Interaction of bilirubin with human erythrocyte membranes.

The kinetics of [3H]bilirubin binding to human erythrocyte ghost membranes was investigated. The binding occurred rapidly and was saturable with respect to [3H]bilirubin and membrane concentration. The apparent dissociation constant (Kd) and maximum binding (Bmax.) for bilirubin of the membranes were 2.3 microM and 0.93 nmol/mg of protein respectively. Low-affinity binding, non-saturable at 400 microM, was observed. Thermal dependency of the saturable binding showed a U-shaped curve with the lowest value around 37 degrees C. Affinity labelling of the membrane proteins using [3H]bilirubin-Woodward's reagent K complex did not define individual proteins. The Kd (12 microM) and Bmax. (4.4 nmol/mg of protein) for bilirubin of the tryptic membranes increased 5.0 and 5.2 times the respective control values (2.4 microM and 0.85 nmol/mg of protein). Heat-treatment of the membranes for 3 min at 100 degrees C increased the saturable binding as much as by 222%. These results indicate that there exist saturable bilirubin-binding sites on the erythrocyte membranes and also suggest that they are not composed of proteins.     

Effect of phospholipase C, trypsin and neuraminidase on binding of bilirubin to mammalian erythrocyte membranes.

Binding of bilirubin to erythrocyte membranes of human, buffalo, sheep and goat was studied after phospholipase C, trypsin and neuraminidase treatment. Phospholipase C and trypsin treatment of membranes greatly enhanced the bilirubin binding in all mammalian species, whereas, neuraminidase treatment resulted into a small increase in the membrane-bound bilirubin. Human erythrocyte membranes bound the highest amount of bilirubin, whereas buffalo, sheep and goat erythrocyte membranes showed different mode of bilirubin binding. The order of bilirubin binding to unmodified as well as neuraminidase-treated erythrocyte membranes was: human>sheep>buffalo>goat; the order was: human>buffalo>sheep>goat; in phospholipase C- and trypsin-treated erythrocyte membranes. These binding results indicate that membrane phospholipids are directly involved in the interaction of bilirubin with the membranes as the differences observed in the membrane-bound bilirubin among mammalian species were directly correlated with the sum of choline phospholipids, especially phosphatidylcholine and sphingomyelin content of the erythrocyte membranes. The negatively charged phosphate moiety of phospholipids of the membranes appears to inhibit a large amount of bilirubin binding to the membrane as its removal by phospholipase C greatly enhanced the binding. Furthermore, membrane proteins and carbohydrate also seem to play a significant regulatory function on the binding as their degradation and/or removal in the form of glycopeptides by trypsin expose a large number of bilirubin binding sites.     

Modification of phospholipids in erythrocyte membranes by phospholipase D. A fluorescence and ESR spectroscopic study

The conversion of more than 65% of the phospholipids in human erythrocyte membranes to phosphatidyl-methanol and phosphatidic acid by incubation with phospholipase D and methanol increased the dissociation constant of the fluorescence probe ANS compared to untreated membranes, but did not affect the number of binding sites and the limiting fluorescence enhancement at maximal binding (Imax). On the contrary, the cationic fluorescence probe dansylcadaverin showed additional binding sites without a change in Kd and an increase of Imax upon incubation with phospholipase D treated erythrocyte membranes compared to incubations of membranes with the original phospholipid pattern. The characteristic temperature-dependence of the quenching of the membrane protein fluorescence by a membrane-bound nitroxide-labeled stearic acid was not influenced by the modification of the phospholipids. A slight reduction of the order parameter, S, determined by ESR-spectroscopy with the same nitroxide spin-labeled fatty acid incorporated into modified membranes compared to controls was found at 40 degrees C, but not at 25 degrees C. The results were interpreted as an indication of membrane domains that retained their physical properties and lipid composition during the incubation with phospholipase D.     

Gonadotropin receptors in plasma membranes of bovine corpus luteum. I. Effect of phospholipases on the binding of 125I-choriogonadotropin by membrane-associated and solubilized receptors.

The ability of bovine corpus luteum plasma membranes to bind 125I-choriogonadotropin has been examined after prior treatment of the membranes with phospholipases A, C, and D. Treatment of the purified membranes with low concentrations of phospholipases A and C resulted in the inhibition of the binding of 125I-choriogonadotropin to its receptors, whereas phospholipase D had no effect. Receptor activity was decreased by low concentrations of phospholipase A from either bee venom, Vipera russelli or Crotalus terrificus terrificus. Similarly, low concentrations of phospholipase C from Clostridium perfringens and Clostridium welchii also inhibited the binding activity while comparatively higher concentrations of phospholipase C from Bacillus cereus were required to achieve comparable inhibition. The time required to produce 50% inhibition of in vitro binding by phospholipases A and C was found to be 6 and 23 min, respectively. Upon either removal or chelation of calcium ions by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) both enzymes were completely inhibited as evidenced by the complete retention of the membrane binding activity. The decrease in the specific binding of choriogonadotropin to membranes after phospholipase digestion resulted in a decrease in the number of binding sites and was not accompanied by a change in the affinity of the hormone-receptor complex. The rates of association and dissociation of the 125I-choriogonadotropin-receptor complex and the equilibrium dissociation constant (Kd) were nearly identical in untreated and phospholipase-treated membranes. Phospholipases did not have any effect on the preformed hormone-receptor complex or on solubilized receptor. Filtration through Sepharose 6B of solubilized 125I-choriogonadotropin-receptor complex from untreated membranes or membranes which had been pretreated with phospholipase C prior to carrying out hormone binding did not alter the profile (Kav 0.38). Gel filtration of membranes treated with phospholipase A showed two peaks of bound radioactivity with distribution coefficients (Kav) of 0.08 and 0.35, respectively.     

Phospholipase A2 from sheep erythrocyte membranes. Ca2+ dependence and localization.

The calcium dependence and the time course of phosphatidylethanolamine and phosphatidylcholine degradation by sheep erythrocyte membrane suspensions in presence of Triton X-100 were investigated. One enzyme with phospholipase A2 specificity was found to be responsible for both phosphatidyl-ethanolamine and phosphatidylcholine degradation. The localization of this enzyme in the membrane of the sheep erythrocyte was investigated by proteolytic treatment of sealed erythrocyte ghosts from the outside and of ghosts which had both sides of the membrane exposed to chymotrypsin. The inability of sealed ghosts to take up chymotrypsin was followed by flux measurements of [14C]dextran carboxyl previously trapped in the ghosts. No efflux of the marker was found during the proteolytic treatment. By comparing the residual phospholipase activities in the membranes from both ghost preparations, we concluded that the phospholipase is oriented to the exterior of the sheep erythrocyte.     

Saturable binding to cell membranes of the presynaptic neurotoxin, beta-bungarotoxin.

Brief exposure to the protein neurotoxin, beta-bungarotoxin, is known to disrupt neuromuscular transmission irreversibly by blocking the release of transmitter from the nerve terminal. This neurotoxin also has a phospholipase A2 activity, although phospholipases in general are not very toxic. To determine if the toxicity of this molecule might result from specific binding to neural tissue, we have looked for high affinity, saturable binding using 125I-labelled toxin. At low membrane protein concentration 125I-labeled toxin binding was directly proportional to the amount of membrane; at fixed membrane concentration 125I-labeled toxin showed saturable binding. It was unlikely that iodination markedly changed the toxin's properties since the iodinated toxin had a comparable binding affinity to that of native toxin as judged by competition experiments. Comparison of toxin binding to brain, liver and red blood cell membranes showed that all had high affinity binding sites with dissociation constants between one and two nanomolar. This is comparable to the concentrations previously shown to inhibit mitochondrial function. However, the density of these sites showed marked variation such that the density of sites was 13.0 pmol/mg protein for a brain membrane preparation, 2.4 pmol/mg for liver and 0.25 pmol/mg for red blood cell membranes. In earlier work we had shown that calcium uptake by brain mitochondria is inhibited at much lower toxin concentrations than is liver mitochondrial uptake. Both liver and brain mitochondria bind toxin specifically, but the density of 125I-labeled toxin binding sites on brain mitochondrial preparations (3.3 +/- 0.3 pmol/mg) exceeded by a factor of ten the density on liver mitochondrial preparations (0.3 +/- 0.05 pmol/mg). It is also shown that labeled toxin does not cross synaptosomal membranes, suggesting that mitochondria may not be the site of action of the toxin in vivo. We conclude that beta-bungarotoxin is an enzyme which can bind specifically with high affinity to cell membranes.     

Effect of pH and temperature on the binding of bilirubin to human erythrocyte membranes

Effect of pH and temperature on the binding of bilirubin to human erythrocyte membranes was studied by incubating the membranes at different pH and temperatures and determining the bound bilirubin. At all pH values, the amount of membrane-bound bilirubin increased with the increase in bilirubin-to-albumin molar ratios (B/As), being highest at lower pH values in all cases. Further, linear increase in bound bilirubin with the increase in bilirubin concentration in the incubate was observed at a constant B/A and at all pH values. However, the slope value increased with the decrease in pH suggesting more bilirubin binding to membranes at lower pH values. Increase in bilirubin binding at lower pH can be explained on the basis of increased free bilirubin concentration as well as more conversion of bilirubin dianion to monoanion. Temperature dependence of bilirubin binding to membranes was observed within the temperature range of 7 degrees -60 degrees C, showing minimum binding at 27 degrees C and 37 degrees C which increased on either side. Increase in bilirubin binding at temperatures lower than 20 degrees C and higher than 40 degrees C can be ascribed to the change in membrane topography as well as bilirubin-albumin interaction.     

Binding sites for calcium-activated neutral protease on erythrocyte membranes are not membrane phospholipids

In order to explore the binding sites for calcium-activated neutral protease (CANP) with high calcium sensitivity (muCANP) on the inner surface of human erythrocyte membranes, we analyzed the binding of muCANP to two kinds of membranes modified by treatment with phospholipase C or Triton X-100. Binding analyses were performed using an immunoblot technique. The amount of muCANP bound to phospholipase C-treated inside-out vesicles was essentially the same as that bound to untreated inside-out vesicles. It was also observed that muCANP binds to Triton X-100-treated membranes, in which most of the integral proteins and glycerophospholipids are removed while the lining proteins remain intact. In both types of modified membrane, the bound muCANP was rapdily converted to an active form by autolysis at physiological free Ca2+ concentrations. These results indicate that the binding sites for muCANP on the inner surface of erythrocyte membranes consist of components other than membrane phospholipids. In addition, it is suggested that one of the binding sites for muCANP is some lining protein.     

Identification of the hemoglobin binding sites on the inner surface of the erythrocyte membrane

The hemoglobin binding sites on the inner surface of the erythrocyte membrane were identified by measuring the fraction of hemoglobin released following selective proteolytic or lipolytic enzyme digestion. In addition, binding stoichiometry to and fractional hemoglobin release from inside-out vesicle preparations of human and rabbit membranes were compared since rabbit membranes differ significantly from human membranes only in that they lack glycophorin. Our results show that rabbit inside-out vesicles bind about 65% less human or rabbit hemoglobin under conditions of optimal and stoichiometric binding, despite being otherwise similar in composition. We suggest that this difference is either directly or indirectly due to the absence of glycophorin in rabbit membranes. Further supportive evidence includes demonstrating (a) that neuraminidase treatment of human membranes did not affect hemoglobin binding and (b) that reconstitution of isolated glycophorin into phospholipid vesicles increased the hemoglobin binding capacity in a manner proportional to the fraction of glycophorin molecules oriented with their cytoplasmic sides exposed to the exterior of the vesicle. Proteolysis of human inside-out vesicles either before or after addition of hemoglobin reduced the binding capacity by about 25%. This is consistent with the known proportion of total hemoglobin binding sites involving band 3 protein and the selective lability of the cytoplasmic aspect of band 3 protein to proteolysis. Phospholipid involvement in hemoglobin binding was determined using various phospholipase C preparations which differ in their reactivity profiles. Approximately 38% of the bound hemoglobin was released upon cleavage of phospholipid headgroups. These results suggest that the predominant sites of binding for hemoglobin on the inner surface of the red cell membrane are the two major integral membrane glycoproteins.     

Evidence for tubulin-binding sites on cellular membranes: plasma membranes, mitochondrial membranes, and secretory granule membranes

We describe the interaction of pure brain tubulin with purified membranes specialized in different cell functions, i.e., plasma membranes and mitochondrial membranes from liver and secretory granule membranes from adrenal medulla. We studied the tubulin-binding activity of cellular membranes using a radiolabeled ligand-receptor assay and an antibody retention assay. The tubulin-membrane interaction was time- and temperature-dependent, reversible, specific, and saturable. The binding of tubulin to membranes appears to be specific since acidic proteins such as serum albumin or actin did not interfere in the binding process. The apparent overall affinity constant of the tubulin- membrane interaction ranged between 1.5 and 3.0 X 10(7) M-1; similar values were obtained for the three types of membranes. Tubulin bound to membranes was not entrapped into vesicles since it reacted quantitatively with antitubulin antibodies. At saturation of the tubulin-binding sites, the amount of reversibly bound tubulin represents 5-10% by weight of membrane protein (0.4-0.9 nmol tubulin/mg membrane protein). The high tubulin-binding capacity of membranes seems to be inconsistent with a 1:1 stoichiometry between tubulin and a membrane component but could be relevant to a kind of tubulin assembly. Indeed, tubulin-membrane interaction had some properties in common with microtubule formation: (a) the association of tubulin to membranes increased with the temperature, whereas the dissociation of tubulin- membrane complexes increased by decreasing temperature; (b) the binding of tubulin to membranes was prevented by phosphate buffer. However, the tubulin-membrane interaction differed from tubulin polymerization in several aspects: (a) it occurred at concentrations far below the critical concentration for polymerization; (b) it was not inhibited at low ionic strength and (c) it was colchicine-insensitive. Plasma membranes, mitochondrial membranes, and secretory granule membranes contained tubulin as an integral component. This was demonstrated on intact membrane and on Nonidet P-40 solubilized membrane protein using antitubulin antibodies in antibody retention and radioimmune assays. Membrane tubulin content varied from 2.2 to 4.4 micrograms/mg protein. The involvement of membrane tubulin in tubulin-membrane interactions remains questionable since erythrocyte membranes devoid of membrane tubulin exhibited a low (one-tenth of that of rat liver plasma membranes) but significant tubulin-binding activity. These results show that membranes specialized in different cell functions possess high- affinity, large-capacity tubulin-binding sites...     

Differential accessibility of bilirubin to erythrocyte membrane vesicles bearing different structural features

Interaction of bilirubin with different types of erythrocyte membrane vesicles such as unsealed, heterogeneous, sealed and inside-out membrane vesicles prepared from human and goat erythrocytes was studied. Out of various types of membrane vesicles, in both species, unsealed membrane vesicles bound quantitatively higher amounts of bilirubin followed by heterogeneous and sealed membrane vesicles whereas inside-out membrane vesicles bound the lowest amount of bilirubin. These differences in the amount of bound bilirubin to different membrane vesicles were correlated well with the percentage accessibility of sialic acid to neuraminidase in these membranes suggesting that bilirubin bound preferentially to the outer layer of erythrocyte membranes than the inner layer. Further, membrane vesicles prepared from human erythrocytes bound higher amounts of bilirubin than those prepared from goat erythrocytes. This can be ascribed to different phospholipid composition of these membranes.     

Synexin binds in a calcium-dependent fashion to oriented chromaffin cell plasma membranes

Oriented plasma membrane fragments from chromaffin cells, isolated on polylysine-coated polyacrylamide beads, bind synexin in a calcium dependent manner. Synexin binding was also detected on beads coated with chromaffin granule membranes, but not to beads coated with erythrocyte membranes or to uncoated beads. Synexin binding to plasma membrane coated beads showed a specific requirement for calcium (K1 2 = 200 microM) and was insensitive to other divalent cations such as magnesium, strontium and barium. Synexin binding to either plasma membrane or granule membrane coated beads was saturable, was partially reversible by EGTA and was directly observed by SDS-polyacrylamide gel electrophoresis.     

Interaction of p-aminobenzoic acid with erythrocyte membrane: photoaffinity labeling of the binding sites

p-Aminobenzoic acid (PABA) was found to prevent eichinocytosis of red cells in vitro. Equilibrium binding studies with right-side-out membrane vesicles revealed a similar number of binding sites and Kd values for both normal and sickle cell membranes. A [14C]Azide analog of PABA was synthesized as a photoaffinity label to probe its sites of interaction on the erythrocyte membranes. Competitive binding studies of PABA with its azide indicated that both the compounds share common binding sites on the membrane surface. The azide was found to covalently incorporate into the membrane components upon irradiation; 52-35% of the label was associated with the proteins and the remaining with the lipids. Electrophoretic analysis of photolabeled membranes revealed that the azide interacts mainly with Band 3 protein in the case of intact erythrocytes and right-side-out sealed vesicles; however, if unsealed ghosts are used, other membrane proteins besides Band 3 are photolabeled. PABA was found to inhibit both high and low affinity calcium-binding sites situated on either surface of the membrane apparently in a non-competitive manner. However, calcium binding stimulated by magnesium and ATP was only slightly affected. Calcium transport into inside-out vesicles was inhibited by PABA, but it did not affect the calcium ATPase activity.     

Demonstration of corticotropin-releasing factor binding sites on human and rat erythrocyte membranes and their modulation by chronic ethanol treatment in rats

In a previous study we reported the presence of specific corticotropin-releasing factor (CRF) binding sites in peripheral tissues of the rat (Endocrinology, 116, 2152, 1985). Using 125I-labeled rat or human CRF, specific CRF binding sites were identified on rat and human erythrocytes, but not on lymphocytes or platelets. Furthermore, identical CRF binding was observed in the presence of intact erythrocytes or lysed erythrocyte membranes. Maximal binding of 125I-CRF occurred within 25 min at 4 degrees C and was saturable. Scatchard analysis of CRF binding to erythrocyte membranes revealed the existence of a single class of binding site. Chronic exposure of rats to ethanol vapor, known to lower specific CRF binding to pituitary tissue by 35%, also decreased 125I-rat CRF binding to erythrocyte membranes by approximately 45%, which was due to a decrease in the number of CRF binding sites. The parallel decrease of CRF binding to rat-erythrocyte and pituitary membranes following chronic ethanol treatment suggests that CRF binding to erythrocyte and pituitary membranes is modulated in a similar direction, which further suggests that the determination of CRF binding to erythrocytes may provide an important clinical tool to indirectly assess CRF-receptor levels in the pituitary gland and thereby enhance our understanding of ethanol-induced disorders of the hypothalamic-pituitary-adrenal axis in patients.     

Studies on the characterization of the sodium—potassium transport adenosinetriphosphatase: XIII. On the organization and role of phospholipids in the purified enzyme

Spin-labeling with 7- and 12-nitroxide-labeled stearic acids and high- and low-angle X-ray diffraction provide strong evidence that the phospholipid in the purified (sodium + potassium)-activated adenosinetriphosphatase from the rectal gland of Squalus acanthias possesses bilayer character. Treatment with phospholipase A produced a change in the ESR spectrum of the stearate-12-nitroxide-labeled enzyme compatible with that seen with erythrocyte and nonmyelinated nerve axon plasma membranes and may be explained by a condensation of the bilayer or the absorption of the spin label in more hydrophilic membrane sites.Phospholipase A treatment also produced a change in the ESR spectrum of spinlabeled NEM-treated enzyme which suggests an increased exposure of buried sulfhydryl groups to a more polar environment. It is suggested that inactivation of the NaK ATPase by phospholipase A digestion may be due to a conformational change in the protein as a result of destabilization of the protein associated with disruption of the phospholipid bilayer.     

Carboxypeptidase Y digestion of band 3, the anion transport protein of human erythrocyte membranes

The exposure of the carboxyl-terminal of the Band 3 protein of human erythrocyte membranes in intact cells and membrane preparations to proteolytic digestion was determined. Carboxypeptidase Y digestion of purified Band 3 in the presence of non-ionic detergent released amino acids from the carboxyl-terminal of Band 3. The release of amino acids was very pH dependent, digestion being most extensive at pH 3, with limited digestion at pH 6 or above. The 55,000 dalton carboxyl-terminal fragment of Band 3, generated by mild trypsin digestion of ghost membranes, had the same carboxyl-terminal sequence as intact Band 3, based on carboxypeptidase Y digestion. Treatment of intact cells with trypsin or carboxypeptidase Y did not release any amino acids from the carboxyl-terminal of Band 3. In contrast, carboxypeptidase Y readily digested the carboxyl-terminal of Band 3 in ghosts that were stripped of extrinsic membrane proteins by alkali or high salt. This was shown by a decrease in the molecular weight of a carboxyl-terminal fragment of Band 3 after carboxypeptidase Y digestion of stripped ghost membranes. No such decrease was observed after carboxypeptidase Y treatment of intact cells. In addition, Band 3 purified from carboxypeptidase Y-treated stripped ghost membranes had a different carboxyl-terminal sequence from intact Band 3. Cleavage of the carboxyl-terminal of Band 3 was also observed when non-stripped ghosts or inside-out vesicles were treated with carboxypeptidase Y. However, the digestion was less extensive. These results suggest that the carboxyl-terminal of Band 3 may be protected from digestion by its association with extrinsic membrane proteins. We conclude, therefore, that the carboxyl-terminal of Band 3 is located on the cytoplasmic side of the red cell membrane. Since the amino-terminal of Band 3 is also located on the cytoplasmic side of the erythrocyte membrane, the Band 3 polypeptide crosses the membrane an even number of times. A model for the folding of Band 3 in the erythrocyte membrane is presented.     

Evidence for a direct,nucleotide-sensitive interaction between actin and liver cell membranes

We have investigated the association of actin with membranes isolated from rat liver. A plasma membrane-enriched fraction prepared by homogenization in a low salt/CaCl2 buffer was found to contain a substantial amount of residual actin which could be removed by treatment with 1 M Na2CO3/NaHCO3, pH 10.5. Using a sedimentation binding assay that uses gelsolin to shorten actin filaments and render membrane binding saturable (Schwartz, M. A., and E. J. Luna. 1986. J. Cell Biol. 102:2067-2075), we found that membranes stripped of endogenous actin bound 125I-actin in a specific and saturable manner. Scatchard plots of binding data were linear, indicating a single class of binding sites with a Kd of 1.6 microns; 66 micrograms actin bound/mg membrane protein at saturation. Binding of actin to liver cell membranes was negligible with unstripped membranes, was competed by excess unlabeled actin, and was greatly reduced by preheating or proteolytic digestion of the membranes. Kinetic measurements showed that binding had an initial lag phase and was strongly temperature dependent. The binding of actin to liver cell membranes was also found to be competitively inhibited by ATP and other nucleotides, including the nonhydrolyzable analogue AMP-PNP. We conclude that we have reconstituted an interaction between actin and integral membrane proteins from the rat liver. This interaction exhibits a number of distinctive features which have not been observed in other actin- membrane systems.     

Gonadotropin receptors in plasma membranes of bovine corpus luteum. II. Role of membrane phospholipids.

The role of phospholipids in the binding of 125I-choriogonadotropin to bovine corpus luteum plasma membranes has been investigated with the use of purified phospholipase A and phospholipase C to alter membrane phospholipids. The phospholipase C-digested plasma membrane preparation showed 85 to 90% inhibition of 125I-choriogonadotropin binding activity when 70% of the membrane phospholipid was hydrolyzed. Similarly treatment of plasma membranes with phospholipase A resulted in 45 to 55% hydrolysis of membrane phospholipid and almost 75% inhibition of receptor activity. Both these enzymes hydrolyzed membrane-associated phosphatidylcholine to a greater extent than phosphatidylethanolamine and phosphatidylserine. Phosphorylaminoalcohols of phospholiphase C end products were completely released into the medium, while phospholipase A by-products remained associated with plasma membranes. Addition of a phospholipids suspension or liposomes to plasma membranes pretreated with phospholipase A and C did not restore gonadotropin binding activity. Soluble phosphorylcholine, phosphorylethanolamine, and phosphorylserine and insoluble diglyceride products of phospholipase C action had no effect on receptor activity. In contrast, end products of the phospholipase A action, such as lysophosphatides and fatty acids, inhibited both on the membrane-associated and solubilized receptor activity. Lysophosphatidylcholine was the most effective end product inhibiting the binding of gonadotropin to the receptor, followed by lysophosphatidylethanolamine and lysophosphatidylserine. The inhibitory effects of phospholipase A or lysophosphatides were completely reversed upon removal of membrane-bound phospholipid end products by washing the membranes with defatted bovine serum albumin. However, phospholipase C inhibition could not be overcome by defatted albumin washings. Solubilization of plasma membranes with detergents which had been pretreated with phospholipase C partially restored the inhibited activity. It is concluded that the phospholipase-mediated inhibition of gonadotropin binding activity was due to hydrolysis and alterations of the phospholipid environment in the case of phospholipase C and by direct inhibition by end products in the case of phospholipase A.     

A comparative study on the extraction of membrane-bound bilirubin from erythrocyte membranes using various methods.

In this study, we used three different methods for the extraction of membrane-bound bilirubin (EMB) from erythrocyte membranes. Use of 2.5% albumin, pH 7.4, for elution of EMB resulted in only 34% of the total EMB which was estimated after the solubilization of bilirubin-loaded erythrocyte membranes (BLEMs) with 1% SDS. On the other hand, incubation of BLEMs with 38 mM sodium carbonate solution containing 5 mM EDTA, pH 11.0, yielded 77% of the total EMB. Application of Fog's reaction for the estimation of EMB directly on the BLEMs resulted in the estimation of 75% of the total EMB. These results suggest that either of the above methods, i.e. use of albumin or high pH, or direct Fog's reaction cannot estimate the total EMB correctly. Increase in ionic strength from 0.15 to 0.45 did not release any EMB from erythrocyte membranes. Therefore, the best method for the estimation of total EMB is the solubilization of membrane with 1% SDS followed by Fog's reaction method.     

Some properties of prostaglandin E2 receptors in rabbit alveolar bone cell membranes

[3H]prostaglandin E2 (PGE2) binding receptors exist in rabbit alveolar bone cell membranes. The presence of high (Kd = 3.9 X 10(-9) M) and low (Kd = 8.8 X 10(-8) M) affinity binding sites of [3H]PGE2 was demonstrated. The saturation values of [3H]PGE2 for high and low affinity binding sites were 0.13 pmol/mg protein and 1.22 pmol/mg protein, respectively. The digestion of the membranes with pronase, phospholipase C, D and neuraminidase led to a decrease of [3H]PGE2 binding but phospholipase A2 did not.