Concanavalin A binds of purified prolyl hydroxylase and partially inhibits its enzymic activity.

Prolyl hydroxylase [(EC 1.14.11.2; prolyl-glycyl peptide, 2-oxoglutarate dioxygenase (4-hydroxylating)] was electrophoresed on polyacrylamide gels and the enzyme in the gels was shown to bind [acetyl-³H]concanavalin A. The enzyme-lectin complex was dissociated by treating the gel with methyl α-D-mannopyranoside, a sugar known to inhibit binding of concanavalin A to glycoproteins. Furthermore, prolyl hydroxylase activity was partially inhibited by concanavalin A when the enzyme was assayed in the absence of bovine serum albumin, a protein which enhances enzymic activity. The inhibition of enzyme activity was prevented by sugars known to react with concanavalin A.     

Acid proteases from species of Mucor. III. Interaction with concanavalin A and concanavalin A Sepharose.

The reaction of Mucor miehei protease with concanavalin A was followed by a turbidimetric assay in the pH range 5-8. At pH 4.0, no turbidity developed but binding of the enzyme to concanavalin A could be demonstrated by gel filtration. Two fractions of apparent molecular weight 65000 and 52000 were isolated, the 65000 molecular weight species apparently representing a protomer of concanavalin A (24000) bound to the enzyme. An analysis of the circular dichroism spectrum of this complex suggested that protomer binding results in a conformational change in the enzyme which is associated with a 30% increase in proteolytic activity. At pH 6.0, the enzyme was strongly bound to columns of concanavalin A Sepharose but could be removed by including alpha-methyl D-glucoside and NaC1 in the elution buffer. Some column degradation occurred at room temperature but was not detectable at 4 degrees C where rapid elution of the enzyme resulted in a greater than 90% yield of highly active protein. Periodate-oxidized Mucor miehei protease and Mucor renin did not react with concanavalin A and were not bound to the affinity column.     

Concanavalin A alters the turnover rate of tyrosine aminotransferase in cultured hepatoma cells

Concanavalin A added to monolayer cultures of Reuber H-35 hepatoma cells caused a rapid inactivation of tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase, E.C. 2.6.1.5) and loss of reactivity with antibody against the native, dimeric enzyme. Analysis of treated cells with an antibody raised against carboxymethylated, denatured enzyme showed that the inactivated enzyme was reactive with this reagent, which does not react with the native enzyme. Subsequent addition of alpha-methyl-D-mannopyranoside to remove concanavalin A restored both enzyme activity and reactivity to antibody against native enzyme. After long-term treatment with concanavalin A, the restored enzyme levels were significantly higher than in controls treated with the sugar but not the lectin. Analysis of the turnover of the enzyme by two methods revealed that the rate of its degradation is reduced about 2-fold in concanavalin A-treated cells. Treatment with H-35 cells with concanavalin A thus effects an alteration in conformation of tyrosine aminotransferase, rendering it somewhat less sensitive to intracellular degradation.     

Irreversible protein binding of norethisterone (norethindrone) epoxide.

14,15-3H-Norethisterone-4 beta, 5 beta-epoxide, a metabolite of norethisterone, was incubated with several proteins and nucleic acids. After 30 min incubation 0.19 nmol of the epoxide were irreversibly bound per mg albumin which contains free sulfhydryl groups; proteins without SH-groups, such as concanavalin A, gamma-globulin, DNA and RNA, did not irreversibly bind norethisterone epoxide. A superoxide (O2) generating enzyme system comprised of xanthine oxidase and hypoxanthine was capable of catalyzing the irreversible binding of the parent compound, norethisterone, to albumin, indicating that an oxidation product was formed which reacted with the protein. When norethisterone epoxide was incubated for 60 min with hepatic microsomes of rats in absence of NADPH, about 2.0 nmol of the epoxide were irreversibly incorporated per mg microsomal protein. This binding was increased to 5.2 nmol by addition of a NADPH regenerating system. Addition of glutathione and cytosol decreased only the NADPH-dependent protein binding; phenobarbital pretreatment of rats induced this NADPH-dependent binding of norethisterone epoxide to microsomal protein by a factor of 2. In presence of NADPH, binding of the epoxide to microsomal protein depended on substrate concentration used. The results indicate that norethisterone epoxide is able to chemically react with proteins. In addition, hepatic microsomal enzymes convert the epoxide to another metabolite which also can react with proteins.     

Sulfhydryl group modification of sarcoplasmic reticulum membranes.

Modification of calcium-translocating sarcoplasmic reticulum membranes (SR) with 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) reveals four classes (kinetic sets) of sulfhydryl groups. Of the 25 mol/1.5 X 10(5) G OF SR protein (i.e., containing 1 mol of ATPase protein) estimated in the presence of sodium dodecyl sulfate, 8 mol are unreactive, while 7, 8, and 2 mol display pseudo-first-order rate constants (k1) of 0.16, 0.68, and 8.3 min(-1), respectively (25 decrees C, pH 7.8, 4 MM Nbs2). Under these conditions, the Ca-ATPase activity is lost with k1 = 0.73 min(-1), whereas the Ca-independent ATPase activity is essentially unchanged. These results are little changed by the presence of Mg2+ or Ba2+ in the modification mixture, while Ca2+ or Sr2+ causes all 16-17 reactable sulfhydryls to be modified with k1 = 0.50 and 0.53 min(-1), respectively. The corresponding values for the loss of Ca-ATPase activity are 0.53 and 0.67 min(-1); this suggests that blocking of only one of the 16-17 SH groups inactivates the enzyme, i.e., that there is a single "essential" SH group. The midpoint of the transition between the Ca2+-free and Ca2+-modification patterns occurs at a free Ca2+ concentration of about 0.9 muM, implying that it is Ca2+ binding at the active sites (KD = 0.1 muM), rather than at the low-affinity nonspecific sites, that effects a conformation change in the ATPase protein (which contains greater than 90% of the cysteines). A calcium-induced conformation change is also suggested by increased ultraviolet absorbance spectrum of the purified ATPase protein upon calcium binding. If protein-lipid interaction is disrupted with deoxycholate or Triton X-100 (which does not destroy the Ca-ATPase activity and hence presumably leaves the tertiary structure of the ATPase protein largely intact), 95% of the sulfhydryls react with Nbs2 considerably faster; thus, at 2 mg/ml o- deoxycholate, 14 groups react with k1 greater than 20, 5 with k1 = 2.3, and 5 with k1 = 0.4 min(-1). These results suggest that the inaccessibility of SH groups in the absence of detergents is due to extensive interaction of the bilayer phospholipids with the ATPase protein.     

Interaction of monolayers of concanavalin A with mono- and polysaccharides. A study by means of infrared-attenuated total reflectance spectroscopy

The effects of pH, Mn2+ and Ca2+ and urea denaturation on the interaction of monolayers of concanavalin A on saline with the polysaccharide dextran B-1355 and the monosaccharides methyl alpha-D-mannopyranoside and D-galactose have been investigated. Infrared absorption spectra of compressed monolayers of the protein and the protein-dextran complex coated on a germanium plate have been obtained by means of attenuated total reflectance spectroscopy. Except in one case of denaturation, the amide I absorption of concanavalin A peaked around 1631 cm-1, indicating a predominance of the beta-pleated sheet conformation, in agreement with its secondary structure in the solution and crystalline phases. The contribution to the absorbance of the concanavalin A-dextran films at 3300 cm-1 due to absorption by the O-H stretching modes of the polysaccharide is a measure of its binding. Increasing the pH from 6.1 to 7.5 appreciably reduced the dextran binding, at pH 9.3 the binding was zero. Adding 1 mM Mn2+ and Ca2+ to the subphase at pH 7.5 restored both the dextran binding and the affinity of concanavalin A for methyl alpha-D-mannopyranoside to that of the native protein at pH 6.1. At this latter pH, the weak binding of dextran to monolayers of demetallized concanavalin A (apo-concanavalin A) was also restored to that for the native molecule by the addition of these divalents. This indicates the requirement of concanavalin A for these ions to maintain the integrity of the saccharide-binding site. The loss of dextran binding with urea denaturation was also observed. These results parallel those for solutions of the protein, indicating the validity of the monolayer system for the study of these interactions.     

Concanavalin A prevents phorbol-mediated redistribution of protein kinase C and beta-adrenergic receptors in rat glioma C6 cells

Exposure of rat glioma C6 cells to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) caused an activation of protein kinase C wherein the enzyme rapidly became membrane-bound (T 1/2 of 15 min). This translocation of protein kinase C from cytosol to membrane was followed by a sequestration of cell surface beta-adrenergic receptors and a loss of isoproterenol-stimulated adenylate cyclase activity. We had reported previously that prior exposure of rat glioma cells to concanavalin A prevents the TPA-mediated sequestration of receptors and desensitization of adenylate cyclase (Kassis et al., 1985). We now show that the concanavalin A treatment also prevents the translocation and activation of protein kinase C. These results are further evidence that in the TPA-treated cells, sequestration of beta-adrenergic receptors is mediated by membrane-bound protein kinase C.     

Binding of bovine brain tissue factor to concanavalin A-Sepharose. Partial purification of coagulant, arylamidase, and alkaline phosphatase activities.

Tissue factor coagulant activity is adsorbed onto concanavalin A-Sepharose from sodium deoxycholate extracts of delipidated bovine brain powders. Coagulant activity is eluted with alpha-methyl-D-glucoside in sodium deoxycholate with 2--25-fold purification. This material has the same coagulant specific activity as that previously prepared in this laboratory. Alkaline phosphatase and alanyl-beta-naphthylamidase activities in the detergent extract also bind to concanavalin A-Sepharose and elute under the same conditions with 4- and 7-fold purification. In addition to these biological activities, the eluate was composed of protein (67.7%), neutral and amino sugars and sialic acid (22.3%), phospholipid (4.5%), uronic acid (3.8%) and nucleic acid (1.7%). This preparation is slightly enriched in carbohydrates compared to previous preparations. Concanavalian A-Sepharose therefore appears to be useful material for partial purification of several mammalian plasma membrane components with retention of biological function.     

Modification of amino acids and bovine pancreatic ribonuclease A by kethoxal.

Kethoxal (3-ethoxy-2-ketobutanal) reacts with the guanidino group of Nalpha-acetylarginine to produce four derivatives, reactive to periodate, stable at pH 7, with 15% reverting to arginine on acid hydrolysis. Other amino acids with blocked alpha-amino groups do not react, except the epsilon-amino of lysine (slowly). The pK of the mixed Kethoxal-Nalpha-acetylarginine derivatives is 5.8-6.1. Kethoxal reacts at neutral pH with arginyl residues of bovine pancreatic ribonuclease A. In the presence of an active-site ligand, arginine-39 and arginine-85 react at about equal rates. The loss of enzymic activity at pH 7 is proportional to the combined loss of these residues. The enzymic activity toward RNA is 20-25% of that of native RNAase at pH 7, and 90-100% at pH 5. In the absence of an active site ligand, arginine-10 is also modified with the loss of almost all enzymic activity, although arginine-10 is not an active-site residue. Arginine-33 is unreactive. Kethoxal-modified RNAase undergoes cross-linking in solution at pH 7 or in the freeze-dried state, Incubation at pH 9 in the presence of homoarginine results in partial regeneration of arginyl residues and activity at pH 7. Kethoxal modification of arginines-39 and -85 appears to raise the pK of lysine-41 by about 1 unit, as indicated ty the pH dependence of arylation by 2-carboxy-4,6-dinitrochlorobenzene. The claims of Patthy and Smith (J. Biol, Chem. (1975) 250, 565-569), and of Takahashi (J. Biol. Chem. (1968) 243, 6171-6179) that arginine-39 is a more important functional residue than is arginine-85 are questioned.     

Selective expression of type I and type II cyclic AMP-dependent protein kinases in subcellular fractions of concanavalin A-stimulated rat thymocytes

Total protein kinase activity and the expression of the type I and type II cyclic adenosine 3′:5′-monophosphate-dependent protein kinases were studied in subcellular fractions of rat thymocytes and the effect of concanavalin A treatment on protein kinase activity was assessed. At a concentration of 100 μ/ml of concanavalin A a marked decline of total nuclear protein kinase activity occurred which lasted approximately 20 to 90 min. Concomitantly, a twofold increase of total protein kinase activity in the 900g supernatant fraction was observed which lasted from 5 to 30 min. Studies using the heat-stable protein kinase inhibitor revealed that the concanavalin A-mediated activity changes were primarily due to changes of cAMP-dependent protein kinase activity, whereas cAMP-independent protein kinase activity remained unchanged. Analysis of the type I and type II cAMP-dependent protein kinase isozyme pattern before and after concanavalin A treatment revealed a selective change of the relative expression of isozyme activities. Whereas type I protein kinase was the major nuclear isozyme before concanavalin A treatment, nuclear type II cAMP-dependent protein kinase increased markedly with a concomitant loss of type I isozyme expression. In the 900g supernatant fraction, containing primarily the type II isozyme in unstimulated cells, concanavalin A treatment caused an increase of the expression of the type I isozyme. The concanavalin A-mediated relative changes of cAMP-dependent protein kinase isozyme expression were confirmed by photoaffinity labeling of the regulatory subunits R^I and R^II before and after concanavalin A stimulation. The intracellular concanavalin A-mediated isozyme changes were time dependent, exhibiting maximal effects about 20 min after concanavalin A addition. These results indicate that selective regulation of intracellular cAMP-dependent protein kinase isozyme expression may be a mechanism related to isozyme-specific phosphorylation of specific intracellular substrates in concanavalin A-activated thymocytes.     

Determination and characterization of neuron specific protein (NSP) associated enolase activity.

Fractionation of the 40–80% (NH₄)₂SO₄ fraction of a soluble rat brain extract on DEAE cellulose resolves three species of enolase activity, two of which react with antiserum to neuron specific protein from rat (NSP-R) and one which does not react. Direct assay of pure neuron specific protein from rat, cat and human brain (NSP-R, NSP-C, NSP-H) as well as bovine brain 14-3-2, using 2 different assay systems demonstrate that all these preparations display enolase activity of between 40 and 70 units/mg. This activity is Mg⁺⁺ dependent and inhibited by fluorophosphate in all cases. Kinetic parameters such as Km for Mg⁺⁺, 2 PGA, and pH optimum were determined for the 2 different NSP preparations and also for bovine brain 14-3-2 protein.     

Purification of glycoproteins by selective transport using concanavalin-mediated reverse micellar extraction.

A novel methodology for coupling liquid-liquid extraction with affinity interaction has been developed to selectively and efficiently purify and separate glycoproteins. The basis for the separation is the selective extraction of glycoproteins from an aqueous solution into a reverse micellar organic phase by using concanavalin A (a sugar-binding lectin) as a facilitative carrier. Specifically, horseradish peroxidase (a common glycoprotein) can be bound to concanavalin A in an aqueous phase and then extracted into an AOT-isooctane organic phase with negligible loss in enzyme activity. Virtually no extraction of peroxidase occurs in the absence of concanavalin A. Electron spin resonance studies have shown that the large lectin-glycoprotein complex (96,000 daltons) resides in a nonaqueous environment within the reverse micelle, perhaps at the surfactant, water-pool interface; hence, extraction of the large complex is feasible. The facilitative extraction has been extended to selective transport of peroxidase from a mixture of peroxidase and alkaline phosphatase (a nonglycosylated protein). This results in an efficient separation strategy with a separation factor of 16.     

Concanavalin A: a useful ligand for glycoenzyme immobilization--a review.

Concanavalin A is finding increasing applications as a useful ligand in glycoenzyme immobilization. An attempt therefore, has been made to summarize the work available in the area. Glycoenzymes that are recalcitrant to immobilization procedures involving covalent coupling to solid supports can be immobilized in high yields by binding to matrices precoupled with concanavalin A. In addition, glycoenzymes associated with concanavalin A matrices usually exhibit high retention of activity and enhanced stability against various forms of inactivation. Binding of the glycoenzymes on the concanavalin A supports, being noncovalent, can be reversed by incubating the preparation with a high concentration of sugars/glycosides or at acidic pH. The association can be, however, rendered covalent by crosslinking the preparations with bifunctional reagents like glutaraldehyde. Crosslinking may be accompanied by further increase in stability, albeit at the expense of the loss of some enzyme activity. Several laboratory-size reactors containing concanavalin A matrix-bound glycoenzyme have been successfully operated for reasonably long durations with only small losses in catalytic activity. Insoluble glycoenzyme preparation can also be obtained by precipitating them from solution as concanavalin A complexes. Such complexes have small particle dimensions but can be successfully used in column reactors after a subsequent immobilization step. Insoluble concanavalin A-flocculates containing various microorganisms and glycoenzymes that successfully carry out multistep transformations have also been obtained by several investigators.     

Evidence for the glycoprotein nature of kidney renin.

The glycoprotein nature of renin isolated from either rabbit or human kidney has been demonstrated by affinity chromatography on concanavalin A-Sepharose. The bulk of rabbit renin activity bound to concanavalin A is released by 20 to 50 mM alpha-methyl-D-mannoside. Adsorption of renin is prevented by periodate oxidation prior to chromatography. Mild acid treatment (pH 2.5) prior to chromatography does not alter the concanavalin A binding profile although the pI values of native rabbit renin (5.1-5.6) are shifted into a broader distribution (4.7-6.4). The molecular weight values of rabbit renin obtained by gel filtration and those from zone centrifugation are identical (37000 +/- 1000), consistent with a low percent of carbohydrate in the glycoprotein. A hydrophobic contribution to the binding of renin by concanavalin A is evident since, in the presence of mM Ca2+ and Mn2+, higher concentrations of alpha-methyl-D-mannoside are required to affect the same release of renin at 23 degrees C compared to that at 4 degrees C. Furthermore, 25% ethylene glycol releases renin in the absence of alpha-methyl-D-mannoside. It is concluded that renin contains a small number of carbohydrate residues in relatively close proximity to a hydrophobic surface which enhances the interaction with concanavalin A.     

Binding of bovine brain tissue factor to concanavalin asepharose. Partial purification of coagulant,arylamidase, and alkaline phosphatase activities

Tissue factor coagulant activity is adsorbed onto concanavalin A-Sepharose from sodium deoxycholate extracts of delipidated bovine brain powders. Coagulant activity is eluted with α-methyl-d-glucoside in sodium deoxycholate with 2–25-fold purification. This material has the same coagulant specific activity as that previously prepared in this laboratory. Alkaline phosphatase and alanyl-β-naphthylamidase activities in the detergent extract also bind to concanavalin A-Sepharose and elute under the same conditions with 4- and 7-fold purification.In addition to these biological activities, the elute was composed of protein (67.7%), neutral and amino sugars and sialic acid (22.3%), phospholipid (4.5%), uronic acid (3.8%) and nucleic acid (1.7%). This preparation is slightly enriched in carbohydrates compared to previous preparations.Concanavalin A-Sepharose therefore appears to be useful material for partial purification of several mammalian plasma membrane components with retention of biological function.     

A dual role for calcium in regulation of superoxide generation by stimulated rat alveolar macrophages

Superoxide production in alveolar macrophages is stimulated by agonists which act through Ca2+-mediated (concanavalin A) and/or protein kinase C (phorbol ester or diacylglycerol analogues) -mediated events. Simultaneous addition of saturating concentrations of concanavalin A and a protein kinase C activator (either phorbol 12-myristate-13-acetate or 1-oleoyl-2-acetyl-sn-glycerol) caused a supra-additive enhancement of the initial rate of O2-. production. This synergism closely correlated with the known time-course of Ca2+ mobilization induced by concanavalin A; however, it occurred under conditions in which protein kinase C activation is reportedly not Ca2+ dependent. Phorbol ester-induced O2-. production was partially inhibited by the Ca2+ ionophore, A23187. Although phorbol ester-stimulated O2-. production initially was enhanced by concanavalin A, the duration of this O2-. production was reduced in comparison to that induced by phorbol ester alone. These results suggest a dual role for intracellular Ca2+ in both stimulatory and inhibitory regulation of O2-. production.     

Lymphocyte function in experimental African trypanosomiasis. VII. Loss of antigen-nonspecific suppressor-T-cell activity

The extent of immunosuppression occurring in mice infected with the pathogenic African trypanosomes was studied. Spleen cells from Trypanosoma rhodesiense-infected C57BL/6J mice were tested for antigen-nonspecific suppressor-T-cell (Ts) activity after concanavalin A (Con A) treatment in vitro. After exposure to Con A, control and infected mouse spleen cells were added to responder spleen cell cultures stimulated with sheep erythrocytes (SRBC). Assays for the resultant plaque-forming cell responses to SRBC revealed that antigen-nonspecific Ts activity was lost during the first week of infection. Changes in infected mouse T-cell subpopulations, including a terminal loss of Lyt 2.2+ cells, accompanied but did not precede the demonstrable loss of Ts function. Splenic suppressor macrophages which arise during infections with T. rhodesiense also did not seem to be associated with the loss of antigen-nonspecific Ts activity. It is concluded that the generalized immunosuppression associated with experimental African trypanosomiasis extends to the mitogen-induced Ts population.     

A labile acid phosphatase isozyme associated with the surface of vegetative Dictyostelium discoideum cells.

A minor acid phosphatase isozyme (acid phosphatase I) of vegetative Dictyostelium discoideum amebae has been shown to be associated exclusively with the external surface of the plasma membrane. The isozyme is not present in phagocytic vacuoles isolated with latex beads. The isozyme disappears from cells removed from nutrient medium and does not reappear during differentiation. When inhibitors of protein synthesis (e.g. cycloheximide, chloral hydrate, concanavalin A) are added to cells growing in nutrient medium, acid phosphatase I is rapidly lost. It appears that the level of protein synthesis need only be moderately reduced (less than 25%) to induce loss of enzyme activity. Treatment with inhibitors of DNA and RNA synthesis for up to 2 h had no effect on isozyme activity. It is postulated that the cells are able to "sense" (through the reduction in levels of protein synthesis) when external conditions become unfavorable, and immediately respond by reducing the activity of enzymes involved in maintaining contact with the extracellular environment. The closed system thought necessary for differentiation would then be created.     

Studies on the reconstitution of bovine erythrocyte superoxide dismutase. V. Preparation and properties of derivatives in which both zinc and copper sites contain copper.

1. We have developed a procedure for preparing derivatives of bovine superoxide dismutase in which primarily the Cu binding sites are occupied by Cu2+ (2 Cu2+-) and in which both the Zn and Cu binding sites are occupied by Cu2+ (4 Cu2+-). 2. The 2 Cu2+ protein shows approximately one-half the superoxide dismutase activity of an equivalent amount of native protein. A two-fold enhancement of the activity of 2 Cu2+-dismutase was observed upon occupation of the Zn sites either with Zn2+ or Cu2+. 3. The electron paramagnetic resonance spectrum of 4 Cu2+ protein was recorded over the temperature range 5-100 degrees K and the results suggest an antiferro-magnetic interaction between Cu2+ in the Zn site and Cu2+ in the Cu site having a coupling constant of approx. 52 cm-1. 4. The binuclear Cu2+ complex was found to accept only one electron from ferrocyanide. 5. One-half the total Cu+ of dithionite reduced 4 Cu+ protein was found to react rapidly with bathocupreine sulfonate whereas the other half reacted slowly. Reduced native protein did not react with bathocupreine sulfonate below 70 degrees C.     

Studies on the glycoprotein component of (Na+ +K+)-ATPase from dog fish salt gland. Binding to concanavalin A and removal of sialic acid by neuraminidase.

1. The presence of concanavalin A binding sugars in the glycoprotein component of a partially purified (Na++K+) ATPase preparation from dog fish salt gland was demonstrated by binding of a Triton X-100 extract of the enzyme and isolated glycoprotein to concanavalin A-Sepharose, and by binding of membrane-associated enzyme to free concanavalin A. 2. The binding of concanavalin A to the glycoprotein in both membrane-associated enzyme and a Lubrol extract of the enzyme had no effect on (Na++K+)-ATPase activity. Binding was completely inhibited by methyl-alpha-mannoside. Also, enzyme activity was not affected by removal of 50% of glycoprotein sialic acid by neuraminidase. These results suggest that the carbohydrate moiety of the glycoprotein does not play a catalytic role in the (Na++K+)-ATPase. 3. When a Triton X-100 extract of (Na++K+)-ATPase was chromatographed on concanavalin A-Sepharose, 37% of total protein was bound to the column and eluted by methyl-alpha-mannoside. The bound fraction was free of lipid, and contained not only the glycoprotein but also the large protein which is the catalytic subunit of the enzyme, and small amounts of other membrane derived proteins. The ratio of large protein to glycoprotein, as measured by the relative Coomassie blue absorbance of the two proteins separated by gel electrophoresis, was the same in the bound fraction as in the membrane. These results suggest that the glycoprotein and lareg protein are either associated together in the membrane or become associated during lipid replacement by Triton.