloride exchange across the human red blood cell membrane

The kinetics of bicarbonate-chloride exchange across the human red cell membrane was studied by following the time course of extracellular pH in a stopped-flow rapid-reaction apparatus during transfer of H+ into the cell by the CO2 hydration-dehydration cycle, under conditions where the rate of the process was determined by HCO3--Cl- exchange flux across the membrane. The flux of bicarbonate increased linearly with [HCO3-] gradient from 0.6 to 20 mM across the red cell membrane at both 37 degrees C and 2 degrees C, and decreased as transmembrane potential was increased by decreasing extracellular [Cl-]. An Arrhenius plot of the rate constants for the exchange indicates that the Q10 is strongly dependent on temperature, being about 1.7 between 24 degrees C and 42 degrees C and about 7 between 2 degrees C and 12 degrees C. These data agree well with the published values for Q10 of 1.2 between 24 degrees C and 40 degrees C and of 8 between 0 degrees C and 10 degrees C. The results suggest that different processes may determine the rate of HCO3--Cl- exchange at low vs. physiological temperatures, and that the functional (and/or structural) properties of the red cell membrane vary markedly with temperature.     

Binding properties of bovine colostral anti-dinitrophenyl (dnp) immunoglobulins g2 (igg2)

The number of combining sites per mole of bovine colostral anti-DNP IgG2 was found to be 2 and the total affinity constant was 0.81 X 10(4) M-1. Unlike bovine colostral IgG1, the nonspecific binding of H3-epsilon-DNP-1-lysine by IgG2 as a function of increased concentrations, did not show a negative, cooperative slope. Spectral measurements made with anti-DNP IgG2 in the reference cell vs. anti-DNP IgG2 plus hapten in the experimental cell revealed a hypochromic, red shift from 363 to 365 nm. If the reference cell contained hapten, a red shift from 280 to 283 nm and an enhancement in the extinction coefficient of IgG2 was observed. These spectral changes were not observed if nonspecific IgG2 was substituted for anti-DNP IgG2 in the experimental cell. The enhancement in the extinction coefficient was interpreted to be due to a possible exposure of previously buried tryosine and tryptophan residues.     

Fluorescent probing of membrane potential in walled cells: diS-C3(3) assay in Saccharomyces cerevisiae

Membrane-potential-dependent accumulation of diS-C3(3) in intact yeast cells in suspension is accompanied by a red shift of the maximum of its fluorescence emission spectrum, lambda max, caused by a readily reversible probe binding to cell constituents. Membrane depolarization by external KCl (with or without valinomycin) or by ionophores causes a fast and reproducible blue shift. As the potential-reporting parameter, the lambda max shift is less affected by probe binding to cuvette walls and possible photobleaching than, for example, fluorescence intensity. The magnitude of the potential-dependent red lambda max shift depends on relative cell-to-probe concentration ratio, a maximum shift (572-->582 nm) being found in very thick suspensions and in cell lysates. The potential therefore has to be assessed at reasonably low cell (< or = 5 x 10(6) cells/ml) and probe (10(-7)M) concentrations at which a clearly defined relationship exists between the lambda max shift and the potential-dependent accumulation of the dye in the cells. The redistribution of the probe between the medium and yeast protoplasts takes about 5 min, but in intact cells it takes 10-30 min because the cell wall acts as a barrier, hampering probe penetration into the cells. The barrier properties of the cell wall correlate with its thickness: cells grown in 0.2% glucose (cell wall thickness 0.175 +/- 0.015 micron, n = 30) are stained much faster and the lambda max is more red-shifted than in cells grown in 2% glucose (cell wall thickness 0.260 +/- 0.043 micron, n = 44). At a suitable cell and probe concentration and under standard conditions, the lambda max shift of diS-C3(3) fluorescence provides reliable information on even fast changes in membrane potential in Saccharomyces cerevisiae.     

Evidence for the denaturation of recombinant hepatitis B surface antigen on aluminium hydroxide gel

Eosinophils, prominent cells in asthmatic inflammation, undergo apoptosis or programmed cell death following deprivation of contact with survival-promoting cytokines such as IL-5 and GM-CSF. The aim of this study was to assess a number of techniques for the quantification of apoptosis in human eosinophils cultured with or without IL-5 or GM-CSF and following staurosporine treatment. The relationship between apoptosis and necrosis in eosinophils was also determined. Eosinophils 'aged' in vitro for 48 h exhibited endonuclease DNA degradation, apoptotic morphology, increased red autofluorescence and externalisation of phosphatidylserine (PS) as assessed by binding of FITC-labelled annexin V. Annexin V-FITC binding was first detectable in eosinophils maintained at 37 degrees C for 5 h post-purification. This method proved to be the most sensitive marker of apoptosis. Morphological assessment of wet preparations of eosinophils by Kimura staining was found to be the next most-sensitive marker followed by increased red autofluorescence. The latter was a relatively insensitive method for the detection of apoptosis. At 5, 20 and 24 h of culture trypan blue exclusion indicated that eosinophil viability was high (85-90% viable cells). However, propidium iodide (PI) staining and flow cytometry revealed that, by 24 h, approximately 75% of cells had compromised membrane integrity. Eosinophils maintained in IL-5 or GM-CSF exhibited a non-apoptotic morphology and levels of annexin V-FITC binding and PI uptake similar to that of freshly isolated cells. Staurosporine (10(-5) M) treatment of eosinophils maintained in IL-5 or GM-CSF resulted in significant levels of apoptotic morphology at 2 h (23.8% +/- 6.9, p < 0.025) which was associated with negligible annexin binding. At 6 h post-staurosporine treatment significant annexin-FITC binding (38% +/- 1.5, p < 0.025) was observed compared with 93% +/- 1.2 of eosinophils displaying apoptotic morphology. Exclusion of PI demonstrated membrane integrity at all time points up to 6 h. Thus, eosinophils aged in vitro in the absence of viability-promoting cytokines exhibit evidence of both apoptosis and necrosis simultaneously. In contrast, staurosporine-treated eosinophils exhibited both membrane integrity and rapid apoptosis-associated morphological changes detected by single step Kimura staining which preceded externalisation of PS.     

White blood cell counts and plasma C3a have synergistic predictive value in patients at risk for acute respiratory distress syndrome

The molecular pathobiology of membrane-associated iron is clearly illustrated by the sickle red blood cell. The cytosolic aspect of the membranes of these cells carries several discrete iron compartments, including denatured hemoglobin and free heme, as well as molecular iron associated with membrane aminophospholipid and denatured globin. Affinity of the membrane for molecular iron is extraordinarily high and predicted to keep cytosolic free iron concentration < 10(-20) M. Membrane iron is bioactive and able to valence cycle, thus serving as a catalyst for generation of highly reactive hydroxyl radical. As a consequence of this oxidative biochemistry at the cytosol/membrane interface, multiple membrane defects arise that are of pathophysiologic importance. Thus, sickle red cells provide a pathobiologic paradigm for the membrane-damaging effect of iron-mediated targeting of oxidative damage at a sub-cellular level. This is relevant to a variety of biologic conditions accompanied by decompartmentalization of iron.     

Studies of the mechanism for enhanced cell surface factor VIIa/tissue factor activation of factor X on fibroblast monolayers after their exposure to N-ethylmaleimide

Fibroblast monolayers constitutively expressing surface membrane tissue factor (TF) were treated with 0.1 mM N-ethylmaleimide (NEM) for 1 min to inhibit aminophospholipid translocase activity without inducing general cell damage. This resulted in increased anionic phospholipid in the outer leaflet of the cell surface membrane as measured by the binding of 125I-annexin V and by the ability of the monolayers to support the generation of prothrombinase. Specific binding of 125I-rVIIa to TF on NEM-treated monolayers was increased 3- to 4-fold over control monolayers after only brief exposure to 125I-rVIIa, but this difference progressively diminished with longer exposure times. A brief exposure of NEM-treated monolayers to rVIIa led to a maximum 3- to 4-fold enhancement of VIIa/TF catalytic activity towards factor X over control monolayers, but, in contrast to the binding studies, this 3- to 4-fold difference persisted despite increasing time of exposure to rVIIa. Adding prothrombin fragment 1 failed to diminish the enhanced VIIa/TF activation of factor X of NEM-treated monolayers. Moreover, adding annexin V, which was shown to abolish the ability of NEM to enhance factor X binding to the fibroblast monolayers, also failed to diminish the enhanced VIIa/TF activation of factor X. These data provide new evidence for a possible mechanism by which availability of anionic phospholipid in the outer layer of the cell membrane limits formation of functional VIIa/TF complexes on cell surfaces.     

Factors affecting the amount and the mode of merocyanine 540 binding to the membrane of human erythrocytes. A comparison with the binding to leukemia cells

In the presence of albumin Merocyanine 540 (MC540) exhibits a very limited binding to the outer surface of the membrane of normal erythrocytes, whereas pronounced binding is observed to leukemia cells. To find out whether this difference is due to differences in the composition or structural organization of the cell membrane we analyzed effects of a number of covalent and non-covalent perturbations of the red cell membrane on the binding and fluorescence characteristics of membrane-bound MC540. It is shown that exposure of the cells to cationic chlorpromazine, neuraminidase or photodynamic treatment with AlPcS4 as sensitizer caused a limited increase (30-50%) of MC540 binding, together with a red shift of the fluorescence emission maximum and an increase of the relative fluorescence quantum yield of membrane-bound MC540. Other forms of perturbation of the membrane structure, like hyperthermia (48 degrees C) and treatments that produce a decrease of phospholipid asymmetry in addition to accelerated flip-flop, did not result in increased MC540 binding, but did cause a red shift of the fluorescence emission maximum and an increase of the relative fluorescence quantum yield. These changes in fluorescence properties indicate a penetration of the dye into more hydrophobic regions in the membrane. MC540, bound to Brown Norway myelocytic leukemia cells, exhibited a red shift of the fluorescence emission maximum and an increased relative fluorescence quantum yield as compared to MC540 bound to untreated erythrocytes. These changes were of the same order of magnitude as in photodynamically treated red blood cells. Dye binding per surface area, however, was about 3-times higher with these leukemia cells than with photodynamically treated red blood cells. This demonstrates that certain perturbations of the erythrocyte membrane evoked a MC540 binding that became qualitatively comparable to the dye binding to leukemia cells, although dye binding per surface area was still significantly lower.     

Permeability of individual human erythrocytes to thiourea

The osmotic swelling to haemolysis of individual red blood cells by isosmotic thiourea has been studied using microcine photography. 2. Crenation occurs immediately upon addition of isosmotic thiourea. The cell becomes a crenated sphere without volume decrease. 3. Subsequently, the cell volume increases linearly with time with maximum swelling occurring at about 102 sec which is 81% of the total haemolysis time. 4. At maximum swelling, the cell volume is 92% greater than the initial cell volume. This volume increase is about double that measured with other permeating substances. 5. The much larger maximum volume implies that thiourea increases the area of the cell membrane. This increase varies from 0 to 75% for individual cells, with a mean of 22%. 6. Membrane expansion varies inversely as the initial cell membrane area and cell volume (r=0-790). 7. Using the increased surface area, increased maximum volume and the swelling time, the mean permeability is calculated to be 5-52 X 10(-7) cm/sec (S.D. of mean=+/-1-19 X 10(-7) cm/sec). The distribution of permeabilities represents a normal distribution. 8. The pre-lytic potassium loss ranged from 0 to 36% with a mean value of 16-5%. This is consistent with values reported in the literature for slow haemolysis. As with other permeants the distribution is skewed towards lower values. 9. Membrane permeability of individual cells varies with the amount of membrane expansion observed. Coefficient of correlation between permeability and expansion index is 0-674. 10. There is no correlation between permeability and the reciprocal of the haemolysis time (r=-0-035). The correlation between permeability and the reciprocal of the swelling time is also poor (r=0-303), probably owing to the variability in membrane expansion by thiourea in individual cells. 11. As has been shown previously for faster permeants, the permeability coefficient cannot be calculated from the haemolysis time. Because thiourea alters the membrane area and the haemolytic volume, the coefficient cannot be calculated from the swelling time unless the changes in the membrane area are also taken into account.     

The binding of phenol red to rabbit renal cortex

The binding of phenol red to the microsomal fraction of rabbit kidney cortex was rapid, reversible and consisted of two independent populations of binding sites: a high affinity and low capacity class which had an association constant of 11.29 - 10(3) M-1 and a binding capacity of 2.41 mumol phenol red bound per g of protein, and a low affinity binding population with an association constant of 0.80 - 10(3) M-1 and a maximal binding capacity of 55.06 mumol per g of protein. Probenecid (0.32 mM) competitively inhibited phenol red binding to only the high affinity binding site, whereas 2,4-dinitrophenol (0.77 mM) competitively inhibited phenol red binding to both the high and the low affinity population of binding sites. The binding of phenol red was highly sensitive to the cationic composition of the medium. The affinity of phenol red to the high and the low affinity binding populations was lowered by decreasing the sodium and potassium concentrations to 19 and 6 mequiv./l, respectively; however, the maximal binding capacity was unchanged. Calcium appeared to have no effect on the phenol red binding to the microsomes. All of these considerations suggest that the high affinity phenol red binding to the microsomal fraction may represent the interaction of phenol red with the physiological receptor necessary for organic acid transport at the peritubular membrane. Phenol red binding to the low affinity binding population may indicate an intracellular binding population which contributes to the intracellular accumulation of weak organic acids.