Interaction of bilirubin with human erythrocyte membranes. Bilirubin binding to neuraminidase- and phospholipase-treated membranes.

Saturable bilirubin binding to human erythrocyte membranes was measured before and after digestion with neuraminidase and phospholipases. Neuraminidase-treated erythrocyte membranes did not show any change in their binding properties, indicating that gangliosides could be excluded as candidates for saturable bilirubin-binding sites on erythrocyte membranes. Although bilirubin-binding properties of the membranes did not change after phospholipase D digestion, either, phospholipase C treatment greatly enhanced bilirubin binding. Thus it is suggested that a negatively charged phosphoric acid moiety of phospholipids on the membrane surface may play a role to prevent a large amount of bilirubin from binding to the membranes. Further saturable bilirubin binding to inside-out sealed erythrocyte membrane vesicles showed values comparable with those of the right-side-out sealed membranes, suggesting that the bilirubin-binding sites may be distributed on both outer and inner surfaces of the membranes, or may exist in the membranes where bilirubin may be accessible from either side.     

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.     

Relationship between erythrocyte membrane phase properties and susceptibility to secretory phospholipase A2

Normally, cell membranes resist hydrolysis by secretory phospholipase A(2). However, upon elevation of intracellular calcium, the cells become susceptible. Previous investigations demonstrated a possible relationship between changes in lipid order caused by increased calcium and susceptibility to phospholipase A(2). To further explore this relationship, we used temperature as an experimental means of manipulating membrane physical properties. We then compared the response of human erythrocytes to calcium ionophore at various temperatures in the range of 20-50 degrees C using fluorescence spectroscopy and two-photon fluorescence microscopy. The steady state fluorescence emission of the environment-sensitive probe, laurdan, revealed that erythrocyte membrane order decreases systematically with temperature throughout this range, especially between 28 and 45 degrees C. Furthermore, the ability of calcium ionophore to induce increased membrane order and susceptibility to phospholipase A(2) depended similarly on temperature. Both responses to calcium influx were enhanced as membrane fluidity increased. Analysis of the spatial distribution of laurdan fluorescence at several temperatures indicated that the ordering effect of intracellular calcium on fluid membranes generates an increase in the number of fluid-solid boundaries. Hydrolysis of the membrane appeared to progress outward from these boundaries. We conclude that phospholipase A(2) prefers to hydrolyze lipids in fluid regions of human erythrocyte membranes, but primarily when those regions coexist with domains of ordered lipids.     

Relation between various phospholipase actions on human red cell membranes and the interfacial phospholipid pressure in monolayers.

The action of purified phospholipases on monomolecular films of various interfacial pressures is compared with the action on erythrocyte membranes. The phospholipases which cannot hyorolyse phospholipids of the intact erythrocyte membrane, phospholipase C from Bacillus cereus, phospholipase A2 from pig pancreas and Crotalus adamanteus and phospholipase D from cabbage, can hydrolyse phospholipid monolayers at pressure below 31 dynes/cm only. The phospholipases which can hydrolyse phospholipids of the intact erythrocyte membrane, phospholipase C from Clostridium welchii phospholipase A2 from Naja naja and bee venom and sphingomyelinase from Staphylococcus aureus, can hydrolyse phospholipid monolayers at pressure above 31 dynes/cm. It is concluded that the lipid packing in the outer monolayer of the erythrocyte membrane is comparable with a lateral surface pressure between 31 and 34.8 dynes/cm.     

Phospholipase A2 hydrolysis of membrane phospholipids causes structural alteration of the nicotinic acetylcholine receptor

Thermal perturbation techniques have been used to probe structural alteration of the nicotinic acetylcholine receptor as a function of perturbations of its native membrane environment. Differential scanning calorimetry and a technique involving heat inactivation of the alpha-bungarotoxin-binding sites on the receptor protein reveal that there is a profound destabilization of the acetylcholine receptor structure when receptor-containing membranes are exposed to phospholipase A2. The characteristic calorimetric transition assigned to irreversible denaturation of the receptor protein and the heat inactivation profile of alpha-bungarotoxin-binding sites are shifted to lower temperatures by approx. 7 and 5 C degrees, respectively, upon exposure to phospholipase A2 at a phospholipase/neurotoxin binding site molar ratio of about 1:100. The effects of phospholipase A2 on receptor structure can be (i) reversed by using bovine serum albumin as a scavenger of phospholipase hydrolysis products of membrane phospholipids, and (ii) stimulated by incorporation into the membranes of free, polyunsaturated fatty acids. In particular, linolenic acid (18:3(n-3] causes detectable destabilization of the alpha-bungarotoxin binding sites on the receptor at free fatty acid/receptor molar ratios as low as 10:1. Furthermore, alteration of receptor structure by added phospholipase occurs very rapidly, which is consistent with the observation of rapid in situ phospholipase A2 hydrolysis of membrane phospholipids, particularly highly unsaturated phosphatidylethanolamine and phosphatidylserine. Based on previously published data on the inhibition of acetylcholine receptor cation-gating activity caused by the presence of either phospholipase A2 or free fatty acids (Andreasen T.J. and McNamee M.G. (1980) Biochemistry 19, 4719), we interpret our data as indicative of a correlation between structural and functional alterations of the membrane-bound acetylcholine receptor induced by phospholipase A2 hydrolysis products.     

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.     

Mechanisms governing the level of susceptibility of erythrocyte membranes to secretory phospholipase A2

Although cell membranes normally resist the hydrolytic action of secretory phospholipase A(2) (sPLA(2)), they become susceptible during apoptosis or after cellular trauma. Experimentally, susceptibility to the enzyme can be induced by loading cells with calcium. In human erythrocytes, the ability of the calcium ionophore to cause susceptibility depends on temperature, occurring best above approximately 35 degrees C. Considerable evidence from experiments with artificial bilayers suggests that hydrolysis of membrane lipids requires two steps. First, the enzyme adsorbs to the membrane surface, and second, a phospholipid diffuses from the membrane into the active site of the adsorbed enzyme. Analysis of kinetic experiments suggested that this mechanism can explain the action of sPLA(2) on erythrocyte membranes and that temperature and calcium loading promote the second step. This conclusion was further supported by binding experiments and assessment of membrane lipid packing. The adsorption of fluorescent-labeled sPLA(2) was insensitive to either temperature or ionophore treatment. In contrast, the fluorescence of merocyanine 540, a probe sensitive to lipid packing, was affected by both. Lipid packing decreased modestly as temperature was raised from 20 to 60 degrees C. Calcium loading enhanced packing at temperatures in the low end of this range, but greatly reduced packing at higher temperatures. This result was corroborated by measurements of the rate of extraction of a fluorescent phosphatidylcholine analog from erythrocyte membranes. Furthermore, drugs known to inhibit susceptibility in erythrocytes also prevented the increase in phospholipid extraction rate. These results argue that the two-step model applies to biological as well as artificial membranes and that a limiting step in the hydrolysis of erythrocyte membranes is the ability of phospholipids to migrate into the active site of adsorbed enzyme.     

Arachidonic acid, bradykinin and phospholipase A2 modify both prolactin binding capacity and fluidity of mouse hepatic membranes

The objective of this study was to determine if arachidonic acid, a precursor of prostaglandin synthesis, bradykinin, a decapeptide known to stimulate membrane phospholipid methylation, arachidonic acid release and prostacyclin synthesis, and enzyme phospholipase A₂, capable of liberating arachidonic acid, alter the fluidity of hepatic membranes which could in turn modify the functionality of prolactin receptors. Liver homogenates of adult C₃H female mice incubated at 28°C for various times with 1–20 μg/ml arachidonic acid, 1–100 μg/ml bradykinin or 0.26–0.00026 U/ml phospholipase A₂ provided the 100,000 × g membrane pellets for subsequent ovine prolactin binding and membrane fluidity studies. Membrane microviscosity was determined by fluorescence polarization techniques using the lipid probe 1,6 diphenylhexatriene. Arachidonic acid, bradykinin and phospholipase A₂ stimulated specific oPRL binding, in a dose-related fashion, with maximum increases of 73%, 21% and 46%, at 4 μg/ml arachidonic acid, 5 μg/ml bradykinin and 0.026 U/ml PLA₂, respectively. This induction, occurring within 30 min of incubation, was found to be due to an increase in the number of receptor sites. Under the same conditions, arachidonic acid, bradykinin and PLA₂ induced 22%, 16%, and 18% decreases in membrane microviscosity, respectively. These data suggest that prostaglandin synthesis modifying agents may modulate the number of prolactin receptors $\text{in vivo}$ by changing the lipid fluidity of the target cell membranes by either of their known effects: arachidonic acid release from the phospholipid matrix, synthesizing appropriate prostaglandins at correct concentration or methylation of membrane phospholipids.     

Relation between various phospholipase actions on human red cell membranes and the interfacial phospholipid pressure in monolayers

The action of purified phospholipases on monomolecular films of various interfacial pressures is compared with the action on erythrocyte membranes. The phospholipases which cannot hydrolyse phospholipids of the intact erythrocyte membrane, phospholipase C from Bacillus cereus, phospholipase A₂ from pig pancreas and Crotalus adamanteus and phospholipase D from cabbage, can hydrolyse phospholipid monolayers at pressure below 31 dynes/cm only.The phospholipases which can hydrolyse phospholipids of the intact erythrocyte membrane, phospholipase C from Clostridium welchii phospholipase A₂ from Naja naja and bee venom and sphingomyelinase from Staphylococcus aureus, can hydrolyse phospholipid monolayers at pressure above 31 dynes/cm. It is concluded that the lipid packing in the outer monolayer of the erythrocyte membrane is comparable with a lateral surface pressure between 31 and 34.8 dynes/cm.     

Factors influencing erythrocyte choline concentrations

Choline concentrations in human erythrocytes increase after freezing and thawing, during incubation in Krebs-phosphate for 30 min or on storage at 0 degrees C for 3-24 hr. The increase is prevented by protein precipitation by 10% perchloric acid, 10% zinc hydroxide, 10% sodium tungstate or boiling in water. It is not prevented by EDTA (10 mM) and is increased by oleate (5 mM). We suggest that the increase is due to the action of phospholipase D on erythrocyte phospholipids.     

Effect of phospholipase A on the structure and functions of membrane vesicles from Mycobacterium phlei.

The phospholipid composition of the electron transport particles and coupling factor-depleted electron transport particles of Mycobacterium phlei are the same, but they differ in contents. The accessibility of partially purified phospholipase A to these membrane phospholipids was found to be different. Treatment of membranes of Mycobacterium phlei with phospholipase A impairs the rate of oxidation as well as phosphorylation. The inhibition of phosphorylation can be reversed by washing the membranes with defatted bovine serum albumin. The reconstitution of membrane-bound coupling factor-latent ATPase activity to phospholipase A-treated depleted electron transport particles and their capacity to couple phosphorylation to oxidation of substrates remained unaffected after phospholipase A treatment. However, the pH gradient as measured by bromthymol blue was not restored after reconstitution of phospholipase A-treated depleted electron transport particles with membrane-bound coupling factor-latent ATPase. These findings show that the phosphorylation coupled to the oxidation of substrates can take place without a pronounced pH gradient in these membrane vesicles. The dye 1-anilino-8-naphthalene sulfonic acid (ANS) exhibited low levels of energized and nonenergized fluorescence in phospholipase A-treated membranes. This decrease in the level of ANS fluorescence in phospholipase A-treated membranes was found to be directly related to the amount of phospholipids cleaved. The decrease in the energy-dependent ANS response in phospholipase A-treated electron transport particles, as compared with untreated electron transport particles, was shown to be a result of a change in the apparent K-d of the dye-membrane complex, and of a decrease in the number of irreversible or slowly reversible binding sites, with no change in the relative quantum efficiency of the dye. The decrease in ANS fluorescence in phospholipase A-treated particles appears to be due to a decrease in the hydrophobicity of the membranes.     

Modification of L-triiodothyronine binding sites from rat erythrocyte membrane by heating and by proteinase treatments

The number of binding sites for L-triiodothyronine in rat erythrocyte membranes was increased 2-fold by incubation at 37 degrees C for 60 min. An increase of approximately 3-fold was found when the incubation was carried out at 50 degrees C. The proteinase inhibitor phenylmethylsulfonyl fluoride abolished the effect. Similar increments in the number of binding sites were obtained by treatment of the membranes with proteinases. The Kd values (0.09 X 10(-10) M and 3.6 X 10(-10) M for the high-affinity and the low-affinity binding sites, respectively) remained unchanged after the treatment, as did the free-SH group requirements, storage stability and stereospecificity. Our results suggest that endogenous proteolytic activity could be involved in the increase of the number of membrane latent sites for L-triiodothyronine.     

The reaction of myelin phospholipids with phospholipase C and D

Phospholipase C and D hydrolyze the membrane-bound phospholipids of isolated, untreated myelin. When the membrane is treated with detergents or solvents which disrupt the membrane structure, the activity of the enzymes against the membrane-bound lipids increases. Myelin in the central nervous system is derived from the cell membrane of the oligodendroglial cell. Because the phospholipids in erythrocyte cell membranes are strikingly resistent to phospholipase C and D hydrolysis the question is raised of whether myelin in situ, as opposed to isolated myelin, is susceptible to phospholipase hydrolysis.     

The transverse distribution of phospholipids in the membranes of Golgi subfractions of rat hepatocytes.

The transverse distribution of phospholipids in the membranes of subfractions of the Golgi complex was investigated by using phospholipase C and 2,4,6-trinitrobenzenesulphonic acid as probes. In trans-enriched Golgi membranes, 26% of the phosphatidylethanolamine is available for reaction with trinitrobenzenesulphonate or for hydrolysis by phospholipase C, and 72% of the phosphatidylcholine is hydrolysed by phospholipase C. In cis-enriched Golgi membranes, 45% of the phosphatidylethanolamine is available for reaction with trinitrobenzenesulphonate and for hydrolysis by phospholipase C, and 95% of the phosphatidylcholine is hydrolysed by phospholipase C. Under the conditions used with either probe the contents of the Golgi vesicles labelled with either [3H]palmitic acid or [14C]leucine were retained. Galactosyltransferase activity of the membrane vesicles was partially inhibited by the experimental procedures used to investigate the transverse distribution of phospholipids. However, the residual activity was latent, suggesting that the vesicles remained closed. Trinitrobenzenesulphonic acid caused no detectable morphological change in either Golgi fraction. Phospholipase C treatment caused morphological changes, including fusion of vesicles and the appearance of 'signet-ring' profiles in some vesicles; however, the vesicles remained closed and the bilayer was retained. It appears, therefore, that neither probe causes major disruption of the Golgi vesicles nor gains access to the inner surface of the membrane bilayer. These observations suggest that phospholipids have a transverse asymmetry in Golgi membranes, that this distribution differs in trans and cis membranes, and that the phospholipid structure of Golgi membranes is inconsistent with a simple flow of membrane bilayer from endoplasmic reticulum to Golgi membranes to plasma membrane.     

ANIONIC SITES OF HUMAN ERYTHROCYTE MEMBRANES : I. Effects of Trypsin, Phospholipase C, and pH on the Topography of Bound Positively Charged Colloidal Particles

The effects of pH, trypsin, and phospholipase C on the topographic distribution of acidic anionic residues on human erythrocytes was investigated using colloidal iron hydroxide labeling of mounted, fixed ghost membranes. After glutaraldehyde fixation at pH 6.5–7.5, the positively charged colloidal particles were bound to the membranes in small randomly distributed clusters. The clusters of anionic sites were reversibly aggregated by incubation at pH 5.5 before fixation at the same pH. These results correlate with the distribution of intramembranous particles found by Pinto da Silva (J. Cell Biol. 53:777), with the exception that the distribution of anionic sites on a majority of the fixed ghosts at pH 4.5 was aggregated instead of dispersed. The randomly distributed clusters could be nonreversibly aggregated by trypsin or phospholipase C treatment of intact ghosts before glutaraldehyde fixation. Previous glutaraldehyde fixation prevented trypsin and pH induced aggregation of the colloidal iron sites. Evidence that N-acetylneuraminic acid groups are the principal acidic residues binding colloidal iron was the elimination of greater than 85% of the colloidal iron labeling to neuraminidase-treated cell membranes. Colloidal iron binding N-acetylneuraminic acid residues may reside on membrane molecules such as glycophorin, a sialoglycoprotein which contains the majority of the N-acetylneuraminic acid found on the human erythrocyte membrane.     

Lipid-protein interactions of erythrocyte membranes: comparison of normal O, Rh (D) positive with the rare O, Rh null

Phospholipase A₂ modification of lipid-protein interactions of normal O,Rh(D) positive erythrocyte membranes increased the fluorescence intensity of the membrane bound probe, 1-anilinonaphthalene-8-sulfonate (ANS) and increased the N-1-[¹⁴C]-ethyl maleimide ([¹⁴C]-NEM) labeling of sulfhydryl groups in two proteins of molecular weight >200,000. In marked contrast, phospholipase A₂ modification of the rare phenotype O,Rh_null membranes resulted in no significant increase in ANS fluorescence or labeling of sulfhydryl groups by [¹⁴C] NEM. Since the O,Rh_null erythrocytes demonstrated an increased osmotic fragility and decreased survival time, the fluorescence and sulfhydryl labeling data support the conclusion that hydrophobic bonding between β-fatty acid side chains and non-polar regions of asymmetric proteins is necessary for maintaining the native structure of the O,Rh(D) positive membrane. Comparative studies with phospholipase C or D implied that ionic bonding played a similar though less important structural role in both membranes.     

Fluidity of natural membranes and phosphatidylserine and ganglioside dispersions. Effect of local anesthetics, cholesterol and protein.

The microviscosity of artificial lipid membranes and natural membranes was measured by the fluorescence polarization technique employing perylene as the probe. Lipid dispersions composed of brain gangliosides exhibited greater microviscosity than phosphatidylserine (268 cP vs 173 cP, at 25 degrees C). Incorporation of cholesterol (30-50%) increased the microviscosity of lipid phases by 200-500 cP. Cholesterol's effect on membrane fluidity was completely reversed by digitonin but not by amphotericin B. Incorporation of membrane proteins into lipid vesicles gave varying results. Cytochrome b5 did not alter membrane fluidity. However, myelin proteolipid produced an apparent increase in microviscosity, but this effect might be due to partitioning of perylene between lipid and protein binding sites since tha latter have a higher fluorescence anisotropy than the lipid. The local anesthetics tetracain and butacaine increased the fluidity of lipid dispersions, natural membranes and intact ascites tumor cell membranes. The effect of anesthetics appears to be due to an increased disordering of lipid structure. The fluidity of natural membranes at 25 degrees C varied as follows: polymorphonuclear leukocytes, 335 cP; bovine brain myelin, 270 cP; human erythrocyte, 180 cP; rat liver microsomes, 95 cP; rat liver mitochondria, 90 cP. In most cases the microviscosity of natural membranes reflects their cholesterol: phospholipid ratio. The natural variations in fluidity of cellular membranes probably reflect important functional requirements. Similarly, the effects of some drugs which alter membrane permeability may be the result of their effects on membrane fluidity.     

Mechanisms of lipid peroxidation in erythrocytes of vitamin E-deficient rats and in phospholipid model systems

Erythrocytes from vitamin E-deficient and control rats were peroxidized by glucose oxidase-glucose or dialuric acid. Losses of polyunsaturated fatty acids from membrane phospholipids, and of dimethylacetals from plasmalogens, were quantitated by gas-liquid chromatography. Similar treatment of solubilized or micellar phospholipids or plasmalogens in vitro showed that in both erythrocytes and micellar systems, arachidonic acid and the 16-carbon plasmalogen are most susceptible to peroxidation by either reagent. The same narrow concentration range of dialuric acid found effective in peroxidizing erythrocytes from tocopherol-deficient rats was also found effective in peroxidizing micellar phospholipids in vitro.Partially peroxidized erythrocytes from tocopherol-deficient rats were subjected to treatment with phospholipase A or phospholipase C. Hemolysis by either phospholipase was accelerated in partially peroxidized cells as compared to controls, suggesting that peroxidation exposes both polar and nonpolar lipid sites in the erythrocyte membrane.     

Rat brain membrane-bound delta opioid receptor: loss and reactivation of binding on dialysis and aging at low temperature.

A change in the environment of rat brain membranes by dialysis from phosphate buffered saline (PBS) to 10 mM potassium phosphate (pH 7.2) led to a 35% loss in delta opioid receptor binding, while alteration of membrane structure on freezing at -20 degrees C for 55 days led to 85% loss of receptor binding. The dialysate, 200 mM KCI and NaCl restored receptor binding lost on dialysis. This K+ and Na+ restabilization of the receptor can be through cation-pi bonding, interactions that are suited to the lipid bilayer. In membranes stored at -20 degrees C, the loss of binding is attributed to increased membrane fluidity by phospholipase A2 action on membrane phospholipids, resulting in an increase of free fatty acids. K+ but not Na+ restabilization of these membrane receptors may be due to the ability of K+ to decrease membrane fluidity.     

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.