Analysis of erythrocyte protein methyl esters by two-dimensional gel electrophoresis under acidic separating conditions

A two-dimensional polyacrylamide gel electrophoresis system which is suitable for the analysis of protein methylation reactions in cells incubated with L-[methyl-3H]methionine is described. The procedure separates proteins under primarily acidic conditions by isoelectric focusing in the first dimension and by sodium dodecyl sulfate electrophoresis at pH 2.4 in the second dimension. The low pH is essential for preserving protein [3H]methyl esters, but it limits the effective separating range of this system to proteins with isoelectric points between 4 and 8. With this system, we have shown that most, if not all, erythrocyte membrane and cytosolic proteins can act as substoichiometric methyl acceptors for an intracellular S-adenosylmethionine-dependent carboxyl methyltransferase and that protein carboxyl methylation reactions may be the major methyl transfer reaction in erythrocytes. These results are most consistent with the generation of protein substrate sites for the carboxyl methyltransferase by spontaneous deamidation and racemization reactions.     

Carboxyl methylation of cytosolic proteins in intact human erythrocytes. Identification of numerous methyl-accepting proteins including hemoglobin and carbonic anhydrase

Intact human erythrocytes incubated with L-[methyl-3H]methionine incorporated radioactivity into base-labile linkages with membrane and cytosolic proteins which are characteristic of protein methyl esters. Kinetic analysis of the methylation reactions in intact cells shows that individual erythrocytes contain approximately 38,000 and 115,000 protein methyl esters with biological half-lives of 150 min or less in the membrane and cytosolic protein fractions, respectively. Fractionation of the methylated cytosolic species by gel filtration chromatography at pH 6.5 followed by sodium dodecyl sulfate-gel electrophoresis at pH 2.4 reveals that many different cytosolic proteins serve as methyl acceptors and that the degree of modification varies widely for individual proteins. For example, hemoglobin is modified to the extent of 3 methyl groups/10(6) polypeptide chains, while carbonic anhydrase contains 1 methyl group/approximately 16,500 polypeptide chains at steady state. Aspartic acid beta-[3H]methyl ester (Asp beta-[3H]Me) can be isolated from carboxypeptidase Y digests of cytosol proteins. By synthesizing and separating diastereomeric L-Leu-L-Asp beta Me and L-Leu-D-Asp beta Me dipeptides, we show that all of the Asp beta-[3H]Me recovered from cytosolic proteins is in the D-stereoconfiguration. Based on these data and on previous observations that erythrocytes contain a single methyltransferase which also methylates red cell membrane proteins at D-aspartyl residues both in vivo (McFadden, P. N., and Clarke, S. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2460-2464) and in vitro (O'Connor, C. M., and Clarke, S. (1983) J. Biol. Chem. 258, 8485-8492), we propose that protein carboxyl methylation is part of a generalized mechanism for metabolizing damaged proteins. The infrequent and spontaneous occurrence of D-aspartyl residues in proteins adequately explains the broad substrate specificity and limited stoichiometries of protein carboxyl methylation reactions.     

Methylation of erythrocyte membrane proteins at extracellular and intracellular D-aspartyl sites in vitro. Saturation of intracellular sites in vivo

A cytosolic protein carboxyl methyltransferase (S-adenosyl-L-methionine:protein O-methyltransferase, E.C. 2.1.1.24) purified from human erythrocytes catalyzes the methylation of erythrocyte membrane proteins in vitro using S-adenosyl-L-[methyl-3H]methionine as the methyl group donor. The principal methyl-accepting proteins have been identified by sodium dodecyl sulfate-gel electrophoresis at pH 2.4 and fluorography as the anion transport protein (band 3), ankyrin (band 2.1), and integral membrane proteins with molecular weights of 45,000, 28,000, and 21,000. Many of the methylation sites associated with intrinsic membrane proteins may reside in their extracellular portions, since these same proteins are methylated when intact cells are used as the substrate. The maximal number of methyl groups transferred in these experiments is approximately 30 pmol/mg of membrane protein, a value which represents less than one methyl group/50 polypeptide chains of any methyl-accepting species. The number of methylation sites associated with the membranes is increased, but not to stoichiometric levels, by prior demethylation of the membranes. The additional sites are associated primarily with bands 2.1 and 4.1, the principal methyl acceptors in vivo, suggesting that most methylation sites are fully modified in vivo. Extracellular methylation sites are not increased by demethylation of membranes. The aspartic acid beta-methyl ester which can be isolated from carboxypeptidase Y digests of [3H]methylated membranes is in the unusual D-stereoconfiguration. Similar results have been obtained with [3H]methylated membranes isolated from intact cells (McFadden, P.N., and Clarke, S. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2460-2464). It is proposed that the methyltransferase recognizes D-aspartyl residues in proteins and is involved with the metabolism of damaged proteins in vivo.     

Membrane protein carboxyl methylation increases with human erythrocyte age. Evidence for an increase in the number of methylatable sites

The level of carboxyl methylation of membrane proteins has been measured in intact human erythrocyte populations of different ages separated by density gradient centrifugation. Age separation was confirmed by measurement of cytosolic pyruvate kinase specific activity in each fraction. When cells of different ages were incubated with L-[methyl-3H]methionine, the steady state level of 3H radioactivity covalently bound to membrane proteins is observed to be at least 3-fold higher in older erythrocytes. Because the specific radioactivity of the methyl group donor S-adenosyl-L-[methyl-3H]methionine was identical in all age fractions, this represents an increase in the extent of modification of membrane proteins by carboxyl methylation. Of the three major methylated erythrocyte membrane proteins, this increase in carboxyl methylation with age is 4 to 7-fold for bands 2.1 and 3, while the increase in band 4.1 is 3 to 4-fold. This increase in the steady state level of methylation with age cannot be explained by changes in either the intrinsic rate of methyl transfer or by changes in the rate constant of methyl turnover. We, therefore, propose that the age-dependent change in carboxyl methylation is due to an increase in the number of available acceptor sites as the erythrocyte ages in vivo. Since methylation of acidic residues on erythrocyte membrane proteins has been detected exclusively on D-aspartic acid residues (McFadden, P. N., and Clarke, S. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2460-2464), these results are consistent with an accumulation of D-aspartic acid in membrane protein due to spontaneous racemization a the cell ages. The relationship of these observations to possible functions of erythrocyte membrane protein carboxyl methylation is discussed.     

A modified theta-toxin produced by limited proteolysis and methylation: a probe for the functional study of membrane cholesterol.

A derivative of cytolytic theta-toxin from Clostridium perfringens was prepared by limited proteolytic digestion of the native toxin followed by methylation. Among the chloroform/methanol-extractable, lipid components of sheep and human erythrocytes, the proteinase-nicked and methylated derivative (MC theta) specifically binds to cholesterol. While MC theta retains binding affinity comparable to that of intact toxin, it causes no obvious membrane damage, resulting in no hemolysis at temperatures of 37 degrees C or lower. Using MC theta, we demonstrated the possible existence of high- and low-affinity sites for theta-toxin on sheep erythrocytes at both 37 degrees C and 10 degrees C. The number of high-affinity sites on sheep erythrocytes was estimated to be approximately 3-times larger at 37 degrees C than that at 10 degrees C. In addition, high- and low-affinity sites were demonstrated in human erythrocytes and a lymphoma B cell line, BALL-1 cells. Both binding sites disappear upon simultaneous treatment of cells with sublytic doses of digitonin, suggesting that cholesterol is an essential component of both the high- and low-affinity sites and that the mode of cholesterol existence in plasma membranes is heterogeneous in these cells. Because of its high affinity for membrane cholesterol without causing any obvious membrane changes at physiological temperatures, MC theta may provide a probe for use in the functional study of membrane cholesterol.     

Recognition of D-aspartyl residues in polypeptides by the erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase. Implications for the repair hypothesis.

We provide here the first direct evidence that D-aspartyl residues in peptides are substrates for the L-isoaspartyl/D-aspartyl protein carboxyl methyltransferase (EC 2.1.1.77). We do this by showing that D-aspartic acid beta-methyl ester can be isolated from carboxypeptidase Y digests of enzymatically methylated D-aspartyl-containing synthetic peptides. The specificity of this reaction is supported by the lack of methylation of L-aspartyl-containing peptides under similar conditions. Methylation of D-aspartyl residues in synthetic peptides was not observed previously because with Km values ranging from 2.5 to 4.8 mM, these peptides are recognized by the methyltransferase with 700-10,000-fold lower affinity than are their L-isoaspartyl-containing counterparts. The physiological significance of D-aspartyl methylation was investigated in two ways. First, analysis of in situ methylated human erythrocyte proteins showed that at least 22% of the methyl groups associated with the proteins ankyrin and band 4.1 are on D-aspartyl residues, suggesting that D-aspartyl methylation is an important function of the methyltransferase in vivo. Second, mathematical modeling of the protein aging and methylation reactions occurring in intact erythrocytes indicated that the accumulation of D-aspartyl residues can be reduced as much as 2-5-fold by the methyltransferase activity. Although this reduction is much less than that predicted for L-isoaspartyl residues, it may be significant in maintaining functional proteins throughout the 120-day life span of these cells.     

Phospholipid methylation by intact rat Leydig cells

Phospholipid methylation by intact Leydig cells was investigated by determining the incorporation of radioactivity from [3H-methyl] methionine into phospholipids. Leydig cells incorporated significantly more radioactivity into phospholipids than did unpurified testicular cells, non-Leydig testicular cells, or red blood cells. Approximately 40% of the radioactivity was found in phosphatidylcholine, indicating that the methyltransferase pathway for the synthesis of this phospholipid is highly active in rat Leydig cells. Addition of luteinizing hormone to cells preloaded with [3H-methyl] methionine did not alter the rate of phospholipid methylation. However, phospholipid methylation by Leydig cells desensitized by the injection of human chorionic gonadotropin 1 to 7 days previously was reduced by approximately 60%. Inhibition of phospholipid methylation to 75% of normal with homocysteine thiolactone did not affect luteinizing hormone-stimulated androgen production. Further inhibition of phospholipid (and protein) methylation by treatment with homocysteine thiolactone and 3-deazaadenosine significantly reduced luteinizing hormone-stimulated androgen production. The results of this study demonstrate that the methyltransferase pathway for the synthesis of phosphatidylcholine is highly active in intact Leydig cells but is reduced in desensitized Leydig cells. There does not appear to be a close association between the activity of this pathway and the ability of luteinizing hormone to acutely stimulate androgen production.     

Studies on the binding of staphylococcal 125I-labeled alpha-toxin to rabbit erythrocytes.

Staphylococcal alpha-toxin, a hemolytic exotoxin, can be iodinated using the lactoperoxidase method. 125 I-Labeled alpha-toxin binds to rabbit erythrocytes in an apparently irreversible and highly specific manner. The binding of 125 I-labeled alpha-toxin to erythrocytes of rabbit and human reflects the species specificity of native alpha-toxin. Binding of 125I-labeled alpha-toxin is blocked by the presence of native alpha-toxin, 127I-labeled alpha-toxin, or anti-alpha-toxin antibody. Simultaneous assays of 125I-labeled alpha-toxin binding and leakage of intracellular 86Rb+ suggest that toxin binding and membrane damage are separate, sequential functions. Both the rate and extent of binding are temperature dependent. Rabbit erythrocytes possess 5 X 10(3) binding sites/cell, while human erythrocytes possess no detectable binding sites. Treatment of rabbit erythrocytes with 125I-labeled alpha-toxin appears to decrease the number of unoccupied binding sites. Chaotropic ions can inhibit 125I-labeled alpha-toxin binding and cause bound 125I-labeled alpha-toxin to dissociate from rabbit erythrocyte membranes. Treatment of intact rabbit erythrocytes with pronase reduces both the binding capacity of the cells for 125I-labeled alpha-toxin, and the cells' sensitivity to hemolysis by native alpha-toxin. It is proposed that the primary binding site for alpha-toxin in biomembranes is a surface membrane protein.     

Enzymatic methylation of band 3 anion transporter in intact human erythrocytes

Band 3, the anion transport protein of erythrocyte membranes, is a major methyl-accepting substrate of the intracellular erythrocyte protein carboxyl methyltransferase (S-adenosyl-L-methionine: protein-D-aspartate O-methyltransferase; EC 2.1.1.77) [Freitag, C., & Clarke, S. (1981) J. Biol. Chem. 256, 6102-6108]. The localization of methylation sites in intact cells by analysis of proteolytic fragments indicated that sites were present in the cytoplasmic N-terminal domain as well as the membranous C-terminal portion of the polypeptide. The amino acid residues that serve as carboxyl methylation sites of the erythrocyte anion transporter were also investigated. 3H-Methylated band 3 was purified from intact erythrocytes incubated with L-[methyl-3H]methionine and from trypsinized and lysed erythrocytes incubated with S-adenosyl-L-[methyl-3H]methionine. After proteolytic digestion with carboxypeptidase Y, D-aspartic acid beta-[3H]methyl ester was isolated in low yields (9% and 1%, respectively) from each preparation. The bulk of the radioactivity was recovered as [3H]methanol, and the amino acid residue(s) originally associated with these methyl groups could not be determined. No L-aspartic acid beta-[3H]methyl ester or glutamyl gamma-[3H]methyl ester was detected. The formation of D-aspartic acid beta-[3H]methyl esters in this protein in intact cells resulted from protein carboxyl methyltransferase activity since it was inhibited by adenosine and homocysteine thiolactone, which increases the intracellular concentration of the potent product inhibitor S-adenosylhomocysteine, and cycloleucine, which prevents the formation of the substrate S-adenosyl-L-[methyl-3H]methionine.     

Arginine methylation and binding of Hrp1p to the efficiency element for mRNA 3'-end formation

Hrp1p is a heterogeneous ribonucleoprotein (hnRNP) from the yeast Saccharomyces cerevisiae that is involved in the cleavage and polyadenylation of the 3'-end of mRNAs and mRNA export. In addition, Hrplp is one of several RNA-binding proteins that are posttranslationally modified by methylation at arginine residues. By using functional recombinant Hrp1p, we have identified RNA sequences with specific high affinity binding sites. These sites correspond to the efficiency element for mRNA 3'-end formation, UAUAUA. To examine the effect of methylation on specific RNA binding, purified recombinant arginine methyltransferase (Hmt1p) was used to methylate Hrp1p. Methylated Hrp1p binds with the same affinity to UAUAUA-containing RNAs as unmethylated Hrpl p indicating that methylation does not affect specific RNA binding. However, RNA itself inhibits the methylation of Hrp1p and this inhibition is enhanced by RNAs that specifically bind Hrpl p. Taken together, these data support a model in which protein methylation occurs prior to protein-RNA binding in the nucleus.     

Characterization of a thyroid-hormone-binding site on nuclear envelopes and nuclear matrices of the male-rat liver

Nuclear envelopes and nuclear matrices were isolated from the male-rat liver. Incubation of 125I-labelled 3,3',5-tri-iodothyronine (T3) with the nuclear-envelope fraction resulted in specific binding of T3 to the membranes. Maximum specific binding occurred at 30 degrees C after 2h incubation. Storage for 1 week at -80 degrees C resulted in no loss of binding. Scatchard analysis revealed a class of binding sites with KD 86 nM. 3,3',5'-Tri-iodothyronine was as effective a competitor of [125I]T3 binding to nuclear envelopes as was L-T3 itself, and tri-iodothyroacetic acid was 70% as potent as T3. L- and D-thyronine did not compete for [125I]T3 binding. Incubation of nuclear envelopes with 0.6 M-NaCl before addition of T3 resulted in the complete loss of specific binding sites, whereas exposure of the membranes to 2.0 M-NaCl after incubation with T3 did not extract binding sites. Nuclear matrices, after incubation with [125I]T3 under the same conditions, were shown to possess a class of binding sites with a similar KD but with approx. 30% of the maximum binding capacity. Nuclear envelopes from hypothyroid animals may possess slightly lower numbers of binding sites compared with nuclear envelopes from the intact animal, whereas nuclear matrices from hypothyroid animals have the same number of binding sites as do nuclear envelopes from the intact animal. In conclusion, nuclear envelopes and nuclear matrices have a class of binding sites with relatively high affinity for T3. It is distinct from nuclear and cytosolic binding sites.     

Selective carboxyl methylation of structurally altered calmodulins in Xenopus oocytes

The eucaryotic protein carboxyl methyltransferase specifically modifies atypical D-aspartyl and L-isoaspartyl residues which are generated spontaneously as proteins age. The selectivity of the enzyme for altered proteins in intact cells was explored by co-injecting Xenopus laevis oocytes with S-adenosyl-L-[methyl-3H]methionine and structurally altered calmodulins generated during a 14-day preincubation in vitro. Control experiments indicated that the oocyte protein carboxyl methyltransferase was not saturated with endogenous substrates, since protein carboxyl methylation rates could be stimulated up to 8-fold by increasing concentrations of injected calmodulin. The oocyte protein carboxyl methyltransferase showed strong selectivities for bovine brain and bacterially synthesized calmodulins which had been preincubated in the presence of 1 mM EDTA relative to calmodulins which had been preincubated with 1 mM CaCl2. Radioactive methyl groups were incorporated into base-stable linkages with recombinant calmodulin as well as into carboxyl methyl esters following its microinjection into oocytes. This base-stable radioactivity most likely represents the trimethylation of lysine 115, a highly conserved post-translational modification which is present in bovine and Xenopus but not in bacterially synthesized calmodulin. Endogenous oocyte calmodulin incorporates radioactivity into both carboxyl methyl esters and into base-stable linkages following microinjection of oocytes with S-adenosyl-[methyl-3H]methionine alone. The rate of oocyte calmodulin carboxyl methylation in injected oocytes is calculated to be similar to that of lysine 115 trimethylation, suggesting that the rate of calmodulin carboxyl methylation is similar to that of calmodulin synthesis. At steady state, oocyte calmodulin contains approximately 0.0002 esters/mol of protein, which turn over rapidly. The results suggest the quantitative significance of carboxyl methylation in the metabolism of oocyte calmodulin.     

High affinity L-triiodothyronine binding to right-side-out and inside-out vesicles from rat and human erythrocyte membrane

3,5,3'-Triiodo-L-thyronine (L-T3)-binding sites from rat and human red cells were characterized as to their distribution between the two surfaces of the membrane. Analysis of L-T3 binding to sealed right-side-out and inside-out vesicles from erythrocyte membrane revealed that high affinity L-T3-binding sites are located on the external side in rat erythrocytes and on the internal side in human red cells. These results were further confirmed by preincubation of intact red cells with p-chloromercuribenzoate, a slowly permeant reagent that interacts reversibly with SH groups of proteins. Following this treatment only the SH groups of L-T3 sites from rat erythrocytes were found to be blocked. Scatchard analysis of the binding data for rat right-side-out and human inside-out vesicles showed high affinity sites with Kd values of 0.2 x 10(-10) and 2 x 10(-10) M, respectively. The results suggest that the orientation of L-T3-binding sites in the erythrocyte membrane is species-dependent.     

Evidence of high and low affinity binding sites for basic fibroblast growth factor in mouse placenta

The placenta has been shown to contain bFGF, but the presence of specific binding sites for this growth factor in this tissue remained to be established. In order to study the role of bFGF in the placenta growth, we looked for specific binding sites on mouse placental cell membranes at days 12, 14, 16, and 18 of pregnancy. At day 12, Scatchard analyses indicated that two classes of specific interaction sites for bFGF were detected. One class of high affinity binding sites was characterized by an apparent Kd of 10 pM and a binding capacity of 10 fmoles per mg of membrane protein. A second class of low affinity binding sites was detected with an apparent Kd of 60 nM and a binding capacity of 26 pmoles per mg of membrane protein. At days 14, 16 or 18, Scatchard analyses only showed low affinity binding sites with an apparent Kd of 24 nM and a binding capacity of 230 pmoles per mg of membrane protein. The characterization of these binding sites was performed by cross linking experiments that revealed two forms of specific complexes. This result suggested that the high affinity binding sites correspond to putative receptors with relative molecular masses equal to 65,000 and 85,000. The dramatic decrease of the high affinity receptor number after the 12th day of pregnancy, which is synchronous with the 9-fold increase of the low affinity binding site number, suggests that the biological activity of bFGF could be regulated by a balance between both the numbers of high and low affinity binding sites on placenta cell membranes. Thus, as it was shown for other growth factors, bFGF could only be involved at specific pregnancy stages.     

Inhibition of phospholipid methyltransferase during zymosan induced secretion of platelet-activating factor in human polymorphonuclear leukocytes

Phagocytosis of zymosan particles coated with complement induces a time and dose dependent inhibition of the enzyme phospholipid methyltransferase in human polymorphonuclear cells. The extent of phospholipid methyltransferase inhibition induced by various concentrations of zymosan strongly correlates with the secretory process: liberation of platelet-activating factor (PAF) and β-glucuronidase. Zymosan also decreases the incorporation of ³H-methyl group into phospholipids in cells pre-labeled with (³H-methyl)-methionine. Finally, preincubation of cells with 3-deaza-adenosine and homocysteine thiolactone, inhibitors of phospholipid methyltransferase, decrease the incorporation of ³H-methyl group into phospholipids in cells pre-labeled with (³H-methyl)-methionine and modulate the release of PAF. These results suggest that phospholipid methylation plays an important role during the transduction of the secretory signal triggered by zymosan in human polymorphonuclear cells.     

Structural elements affecting the recognition of L-isoaspartyl residues by the L-isoaspartyl/D-aspartyl protein methyltransferase. Implications for the repair hypothesis.

We have synthesized a series of L-isoaspartyl-containing (isoD) peptides and characterized their interaction with the human erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase (EC 2.1.1.77). Our findings indicate that this enzyme interacts with 6 residues extending from the isoD-2 to isoD+3 positions in peptide substrates. Although peptides as simple as G-isoD-G are methylated with low affinity (Km = 17.8 mM), a wide variety of L-isoaspartyl-containing sequences in larger peptides are recognized with high affinity (Km less than 20 microM), the best yet discovered being VYP-isoD-HA, with a Km of 0.29 microM. Only two sequence elements have been found that can interfere with the high affinity binding of peptides of 4 or more residues, these being a prolyl residue in the isoD+1 position and negatively charged residues in the isoD+1, isoD+2, and/or isoD+3 positions. We investigated the effect of higher order structure on binding affinity using several L-isoaspartyl-containing proteins. Although conformation did, in some cases, lower the affinity of the methyltransferase for L-isoaspartyl residues, the range of kinetic constants for the methylation of these proteins was similar to that observed with the synthetic peptides. The L-isoaspartyl/D-aspartyl methyltransferase has been proposed to function in vivo to prevent the accumulation of L-isoaspartyl residues that arise spontaneously as proteins age. To examine whether such a mechanism is feasible given the wide range of substrate Km values observed in vitro, we set up a computer simulation to model the degradation and methylation reactions in aging human erythrocytes. Our results suggest that enough methyltransferase activity exists in these cells to significantly lower the expected number of L-isoaspartyl residues, even when these residues have millimolar Km values for methylation.     

Regulation by phorbol esters of asialoglycoprotein and transferrin receptor distribution and ligand affinity in a hepatoma cell line

We have investigated the simultaneous regulation of cell surface distribution and ligand binding of the asialoglycoprotein (ASGP) receptor and the transferrin receptor in a hepatoma cell line by phorbol esters. One hour exposure to phorbol esters causes a redistribution of both receptors to the cell interior as shown by radioligand binding at 4 degrees C and selective immunoprecipitation from the plasma membrane. This effect is temperature- and dose-dependent and is not seen with 4-alpha-phorbol, an inactive tumor promoter. The mechanism and kinetics of the ASGP receptor response to phorbol esters appears to differ from that of the transferrin receptor in this cell line. Within the first 10 min there is a decrease in binding of iodinated ligands for both receptors to the HepG2 cell surface. For the transferrin receptor this results from a net internalization of receptor molecules from the plasma membrane pool, while for the ASGP receptor this decrease is accounted for by a 3.5-fold reduction in ligand binding affinity (6.6 X 10(-8) M to 24.0 X 10(-8) M), with essentially no change in the number of ASGP receptors recoverable from the plasma membrane pool by immunoprecipitation. The altered affinity of the ASGP-R is transient; the Kd returns to control levels by 20 min of continued exposure to the agent. The transferrin receptor shows no change in binding affinity during the course of exposure to phorbol esters. ASGP receptors in cells exposed to phorbol esters for 1 h maintain their competence to deliver exogenous ligand to intracellular sites of degradation and to participate in the recycling pathway of receptor-mediated endocytosis, although at a lower rate than in control cells. We conclude that under identical conditions phorbol esters modulate the binding capacity of two receptors at the cell surface by separate mechanisms. Furthermore, the transient nature of the altered ASGP-R binding affinity suggests that at least two mechanisms, receptor redistribution as well as decreased binding affinity, are operative in the modulation of ASGP-R cell surface binding during the first hour of exposure to the phorbol esters.     

Binding of plasma low density lipoproteins to erythrocytes

Low density lipoproteins (LDL) containing apolipoprotein B bind to intact, freshly isolated erythrocytes. The LDL-erythrocyte interaction is of low affinity, with a Kd of 1.1 x 10(-6) M. Binding is noncooperative. There are about 200 binding sites per cell and, within the limits of experimental uncertainty, these sites comprise a homogeneous class. Binding of LDL is a temperature-independent process. The maximum amount of LDL blood increases following proteolytic digestion of the cells with trypsin or chymotrypsin. The specificity of the binding sites for LDL is not absolute: high density lipoproteins and lipid vesicles composed of phosphatidylcholine or phosphatidylcholine/cholesterol (equimolar) complete with LDL for occupancy of 60% of the binding sites. Modification of 5--6 of the 9 apolipoprotein B arginine residues with 1,2-cyclohexanedione/borate or of 10--15 of the 20 lysine residues by reductive methylation does not alter the ability of LDL to bind to erythrocytes. Native LDL and methylated-LDL alter erythrocyte morphology. However, LDL in which the arginine residues are derivatized with 1,2-cyclohexanedione/borate do not induce the discocyte leads to echinocyte transformation. Chemically modified and native LDL exchange cholesterol with erythrocytes at equal rates and to nearly equal extents. Taken together, the data suggest that the binding sites for LDL on the erythrocyte membrane are distinct from the LDL receptors at the surface of other cells--e.g., fibroblasts and lymphocytes--which do not bind HDL and which do not recognize LDL with derivatized arginine or lysine residues. It is proposed that the biological function of the erythrocyte binding sites is to mediate the exchange of cholesterol between the cell membrane and lipoproteins.     

Hormone action at the membrane level VI. Binding of (—)-[3H]dihydroalprenolol and (±)-[3H]isoproterenol to turkey erythrocytes and correlation with adenylate cyclase activity

The binding of (±)-[³H]isoproterenol and (—)-[³H]dihydroalprenolol to intact turkey erythrocytes was studied using a rapid centrifugation technique. The binding of both ligands is rapid, dissociable, stereospecific and inhibited by (—)-propranolol. The total number of isoproterenol binding sites is 2800 sites/ cell. This consists of a low and high affinity site both of which show stereospecific binding. The high affinity isoproterenol site has a K_d of 15.5—19.5 nM and has 600 sites/cell. The low affinity isoproterenol site has a K_d of 195 nM and has 2200 sites/cell. The binding of (—)-[³H]dihydroalprenolol shows one type of site with a K_d of 7.8 nM and has 2500 sites/cell. The agonists epinephrine, norepinephrine, soterenol and p-hydroxyphenylisoproterenol which were tested by competition for binding showed a 6—25-fold greater affinity for the high affinity site determined by (±)-[³H]isoproterenol as compared to the (—)-[³H]dihydroalprenolol binding site. However, the antagonists propranolol, practolol and metrapolol showed similar affinities for the binding sites as determined by competition of binding of either labeled isoproterenol or dihydroalprenolol. These studies indicate that isoproterenol binding can recognize two independent stereospecific β-adrenergic receptors or can recognize two different conformational states of a single receptor. Provisional calculations are made on the turnover number of adenylate cyclase under physiological conditions using intact erythrocytes. The turnover number is 4000 molecules of cyclic AMP/10 min per high affinity receptor.