Binding of antibodies in liposomes to extracellular matrix antigens

We have incorporated antibodies against fibronectin or laminin into liposomes and studied their interaction with insoluble forms of these antigens. The antibodies, after modification by palmitoylchloride, were incorporated into the lipid bilayer by the cholate dialysis method. The antibodies in the liposomes recognized their specific antigen with little reaction to the alternative attachment protein or to albumin (less than 2%). The binding of antibody-containing liposomes to insoluble antigen was inhibited by soluble antibodies to the respective antigens but not by antibodies to other antigens. The affinity constant of the liposome-antibody complex with the antigen was estimated at 1-10 X 10(-9) M liposomes. Thus, antibodies in liposomes retain their reactivity and specificity, and the reaction constant is comparable to that observed for immune complexes.     

Protein interaction with immobilized ligands: quantitative analyses of equilibrium partition data and comparison with analytical chromatographic approaches using immobilized metal affinity adsorbents

Quantitative or analytical affinity chromatography has been successful primarily for the analysis of biologically determined macromolecular affinity relationships. Quantitative approaches are also needed to better characterize simpler, chemically defined immobilized ligands with potential for selective interaction with specific, predetermined protein surface groups. Protein interaction with immobilized metal is a rather selective and versatile, high-affinity adsorption technique for which there is little quantitative information. Using model protein interactions with immobilized Cu2+ ions, we have compared analytical frontal affinity chromatographic methods to a simple, nonchromatographic protocol for the rapid determination of quantitative affinity relationships. Values obtained for the equilibrium dissociation constant (Kd) and binding capacity (Lt) characterizing the interaction of lysozyme with immobilized Cu2+ were quite similar by frontal analysis (Kd = 37-42 X 10(-6) M; Lt = 6.8-7.4 X 10(-6) mol protein/ml gel) and by equilibrium binding analyses (Kd = 33 +/- 4.7 X 10(-6) M; Lt = 5.8-6.1 X 10(-6) mol protein/ml gel; 14 determinations). The interaction of ovalbumin with immobilized Cu2+ was characterized by an affinity (Kd = 4.2-4.8 X 10(-6) M) and capacity (Lt = 1.5-2.1 X 10(-6) mol protein/ml gel) which were also the same regardless of the method for affinity analysis. These values indicate that the total protein bound at saturation corresponds to as much as 17% of the total immobilized Cu2+ ions (approximately 40 X 10(-6) mol/ml gel). Thus, depending on the fraction of total immobilized Cu2+ available for interaction with a given protein (e.g., lysozyme), the number of individual immobilized ligands actively participating as well as those rendered unavailable upon individual protein binding events may be greater than 1. Linear Scatchard plots obtained for both lysozyme and ovalbumin (purified) suggest the presence of only a single type of immobilized Cu2+-protein interaction operative under the experimental conditions employed. However, Scatchard analyses of data obtained by the nonchromatographic equilibrium binding method also demonstrated the ability to simultaneously resolve the contribution of two components whose presence was predicted by frontal chromatography. Our results support the validity and utility of equilibrium binding data analyzed according to the equations outlined by Scatchard and others as an alternative to analytical chromatographic methods.     

Effect of lipidic factors on membrane cholesterol topology--mode of binding of theta-toxin to cholesterol in liposomes.

We have previously suggested the existence of two distinct states for cholesterol in cell membranes as revealed by high- and low-affinity binding sites for theta-toxin of Clostridium perfringens. In liposomes, phospholipid and cholesterol compositions, but not membrane protein composition, have been shown to be major determinants for the topology of membrane cholesterol. The effects of lipidic factors on cholesterol topology were investigated in detail by analyzing toxin binding to large unilamellar liposomes composed of cholesterol and phospholipids (neutral phospholipids/phosphatidylglycerol = 82:18, mol/mol). The numbers of high- and low-affinity toxin-binding sites depend strictly on the cholesterol mole percentage in liposomes. High-affinity toxin-binding sites appear only in liposomes with high cholesterol contents. Liposomes whose cholesterol/phospholipid ratio is 0.4 or less have no high-affinity sites regardless of their phospholipid compositions, while low-affinity sites appear in liposomes with lower cholesterol contents. The threshold values for the cholesterol mole percentage above which high-affinity toxin-binding sites appear were examined. The values decrease in accordance with the increase in the mole fraction of 18-carbon hydrocarbon chains among the total 14-18 carbon-hydrocarbon chains of the liposomal phospholipids. Furthermore, both the partial replacement of phosphatidylcholine with phosphatidylethanolamine and the digestion of phospholipids with phospholipase C also affect the threshold values. Thus the cholesterol mole percentage, in combination with phospholipid chain length and other factors, determines the topology of membrane cholesterol providing distinctively different affinity sites for theta-toxin.     

Binding of glucagon to lipid bilayers

At physiological pH and temperature, glucagon binds to liposomes composed of egg phosphatidylcholine and cholesterol (2:1 mol/mol) in a highly specific manner. The chemical reactivities of the functional groups were determined over the concentration range of 1.0 X 10(-6)-3.0 X 10(-8) M by the method of competitive labelling with 1-fluoro-2,4-dinitrobenzene as the labelling reagent. At concentrations above 3 X 10(-7) M, the amino terminal histidine and the two tyrosine residues showed a marked decrease in reactivity in the presence of liposomes, but the reactivity of the Lys-12 N epsilon-amino group was unaltered. At lower concentrations the Lys-12 reactivity also decreased markedly, owing to a change in the environment of this group. These results indicated that two different forms of glucagon existed over the concentration range studied. Both in the absence and presence of liposomes the Lys-12 N epsilon-amino groups showed a transition in reactivity at 1.8 X 10(-7) M. In the presence of liposomes the other functional groups also showed a transition in reactivity at 2 X 10(-7) M but the change was much smaller. The pattern of reactivities were consistent with the X-ray crystallographic structure of the type 2 glucagon trimer being the predominant species at 10(-6) M, with free monomeric glucagon occurring at 3 X 10(-8) M. A trimerization constant of 4 X 10(13) M-2 at pH 7.5 and 37 degrees C was determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and partial characterization of basolateral membranes from rat proximal colonic epithelial cells

The isolation of basolateral membranes from rat proximal colonic epithelial cells is described. Cells were harvested using a technique combining chelation of divalent cations with mechanical dissociation. After homogenization, differential centrifugation yielded a 'crude' membrane fraction which was further purified using sucrose density centrifugation. The final membrane fraction was enriched 10-14-fold over homogenate in ouabain-sensitive sodium-potassium dependent adenosine triphosphatase and ouabain-sensitive potassium-dependent phosphatase specific activities. SDS-polyacrylamide gel electrophoresis of this membrane revealed at least 18 protein bands with molecular weights of 14600-200000. Phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, free cholesterol and fatty acids were the major lipid components of this membrane. The predominant fatty acids were palmitic (16:0), oleic (18:1), stearic (18:0) and linoleic (18:2) acid. Membranes and their liposomes were studied, using the lipid soluble fluorophore 1,6-diphenyl-1,3,5-hexatriene (DPH), by steady-state fluorescence polarization. The fluorescence anisotropy was greater in the intact membranes compared to their liposomes, indicating greater fluidity in the liposomes. Compositional studies suggested that the high fluidity of this membrane was due to its low ratios of protein/lipid (w/w), cholesterol/phospholipid (mol/mol), and sphingomyelin/phosphatidylcholine (mol/mol).     

Rat Brain S100b Protein: Purification, Characterization, and Ion Binding Properties. A Comparison with Bovine S100b Protein

We purified to homogeneity rat brain S100b protein, which constitutes about 90% of the soluble S100 protein fraction. Purified rat S100b protein comigrates with bovine S100b protein in nondenaturant system electrophoresis but differs in its amino acid composition and in its electrophoretic mobility in urea-sodium dodecyl sulfate-polyacrylamide gel with bovine S100b protein. The properties of the Ca2+ and Zn2+ binding sites on rat S100b protein were investigated by flow dialysis and by fluorometric titration, and the conformation of rat S100b in its metal-free form as well as in the presence of Ca2+ or Zn2+ was studied. The results were compared with those obtained for the bovine S100b protein. In the absence of KCl, rat brain S100b protein is characterized by two high-affinity Ca2+ binding sites with a KD of 2 X 10(-5) M and four lower affinity sites with KD about 10(-4) M. The calcium binding properties of rat S100b protein differ from bovine S100b only by the number of low-affinity calcium binding sites whereas similar Ca2+-induced conformational changes were observed for both proteins. In the presence of 120 mM KCl rat brain S100b protein bound two Zn2+-ions/mol of protein with a KD of 10(-7) M and four other with lower affinity (KD approximately equal to 10(-6) M). The occupancy of the two high-affinity Zn2+ binding sites was responsible for most of the Zn2+-induced conformational changes in the rat S100b protein. No increase in the tyrosine fluorescence quantum yield after Zn2+ binding to rat S100b was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Action of modifiers of a radiation lesion on the binding of prostaglandin E2 with liver membranes in F1 CBA X C57BL strain mice

Prostaglandin E2 (PGE2) was specifically bound by the membrane fraction prepared from the mouse liver. The binding constants indicate the presence of high-affinity PGE2 binding sites with an apparent dissociation constant (Kd) of 0.82 X 10(-9) M and a capacity of 0.36 X 10(9) M/mg protein and a lower affinity PGE2 binding site with Kd = 15.73 X 10(-9) M and a capacity of 5.31 X 10(9) M/mg protein. The radioprotectors, MEA and APAETP inhibit PGE2 binding and alter its kinetics. Apparently the mechanism of PGE2 binding by membranes is related to interaction of prostaglandins with thiols and sufhydryl groups of membrane lipoproteins, while the radioprotectors modify the functional groups participating in receptor PGE2 binding.     

High-affinity, sodium-gradient-dependent transport of choline into vesiculated presynaptic plasma membrane fragments from the electric organ of Torpedo marmorata and reconstitution of the solubilized transporter into liposomes

Vesiculated fragments of presynaptic plasma membranes have been isolated from the purely cholinergic electromotor nerve terminals of Torpedo marmorata. Synaptosomes, generated from the terminals by homogenization, were separated on a discontinuous Ficoll gradient and then lysed by osmotic shock at 2 degrees C, pH 8.5 in the presence of 0.1 mM MgCl2. These conditions for lysis were optimal for choline transport. Electron micrographs of lysed synaptosomes showed vesiculated membranes with diameters smaller than those of synaptosomes; occasionally, synaptic vesicles were observed attached to them. Intact mitochondria or synaptosomes and basal laminae were not present. High-affinity (KT = 1.7 microM) uptake of choline into these vesiculated membrane fragments showed: an absolute dependence on the Na+ gradient (outside greater than inside), a transient Na+-gradient-dependent accumulation of choline over the equilibrium concentration (over-shoot), electrogenicity and rheogenicity, since the uptake was further stimulated in the presence of a Na+ gradient by valinomycin, dependence on the presence of external Cl-, and partial dependence on a Cl- gradient (outside greater than inside), high-affinity (Ki = 25 nM) inhibition by hemicholinium-3 and temperature sensitivity. The plasma membranes were further purified by centrifugal density gradient fractionation on a 4-12% Ficoll gradient. Several enzymes and polypeptides copurified with the specific binding sites for choline present in the membranes. The fraction with the most binding sites was one denser than 12% Ficoll. This was also the fraction richest in acetylcholinesterase, 5'-nucleotidase and polypeptides of relative molecular mass, Mr (X 10(-3)) of greater than 200, 140, 68 (doublet), 57, 54 and 28. Acetylcholinesterase was positively identified as a Mr 68 000 component by immune blot. By contrast the ouabain-sensitive ATPase showed a negative correlation with choline binding sites. When the solubilized proteins of the vesiculated membranes were transferred to liposomes, they conferred on the latter the capacity to take up choline in a manner closely resembling its transport in natural membranes but with an initial (one minute) rate of uptake approximately 10-times greater per mg of protein. Several proteins were selectively transferred to the liposomes including ones of Mr (X 10(-3)) 34, 42, 47, 54, 60, 68, 92, 160 and greater than 200. The polypeptides of Mr (X 10(-3)) 140, 57 and 28 were lost in the transfer.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nerve Growth Factor Receptors in Human Neuroblastoma Cells

Receptors for the nerve growth factor protein (NGFR) present in the human neuroblastoma cell line LAN-1 were characterized. LAN-1 cells display high-affinity (type I, with KD value of 5.9 X 10(-11) M) and low-affinity (type II, with KD value of 9.2 X 10(-9) M) binding to NGF. NGFR were fractionated by preparative isoelectric focusing in a granulated gel (PEGG). High-affinity binding was found in the 5.9-6.2 pH region of the PEGG, and low-affinity binding in the 4.6-4.8 and 8.8-9.3 pH ranges. After further analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) we observed both 92.5- and 200-kDa molecular species associated with NGF binding activity. The 200-kDa protein was found in fractions displaying high-affinity NGF binding and the 92.5-kDa protein in fractions displaying low-affinity NGF binding. Equilibrium binding analysis of NGF in PEGG fractions confirmed the presence of two specific saturable binding sites with KD values similar to those observed for whole dissociated cells. When NGFR II activity from the acidic region of the PEGG chromatogram was incubated with NGFR II from the basic region of the PEGG chromatogram, there was no change in NGF binding or in the number of apparent NGF receptors. However, incubation of these same fractions with a fraction having only NGFR I showed an apparent increase in high-affinity NGF binding and a decrease in low-affinity NGF binding. Immunoprecipitation of this "mixed" fraction and analysis on SDS-PAGE under reduced and nonreduced conditions showed 200-kDa and 92.5-kDa proteins under nonreduced conditions and a 92.5-kDa protein under reduced conditions. Our findings are consistent with the hypothesis that there are two distinct NGF receptors in NGF-responsive cells. The interconvertibility of low- and high-affinity receptors and the possible existence of a modulator type protein or of "silent" type receptors are also in agreement with our findings.     

Binding of thyroxine and triiodothyronine in solubilized nuclear extract of human leukocytes.

An in vitro study was carried out of the interaction of thyroidal hormones with leukocytes and with a solubilized extract of the nuclear fraction of human leukocytes. The respective association constants characterising the binding of triiodothyronine (T3) and thyroxine (T4) to whole leukocytes are roughly equal (for T3, KA = 3.1 X 10(11) 1/mol and for T4, KA = 4 X 10(11) 1/mol) but a 0.4 KCl extract of the nuclear fraction exhibits a different affinity to T3 (KA = 2.16 X 10(11) 1/mol) in comparison with T4 (KA = 1.3 X 10(10) 1/mol). In the nuclear extract, both hormones are bound with the affinity higher for T3 than for T4. The soluble nuclear binding protein in human leukocytes had a molecular weight 46 000, was chromatographically homogenous in chromatography on Sepharose 2B and Sephadex G200, and exhibited a longlasting stability at -25 degrees C, without any marked change in the binding affinity to thyroidal hormones.     

New, highly efficient formulation of diclofenac for the topical, transdermal administration in ultradeformable drug carriers, Transfersomes

Unmodified and polyethylene glycol (PEG) modified neutral and negatively charged liposomes were prepared by freeze-thaw and extrusion followed by chromatographic purification. The effects of PEG molecular weight (PEG 550, 2000, 5000), PEG loading (0-15 mol%), and liposome surface charge on fibrinogen adsorption were quantified using radiolabeling techniques. All adsorption isotherms increased monotonically over the concentration range 0-3 mg/ml and adsorption levels were low. Negatively charged liposomes adsorbed significantly more fibrinogen than neutral liposomes. PEG modification had no effect on fibrinogen adsorption to neutral liposomes. An inverse relationship was found between PEG loading of negatively charged liposomes and fibrinogen adsorption. PEGs of all three molecular weights at a loading of 5 mol% reduced fibrinogen adsorption to negatively charged liposomes. Protein adsorption from diluted plasma (10% normal strength) to four different liposome types (neutral, PEG-neutral, negatively charged, and PEG-negatively charged) was investigated using gel electrophoresis and immunoblotting. The profiles of adsorbed proteins were similar on all four liposome types, but distinctly different from the profile of plasma itself, indicating a partitioning effect of the lipid surfaces. alpha2-macroglobulin and fibronectin were significantly enriched on the liposomes whereas albumin, transferrin, and fibrinogen were depleted compared to plasma. Apolipoprotein AI was a major component of the adsorbed protein layers. The blot of complement protein C3 adsorbed on the liposomes suggested that the complement system was activated.     

Potentiation of immune response against the RESA peptides of Plasmodium falciparum by incorporating a universal T-cell epitope (CS.T3) and an immunomodulator (polytuftsin), and delivery through liposomes.

Synthetic peptides representing repeat sequences of ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum have shown poor immunogenicity and protection. In this study, the RESA peptides [(EENVEHDA)2 and (DDEHVEEPTVA)2] were chemically linked to a universal T-cell determinant, CS.T3, derived from the CS protein of P. falciparum. Polytuftsin (TKPR)40, a polymer of naturally occurring immunomodulator "tuftsin," was physically mixed with these conjugates. These preparations in alum and liposomes were immunized in four inbred strains of mice with different genetic backgrounds to study the humoral response. In the case of liposome-entrapped preparations, a 10 microg dose of antigen showed the optimum antibody response. Mice immunized with liposome containing RESA peptide(s)-CS.T3 conjugate along with polytuftsin showed the highest antibody levels in all the strains, whereas the RESA peptide(s) alone, adsorbed on alum or entrapped in liposomes, showed either poor or moderate antibody levels. The antibodies raised against liposome-entrapped preparations in both high-responder strain (SJL/J H-2s) and low-responder strain (FVB/J H-2q) showed 2 4-fold lower Kd values as compared to the alum adsorbed preparations, suggestive of high affinity antibodies. All the antigen preparations predominantly induced IgG2a and IgG2b isotype response, suggesting that the T-helper response involved is of the CD4 Thl type. The in vitro merozoite reinvasion inhibition assay showed 50-92% inhibition with sera raised against different antigen formulations. The highest percentage inhibition was observed with the RESA peptide-CS.T3 conjugate containing polytuftsin in liposomes. Thus, the incorporation of peptide antigens inside liposomes not only reduced the antigen dose by 5-fold but also elicited a high titre with high affinity antibodies and the inhibition of merozoites to RBC in vitro. Therefore, we conclude that the incorporation of these synthetic constructs in liposomes could be a useful strategy for the development of a subunit immunogen against malaria.     

Comparison of 9-aminoacridine and atebrine induced changes in optical, electrical and mechanical characteristics of lipid bilayers.

The effects of fluorescent probes 9-aminoacridine (9AA) and atebrine (AT) on physical properties of liposomes and planar bilayer lipid membranes (BLM) were studied. The method of fluorescence spectroscopy and the electrostriction method based on measurement of higher current harmonics were used. At low concentrations (10(-5)-5 x 10(-5) mol/l), 9AA increased fluorescence intensity, while in liposomes from soybean phosphatidylcholine fluorescence quenching occurred at higher probe concentration. Fluorescence quenching occurred over the entire concentration range tested (10(-5)-10(-4) mol/l) in liposomes made from a mixture of egg phosphatidylcholine and cardiolipin. In contrast to 9AA, AT, thanks to its hydrophobic chain, penetrates deeper into the hydrophobic membrane moiety; thus, immobilization of the molecule and an increase in fluorescence intensity was always observed. Probes adsorbed to membranes, leaving their electric capacitance effectively unchanged. Adsorption of charged dye particles induced small changes in transmembrane potential. In the presence of 10(-5) mol/l AT, the modulus of elasticity E perpendicular increased somewhat for soft membranes (E perpendicular approximately 2.5 x 10(7) Pa), whereas it decreased for hard membranes (E perpendicular approximately 5 x 10(7) Pa). pH gradient present on the membrane affected the ability of the dyes to incorporate into the membranes. Our results provide evidence against the proposed model of the quenching mechanism introduced by Rottenberg and Lee (1975).     

Immobilization of alpha-chymotrypsin on sucrose stearate--palmitate containing liposomes

Stable liposomes have been prepared from lipid mixture containing sucrose stearate-palmitate. 1.2 X 10(-4) mol of model enzyme alpha-chymotrypsin per mol of lipid have been coupled to prepared liposomes activated by periodate oxidation of sucrose units.     

An ultrarapid filtration method adapted to the measurements of water and solute permeability of synthetic and biological vesicles.

An ultrarapid filtration method was adapted to the determination of water and solute permeability of membrane vesicles. This method consisted of measuring substance washout from vesicles first loaded with 3H2O or labeled solutes, placed on filters, and rinsed at high rates for short periods. The retention of the vesicles on the filters was analyzed and was found to be a function of the nature and porosity of the filters as well as of the vesicle origin. Washing buffer flow rate and washing duration did not affect vesicle retention. The diffusional water permeability of cholesterol-free liposomes was determined at 16 degrees C. Its value was reduced by a factor of 2.5 when the liposomes were prepared with 20% cholesterol and a threefold increase was noted when the liposomes were preincubated with gramicidin (6 mg/g lipid). Water permeability of liposomes was strongly temperature-dependent: Ea = 15.3 kcal/mol. Diffusional water permeability of pink ghosts was also measured: a value of (4.4 +/- 0.2) X 10(-3) cm/s (n = 3) was obtained at 13 degrees C. This permeability was reduced by 45.2% with 0.4 mM HgCl2. The urea permeability of intestinal and renal brush-border membrane vesicles was (1.15 +/- 0.18) X 10(-6) cm/s (n = 7) and (1.67 +/- 0.08) X 10(-6) cm/s (n = 9), respectively. The renal value was reduced by a factor of 4.4 by 100 mM thiourea. This ultrarapid filtration technique provides an accurate method of transport measurement in sealed membranes such as liposomes and plasma membrane vesicles.     

Protein immobilization on the surface of liposomes via carbodiimide activation in the presence of N-hydroxysulfosuccinimide

A method of the covalent immobilization of proteins on the surface of liposomes, containing 10% (by mol) of N-glutaryl phosphatidylethanolamine, is described. Carboxylic groups of liposomal N-glutaryl phosphatidylethanolamine were activated in the presence of water-soluble carbodiimide and N-hydroxysulfosuccinimide and reacted subsequently with protein amino groups. The liposome-protein conjugates formed contained up to 5 x 10(-4) mol protein/mol lipid. Lectins (RCA1 and WGA) upon immobilization on liposomes retained saccharide specificity and the ability to agglutinate red blood cells. The immobilization of mouse monoclonal IgG in a ratio of 3.5 x 10(-4) mol IgG/mol lipid was achieved. The liposome activation in the absence of N-hydroxysulfosuccinimide resulted in a 2-fold decrease of protein coupling yields.     

Measurement of antibody-dependent binding, proteolysis, and turnover of C1s on liposomal antigens localizes the fluidity-dependent step in C1 activation

The antibody-dependent binding and activation of the first component of human complement (C1) by liposomes containing nitroxide spin-label lipid haptens have been simultaneously measured. The liposomes were either fluid (dimyristoylphosphatidylcholine) or solid (dipalmitoylphosphatidylcholine) at the temperature of the experiments (32 degrees C). In 10 minutes fluid liposomes activate 40% of the C1 whereas solid liposomes only activate 10% of the C1. The fraction of C1 bound at the end of the activation incubation is approx. 2% for fluid liposomes and approx. 4% for solid liposomes. This binding is consistent with the relative amounts of antibody which bind to these two types of liposomes. These results demonstrate turnover of C1 or C1r2s2 on the liposome surface. It is concluded that the differential activation of C1 is due to a difference in the rate of activation of C1 after it is bound to the liposome surface. Lower limits for the activation rate constant for C1 bound to fluid and solid liposomes are estimated to be 8 X 10(-2) s-1 and 1 X 10(-2) s-1, respectively.     

The viroimmunotest for mass determinations of nanogram amounts of antigens

Simple highly sensitive screening variant of viral immunoassay for detection of nanogram quantity of antigen is proposed. The antigen linked with antibodies adsorbed on polystyrene 96-well plates was revealed by conjugate of Fab' fragments of antibodies with phage phi X174. The concentration of the antigen could be detected qualitatively and quantitatively (from 0.3 ng/ml to 1000 ng/ml).     

Human alpha-fetoprotein and albumin: differences in zinc binding

The binding of zinc to both human alpha-fetoprotein (AFP) and albumin isolated from cord serum was studied by Sephadex G-50 gel-filtration chromatography. We found that the total number of binding sites for zinc on AFP and albumin were approximately 16 and 12, respectively. Both graphical analysis and the computer program 'LIGAND' indicate that there are at least two major classes of binding sites for both proteins. Both methods of analysis suggested that there are four to five high-affinity sites for zinc on AFP and only two to three similar sites on albumin. The affinity of zinc for AFP (dissociation constant, Kd, 6-8 X 10(-6) mol/l) was higher than for albumin (Kd, 1-3 X 10(-5) mol/l) for the high-affinity sites. The estimates for the zinc low-affinity binding sites were more uncertain, and several classes of low-affinity binding sites of different affinities might be present in both proteins. The results of our inhibition studies suggest that calcium, copper and lead might also bind with AFP at the zinc-binding sites.     

Purification of bovine adrenal-cortex plasma-membrane vesicles containing a highly corticotropin-sensitive adenylate-cyclase system and angiotensin-II-binding sites.

The purpose of this experimental investigation was to provide a purified plasma membrane fraction containing a highly hormone-responsive adenylate cyclase system. Bovine adrenal cortex was homogenised and a washed pellet (450 000 X g - min) was fractionated by zonal centrifugation in a sucrose and dextran gradient. Adenylate cyclase activity was purified up to 60-fold to a specific activity of 55, 340 and 210 pmol of adenosine 3':5'-monophosphate (cyclic AMP) produced/minute per mg of protein at 38 degrees C for the basal, adrenocorticotrophin and fluoride-activated states, respectively. The time course of the adenylate cyclase activity is linear. The concentration necessary for half-maximal stimulation by adrenocorticotrophin-(1-24)-tetracosipeptide is 0.5 muM. The high hormone-responsiveness of the membrane preparation allows one to demonstrate activation of adenylate cyclase by very weakly agonistic adrenocorticotrophin fragments. The F- activated state can be detergent-dispersed by Lubrol and shows a Km (ATP) different from that of either the basal or adrenocorticotrophin-stimulated state. Other marked enzymes such as 5'-nucleotidase, glucose-6-phosphatase and cytochrome oxidase were followed during purification. The plasma membrane fraction shows rather homogeneous, relatively large vesicles (mean diameter 0.5 mum). It contains high-affinity binding sites for angiotensin II (about 2 pmol per mg protein) with an apparent association constant of 2 X 10(7) (1/mol) at 12 degrees C. The yield, 20 mg of membrane protein per preparation, may make it a tool in either affinity-labelling studies with the peptide hormones mentioned or the starting point for solubilisation and purification of adenylate cyclase.