Identification of the alpha beta monomer of the adipocyte insulin receptor by insulin binding and autophosphorylation.

The insulin receptor consists of an insulin-binding subunit (alpha) of 135,000 daltons. More recently, it has been documented that the receptor undergoes insulin-stimulated autophosphorylation that predominantly labels a 95,000-dalton (beta) subunit. We solubilized rat adipocyte insulin receptors in Triton X-100 and partially purified the protein on a wheat germ agglutinin-Sepharose affinity column. Subsequently, we labeled the two subunits of the receptor independently by using 125I-labeled insulin for the 135,000-dalton alpha-subunit and 32P for the 95,000-dalton beta-subunit. Sucrose density gradient sedimentation and NaDodSO4/PAGE were used to characterize the native, oligomeric structure of the receptor. In 0.1% Triton X-100, the receptor sedimented as a single peak of s20,w = 10.2 S as detected by both 125I and 32P. NaDodSO4/PAGE under nonreducing conditions revealed a large species that appeared to be alpha 2 beta 2 and, to a lesser extent, alpha beta. Treatment of the solubilized, partially purified receptor with 10 mM dithiothreitol led to the partial conversion of the 10.2S species to a smaller one sedimenting at 6.6 S. The composition of this species was determined to be alpha beta by nonreducing NaDodSO4/PAGE. Our results suggest that detergent-solubilized insulin receptors can exist as dimers and monomers. The oligomeric structure of receptors functional in the cell membrane cannot be immediately deduced from these results due to the possibility of artifacts arising from membrane disruption and extraction procedures. However, the ability to label the two subunits of the receptor separately should facilitate a detailed study of its oligomeric structure both in solution and in the membrane.     

High-molecular weight kininogen is present in cultured human endothelial cells: localization, isolation, and characterization

The presence of high-molecular weight (mol wt) kininogen was demonstrated in cultured human endothelial cells derived from the umbilical cord by immunofluorescence techniques. Cultured human endothelial cells contain 58 +/- 11 ng (n = 16) high-mol wt kininogen/10(6) cells as determined by an enzyme-linked immunosorbent assay (ELISA) specific for high-mol wt kininogen. High-mol wt kininogen was isolated from cultured human endothelial cells by immunoaffinity chromatography. Nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that endothelial cell high-mol wt kininogen consisted of five protein bands with mol wts of 95,000, 85,000, 65,000, 46,000, and 30,000 daltons. Immunoblotting of the endothelial cell high-mol wt kininogen by using specific antisera against the heavy and light chain indicated that the 95,000-, 85,000-, and 65,000-dalton bands consisted of the heavy and light chain whereas the 46,000- and 30,000-dalton bands reacted only with the anti-light chain antiserum. Immunoprecipitation studies performed with lysed, metabolically labeled endothelial cells and monospecific antisera directed against high-mol wt kininogen suggested that high-mol wt kininogen is not synthesized by the endothelial cells. Endothelial cells cultured in high-mol wt kininogen-free medium did not contain high-mol wt kininogen. These studies indicate that endothelial cell high-mol wt kininogen was proteolytically cleaved in the culture medium and subsequently internalized by the endothelial cells. Binding and internalization studies performed with 125I-labeled, proteolytically cleaved, high-mol wt kininogen showed that endothelial cells can indeed bind and internalize proteolytically cleaved high-mol wt kininogen in a specific and saturable way.     

Membrane receptor function and the loss of glucagon-stimulated adenylate cyclase activity in hepatomas.

Plasma membranes were prepared from homogenates of two well differentiated hepatomas (Morris rat 7787 and Dalton mouse 9815), two poorly differentiated hepatomas (Morris rat 7288-C and Dalton mouse 129), and normal liver. Adenylate cyclase activity and [125I]iodoglucagon binding were measured in the plasma membrane preparations over a wide range of glucagon concentrations. Nether glucagon-stimulated adenylate cyclase activity nor [125I]iodoglucagon binding could be detected in the poorly differentiated hepatomas. Fluoride and epinephrine stimulated adenylate cyclase activity in all hepatomas. Maximum activity of glucagon-stimulated adenylate cyclase and maximum binding of glucagon in the wall differentiated hepatomas were less than those of normal liver. Plasma membranes from liver and hepatomas were solubilized with Lubrol-PX and, after reducing the concentration of detergent, were incubated with [125I]iodoglucagon and then chromatographed on a column of Bio-Gel A 1,5 m. Two peaks containing both protein and [125I]iodoglucagon were found for normal liver but not for the poorly differentiated hepatomas. Fractions from the Bio-Gel column containing the greatest concentration of protein were also subjected to a binding microassay. Material from the poorly differentiated tumors did not bind glucagon in this system, whereas the solubilized normal liver membranes bound up to 1.4 pmol [125I]iodoglucagon/mg protein. This indicates that there is no detectable glucagon receptor in these undifferentiated tumors.     

Identification of a spectrin-like protein in nonerythroid cells.

We have demonstrated the existence of a spectrin-like protein in a variety of nonerythroid cultured cells. Indirect immunofluorescence studies with monospecific antispectrin IgG indicated the presence of proteins that have common antigenic determinants to spectrin in embryonic chicken cardiac myocytes, mouse fibroblast lines (3T3, simian virus 4-transformed 3T3), and rat hepatoma lines (HTC, HMOA). Two spectrin-like peptides of 240,000 and 230,000 daltons were immunoprecipitated from octyl glucoside-solubilized embryonic chicken cardiac myocytes, along with associated cytoskeletal proteins. Immunoautoradiographic characterization of the myocyte immunoprecipitate showed that only the spectrin-like 240,000- and 230,000-dalton peptides were stained with monospecific antispectrin IgG and 125I-labeled protein A. One-dimensional partial proteolytic mapping of the myocyte 240,000- and 230,000-dalton peptides showed that these peptides share substantial sequence homology with embryonic chicken erythrocyte spectrin 240,000- and 220,000-dalton peptides.     

Characterization of an interferon receptor on human lymphoblastoid cells.

A cell-free assay was developed to measure the binding of iodinated human interferon-alpha 2 to membranes prepared from lymphoblastoid Daudi cells. The kinetics of binding were similar at 0 degrees C and 30 degrees C, with 1.3-fold more interferon bound at the higher temperature. Membrane preparations treated with Triton X-100 proved to be a convenient source of solubilized receptor. An assay was developed to measure the binding of 125I-labeled interferon (125I-interferon) to solubilized receptors, based on the precipitation of interferon-receptor complexes with polyethylene glycol. Optimal binding with this assay was obtained at 0 degrees C. The solubilized receptor was analyzed by zonal sedimentation centrifugation and gel filtration. Sedimentation analysis in H2O and 2H2O gradients provided the sedimentation coefficient and the partial specific volume of the receptor-Triton X-100 complex. Gel filtration chromatography provided the Stokes radius of this complex. From these data we calculated several physical parameters, including Mr = 95,000 for the protein portion of the complex. The receptor is a highly asymmetric and hydrophobic membrane protein. 125I-Interferon could be crosslinked to receptors of intact Daudi cells or of isolated membranes by use of disuccinimidyl suberate. The covalently linked 125I-interferon-receptor complexes were analyzed by gel electrophoresis. A single band with Mr = 140,000 was detected in gel autoradiographs. If one molecule of interferon is present in this complex, the Mr of the receptor is close to 120,000. Possible reasons for the different Mr values obtained with the two analytical procedures used are discussed.     

Biochemical characterization of the pancreatic cholecystokinin receptor using monofunctional photoactivatable probes

Receptor characterization by affinity labeling can be enhanced by taking multiple complementary approaches. To extend our observations on the subunit structure of the rat pancreatic cholecystokinin (CCK) receptor (made using bifunctional cross-linking reagents), we synthesized two monofunctional photoactivatable receptor probes. CCK-8 was acylated with the iodinated aryl azide derivatives, methyl-3-azido-4-hydroxy-5-[125I]iodobenzimidate and N-[4-(4'-azido-3'-[125I]iodophenylazo)benzoyl]-3-aminopropionyl-N- oxy- succinimide. The products were purified by reverse-phase HPLC to a specific radioactivity of 2,000 Ci/mmol. Both analogs demonstrated saturable and specific binding to rat pancreatic plasma membranes. Photoaffinity labeling of pancreatic membranes with these monofunctional probes identified an Mr 85,000-95,000 protein that was not part of a larger disulfide-linked complex. High affinity for CCK was demonstrated by the concentration-dependent inhibition of labeling observed with competing CCK-8 (IC50 = 1 nM). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) this protein co-migrates with the major component we identified using a series of cross-linkable, iodinated decapeptide analogs of CCK, and is different from the major protein labeled using 125I-Bolton Hunter-CCK-33. Thus, these results support the presence of an Mr 85,000-95,000 subunit in the pancreatic CCK receptor, while the small size of these photoaffinity probes and their monovalency suggest that this subunit may contain or be spatially apposed to the active binding site. These probes should be very useful in the further characterization of this and other receptors for this hormone.     

Solubilization of a guanine nucleotide-sensitive parathyroid hormone-receptor complex from canine renal cortex

Canine renal cortical PTH receptors were solubilized after occupancy of membrane-associated receptors with the agonist ligand [125I]bovine (b) PTH-(1-34). Stabilization of binding during solubilization required the use of high concentrations of BSA (optimally 5%) and appropriate detergents (0.5% 3-[(3-cholamidopropyl)dimethylammonio]2-hydroxy-1-propanesulfonate, 0.5% 3-[(3-cholamidopropanyl)dimethylammonio]1-propanesulfonate, or 0.5-1.0% digitonin). The soluble fraction (240,000 X gav supernatant) contained [125I]bPTH-(1-34) associated with macromolecular components as well as unbound [125I]bPTH-(1-34) that dissociated during solubilization. The soluble macromolecular complex had functional properties expected of a ternary complex consisting of [125I]bPTH-(1-34) receptor stimulatory guanine nucleotide-binding protein (Ns). Thus, the dissociation of labeled PTH at 30 C was slow (t1/2 = 75 min); in the presence of GTP (10(-4) M), 75% of the sites displayed rapid dissociation kinetics (t1/2 = 2.3 min). This effect was nucleotide specific, with GTP approximately equal to GTP gamma S approximately equal to GDP greater than GDP beta S greater than ITP approximately equal to guanylylimidodiphosphate much greater than GMP approximately equal to App(NH)p. ATP was ineffective. GTP produced a half-maximal response at a concentration of 200 nM. These results are consistent with the reported nucleotide specificity and affinity of purified Ns. Treatment of membranes with N-ethylmaleimide during the binding reaction rendered the solubilized complex refractory to GTP. Gel filtration chromatography (Sepharose 6B) revealed a GTP-sensitive complex that eluted in the position expected of a detergent-free spherical protein of 180,000 daltons. This complex may consist of the 60,000 to 70,000-dalton PTH-binding subunit (previously identified by photoaffinity labeling) together with Ns.     

Phorbol ester- and diacylglycerol-mediated desensitization of cardiac beta-adrenergic receptors

There are specific phorbol ester receptors on cardiac myocytes which may be identical with the calcium/phospholipid-dependent protein kinase (protein kinase C). Incubation of enzymatically dissociated rat cardiac myocytes with biologically active phorbol esters (such as 4 beta-phorbol-12, 13-dibutyrate and 12-O-tetradecanoyl phorbol-13-acetate) leads to a time- and concentration-dependent loss of beta-adrenergic receptors detectable with the hydrophilic ligand [3H]-CGP-12177. This loss is attributable to a reduction in both maximal beta-receptor numbers and their affinities. The synthetic diacylglycerol, 1-oleyl-2-acetyldiglycerol, which is known to activate protein kinase C, also induces desensitization of beta-receptors. Both phorbol dibutyrate and 1-oleyl-2-acetyldiglycerol have additive effects to isoproterenol, suggesting a separate site of action in promoting beta-receptor desensitization. The effects of phorbol dibutyrate and 1-oleyl-2-acetyldiglycerol are prevented by colchicine (but not its inactive analog, trimethylcolchicinic acid), indicating a microtubule dependence. The loss of membrane-bound beta-receptors after phorbol dibutyrate- or 1-oleyl-2-acetyldiglycerol preincubation is accompanied by an increase in beta-receptors associated with a cytosol-derived vesicular fraction devoid of plasma membrane markers, a finding consistent with an internalization process. These results suggest that protein kinase C activation by diacylglycerols derived from receptor-linked phosphoinositide hydrolysis may be a novel mechanism of cardiac beta-receptor desensitization.     

Subunit interaction of human chorionic gonadotropin (hCG) with rat ovarian luteinizing hormone (LH)/CG receptor.

The subunit interaction of hCG with its rat ovarian LH/CG receptor was studied by cross-linking the solubilized receptor-hormone complex with glutaraldehyde (GA), disuccinimidyl suberate (DSS) or dithiobis(succinimidyl propionate) (DSP) and analyzing the complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. The hormone was labeled either in its alpha-subunit (125I-hCG) or in its beta-subunit (3H-hCG) or the label (3H) was introduced into the receptor molecule instead of the hormone. All of the labeling procedures led to the detection of only the receptor-(alpha,beta)hCG and receptor-(alpha)hCG complexes on the autoradiograms. The sizes of these complexes were 137,000 and 106,000, respectively, under reducing conditions. These results suggest that the receptor binds one hormone molecule, and that hCG interacts with the receptor mainly through its alpha-subunit. In addition, polyclonal antibodies directed against the LH/CG receptor and the alpha- and beta-subunits of hCG were used to detect the non-reduced receptor-(alpha,beta)hCG complex in immunoblotting. As antibodies directed against both the alpha-subunit and the beta-subunit were able to detect the Mr 130,000 complex, it is conceivable that both of the subunits are at least partially exposed on the receptor-hormone complex. 125I-hCG was also cross-linked to the membrane-bound receptor. The membrane-bound complex had an Mr of 144,000 under reducing conditions, i.e. approximately 7000 higher than that of the solubilized complex (Mr 137,000). This may indicate that the membrane-bound receptor is covalently modified or differs in conformation from the solubilized receptor.     

Biochemical characteristics of nuclear 5 alpha-reductase and cytosol 3 alpha-hydroxysteroid dehydrogenase in rat submandibular gland

Biochemical studies of testosterone 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) in rat submandibular gland (SMG) were performed. 14C-labeled testosterone or dihydrotestosterone (DHT) was incubated with subcellular fractions from rat SMG in the presence of 0.2 mM NADPH at 37 degrees C for 20 min in an atmosphere of 95% O2 and 5% CO2. Among the subcellular fractions, the high 5 alpha-reductase activity was detected in the nuclear fraction and 3 alpha-HSD in cytosol. Nuclear 5 alpha-reductase was efficiently solubilized in 2 mg digitonin per mg protein and 0.3 M KCl solution at 4 degrees C for 30 min. The maximum velocities (Vmax) of nuclear and solubilized 5 alpha-reductase activity for testosterone were 71.4 pmol/mg protein per min and 25.4 pmol/mg protein per min. Apparent Michaelis constant (Km) of nuclear and solubilized enzymes for testosterone were calculated as 11.1 microM and 16.7 microM by the Lineweaver Burk plot, respectively. The activity of solubilized 5 alpha-reductase from nuclei was stable by NADPH and KCl, and the molecular weight of the enzyme was estimated as 158 K.D approximately 200 K.D by Bio-Gel A-1.5 m column chromatography. The column chromatography also showed a peak of 3 alpha-HSD activity in cytosol, revealing the molecular weight of approximately 50 K.D. However, the elution peak of the 3 alpha-HSD was effectively decreased by KCl in Tris-HCl buffer. The molecular weight of 5 alpha-reductase and 3 alpha-HSD in SMG were similar to those in prostate. A stable and extractable 5 alpha-reductase was demonstrated in nuclei of rat SMG with possessing a considerable affinity for testosterone and also high 3 alpha-HSD activity for DHT was revealed in cytosol of the tissue.     

Identification and characterization of insulin-like growth factor receptors on adult rat cardiac myocytes: linkage to inositol 1,4,5-trisphosphate formation.

Cultured cardiac myocytes from adult Sprague-Dawley rats express both insulin-like growth factor-I (IGF-I) receptors and insulin-like growth factor-II/mannose 6-phosphate (IGF-II/Man6P) receptors and respond to IGF-I with a dose-dependent accumulation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,4-bisphosphate [Ins(1,4)P2]. Specific binding of [125I]IGF-I to isolated membranes from cultured cardiac myocytes amounted to 1-1.2%. Binding of [125I]IGF-I was inhibited by unlabeled IGF-I at nanomolar concentrations and insulin at much higher concentrations. These data suggest that IGF-I binds to its own receptor on rat cardiac myocytes. Competitive binding studies using isolated membranes from cardiac myocytes and [125I]IGF-II showed 2-4% specific binding. Binding of [125I]IGF-II was inhibited by IGF-II and much less potently by IGF-I and insulin. Immunoglobulin G (IgG) 3637 (an IgG directed against the IGF-II/Man6P receptor) partially inhibited binding of [125I]IGF-II whereas nonimmune IgG did not. Affinity cross-linking studies with [125I]IGF-II and cardiac myocyte membranes and subsequent analysis of the ligand-receptor complex using SDS-PAGE and autoradiography showed a radiolabeled band of approximately 250 kilodalton (kDa). The formation of the [125I]IGF-II-receptor complex was inhibited by incubation with IGF-II and IgG 3637 but not by insulin or nonimmune IgG. Western blotting of protein extracts from cultured cardiac myocytes was performed using IgG 3637 and an immunoperoxidase technique for the visualization of the IGF-II/Man6P receptor protein. A specific band at 220 kDa under nonreducing conditions was detected on the blots, providing further evidence for the expression of the IGF-II/Man6P receptor by cardiac myocytes. The effect of IGFs on the accumulation of inositol phosphates was measured by HPLC analysis of perchloric acid extracts from myo-[3H]inositol-labeled cultured cardiac myocytes. IGF-I (50 ng/ml) stimulated the accumulation both of Ins(1,4,5)P3 and Ins(1,4)P2 after 30 sec by 43% and 63%. IGF-II (up to 500 ng/ml) had no significant effect on inositol phosphate accumulation under the same conditions. However, in the presence of millimolar concentrations of Man6P, IGF-II (500 ng/ml) also increased Ins(1,4,5)P3 accumulation by 59%. We conclude that cardiac myocytes from adult rats express IGF receptors and respond to IGFs with the accumulation of Ins(1,4,5)P3 and Ins(1,4)P2. This effect seems to be mediated by an IGF-I receptor-specific pathway.     

Rapid recovery of cardiac beta-adrenergic receptors after isoproterenol-induced "down"-regulation

Desensitization of beta-receptor-linked adenylate cyclase occurs after prolonged occupancy of the beta-receptors by their agonists. We have followed the development and recovery from "down"-regulation of beta-receptors in enzymatically dissociated cardiac myocytes by using the hydrophilic antagonist [3H]-CGP-12177 to identify surface-bound beta-receptors. After in vitro incubation with (-)-isoproterenol, almost 50% of the beta-receptors are lost within 10 minutes. Isoproterenol-mediated cyclic adenosine monophosphate accumulation by isolated myocytes was also decreased after a 15-minute preincubation with isoproterenol. "Lost" beta-receptors can, however, be recovered when isoproterenol-pretreated, washed cardiac myocytes are incubated at 37 degrees C, 85 +/- 7% of the lost beta-receptors have returned to the cell surface after 20 minutes of incubation. The requirements for such recycling were investigated. Receptor recovery does not depend on de novo protein synthesis, since it is unaffected by prior exposure to cycloheximide. It is, however, dependent on cellular energy, because it is prevented by adenosine triphosphate depletion and involves a lysosomal step since it is inhibited by the lysomotropic agent, chloroquine. In addition, the Golgi apparatus and the microtubules are involved in the beta-receptor recycling to the cell surface, as evidenced by the inhibitory effects of monensin and colchicine, respectively. The mechanism of isoproterenol-induced down-regulation of cardiac beta-receptors involves a rapid, reversible cycling to and from the cytosol and the cell membrane. This intracellular receptor traffic is energy dependent, requires several structures, including lysosomes and microtubules, and may be modified by pathological processes involving the heart.     

The heterogeneity of serum hPRL in a patient with a giant prolactinoma

The heterogeneity of hPRL in the serum of a patient with a giant prolactinoma (hPRL 27 micrograms/ml) was studied employing gel chromatography, RIA and lymphoma cell bioassay. After gel filtration of serum sample on Sephadex G-100 (superfine) column, a few hPRL peaks were detected by RIA. Rechromatography of the second peak, which eluted between the void volume and the small third peak, revealed four hPRL peaks designated big-big hPRL, medium-big hPRL, big hPRL and little hPRL, with BA/RIA ratio 1.3 approximately 2.6, 0.8 approximately 1.2, 0.3 approximately 0.7 and 0.7 approximately 1.5, respectively. The molecular weight of medium-big hPRL was estimated as 80K approximately 90K daltons. Most of big-big, medium-big and big hPRL were converted to little hPRL after mercaptoethanol treatment. These data suggest that most of big-big, medium-big and big hPRL are aggregates of little hPRL.     

3'-Monoiodothyronine degradation in rat liver homogenate: enzyme characteristics and documentation of deiodination by high-pressure liquid chromatography

Characteristics of 3'-monoiodothyronine (3'-T1) degradation were examined in vitro in rat tissue homogenates. In rat liver homogenates, 3'-T1 degradation was optimal at pH 7.4, and was dependent upon time, temperature, and tissue concentration. The Michaeli's constant (Km) = 0.84 mumol/L. 3'-T1 degradation was enhanced by dithiothreitol and inhibited by propylthiouracil, sodium ipodate, ANS, and sodium azide but not by methimazole. Animals that fasted for three days had significant reductions in both hepatic T4 to T3 conversion (199 +/- 12 v 116 +/- 12 pg T3 generated/mg protein; P less than 0.001) and 3'-T1 degradation (588 +/- 31 v 148 +/- 53 pg 3'-T1 degraded/mg protein; P less than 0.001). To document that 3'-T1 degradation was occurring by deiodination, both liver and kidney homogenates were incubated with 125I-3'-T1 (approximately 3 microCi; 13.1 nmol/L). The reaction products were separated on a reverse-phase high pressure liquid chromatography (HPLC) column. In both tissues an iodide peak was generated, and no other radiolabeled peaks appeared except for 125I-3'-T1. These data suggest that 3'-T1 is metabolized by phenolic-ring monodeiodination and is enzymic in nature.     

Identification of distinct receptor complexes that account for high-and low-affinity glucagon binding to hepatic plasma membranes.

We have analyzed ligand-receptor complexes resulting from (i) the incubation of canine hepatic plasma membranes with [125I]iodoglucagon and (ii) subsequent gentle solubilization of receptor-bound ligand with digitonin. The complexes (molecular weight approximately equal to 500,000) retain the radiolabeled ligand during gel filtration and subsequent manipulation at 4 degrees C in the absence of covalent crosslinking. Affinity chromatography of the glucagon-receptor complexes on columns of wheat germ lectin linked to agarose resulted in two fractions, one of which was not bound by the column and the other of which was specifically eluted by N-acetylglucosamine. The presence of GTP during the incubation of plasma membranes with [125I]iodoglucagon caused about a 50% decrease in total ligand binding but affected only the ligand-receptor complexes that bound to wheat germ lectin. Moreover, it was found that the proportion of the two forms of ligand-receptor complexes identified by chromatography on wheat germ lectin depended on the degree of saturation of the membrane receptor. Thus, both the inhibition by glucagon of radiolabeled glucagon binding to membranes and the concomitantly decreased extent of association of the radiolabeled ligand with solubilized receptor complexes could be modeled in terms of two noninteracting receptor populations (having dissociation constants of about 0.35 and 4.94 X 10(-9) M). We conclude that (i) glucagon-receptor complexes formed on canine hepatic plasma membranes exist in two forms that differ after solubilization by digitonin in their avidities for wheat germ lectin, (ii) the high-and low-affinity binding of glucagon characteristic of hepatic plasma membranes arises from distinct receptor populations that probably differ in glycosylation, and (iii) the effect of GTP to decrease binding of glucagon to membranes arises from interactions of the nucleotide with the receptor complex that binds to wheat germ lectin.     

Detection of a 58-kilodalton high density lipoprotein-binding protein in the membrane fraction of luteinized rat ovaries

The membrane fraction from the ovaries of pseudopregnant rats exhibits specific, high affinity binding of high density lipoproteins (HDL). Previous studies have indicated that HDL binding in this tissue is up-regulated by hCG and may be involved in supplying cholesterol as substrate for steroid hormone production. To characterize the HDL-binding activity, we solubilized the membrane proteins using 40 mM beta-octylglucoside and then separated them by electrophoresis on a 7% sodium dodecyl sulfate-polyacrylamide gel. The separated proteins were transferred to nitrocellulose sheets and subsequently incubated in the presence of [125I]apolipoprotein-E-free HDL. Autoradiography of the nitrocellulose revealed that the labeled HDL was bound to a single major band, with an apparent mol wt of 58,000 daltons. Neither reduction with beta-mercaptoethanol nor heat denaturation before separation on the gel affected the molecular size of the band, which indicates that it is probably a single polypeptide chain. The band was up-regulated in this tissue by in vivo treatment with 25 IU hCG in a time-dependent manner similar to the up-regulation of [125I]HDL-binding activity. In contrast to the binding of low density lipoprotein (LDL) to its receptor, the binding of HDL is independent of Ca+2. Incubation of the transferred proteins in the presence of [125I] Incubation of the transferred proteins in the presence of [125I]apolipoprotein-E-free HDL and either 5 mM CaCl2 or 15 mM EDTA had no effect on the appearance of the 58-kDa band. Furthermore, ligand blotting in the presence of a 100-fold excess of LDL did not affect the appearance of the band, whereas a 100-fold excess of apoliprotein-E-free HDL caused the disappearance of the band, indicating specificity for binding of HDL. Treatment of the sample with trypsin before electrophoresis also caused the band to disappear, revealing the protein nature of the band. These experiments indicate that the HDL receptor in luteinized rat ovaries is a 58,000-dalton protein.     

Binding of recombinant interleukin-1 beta to the third complement component and alpha 2-macroglobulin after activation of serum by immune complexes.

Activation of human normal serum with tetanus/antitetanus immune complexes (TAT-IC) resulted in increased binding of 125I-labeled interleukin-1 beta (IL-1 beta) to serum factors, as opposed to untreated serum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography showed labeling of two large molecular mass factors of an apparent molecular weight (Mr) of 200,000 and 400,000, respectively. These complexes could be dissociated by reduction. No complexes were formed when reducing compounds were added to serum-TAT-IC-125I-IL-1 beta mixtures. Complex formation was largely prevented by alkylating compounds. Molecular sieve chromatography of TAT-IC-activated serum confirmed that 125I-IL-1 beta became bound to high Mr serum proteins. Fractions containing high molecular 125I-IL-1 serum protein complexes partially retained IL-1-like activity since they induced proliferation of an IL-1-dependent murine T helper (D10G4) cell lineage. The 125I-IL-1 beta binding factors could be immunoprecipitated from TAT-IC-activated serum 125I-IL-1 beta solutions by antisera to alpha 2-macroglobulin (alpha 2M) or to the third complement component (C3). SDS-PAGE of the immunoprecipitates showed radioactive bands corresponding to the expected Mr resulting from complex formation between 125I-IL-1 beta and these two proteins. Treatment of purified plasma alpha 2M and C3 with trypsin or activation with methylamine, which causes cleavage of the internal thiol ester and the appearance of free thiol groups in these proteins, mediated binding of 125I-IL-1 beta to alpha 2M and C3b. The results suggest that cleavage of the internal thiol ester in C3 and alpha 2M makes these plasma proteins susceptible to binding of 125I-IL-1 beta and that free thiol groups do play a role in the formation of 125I-IL-1 beta plasma protein complexes. Activated C3 and alpha 2M may function as IL-1 beta carrier proteins in biologic fluids, in addition to their other physiologic roles.     

Isolation and characterization of a cell surface albumin-binding protein from vascular endothelial cells.

Albumin-binding proteins identified in vascular endothelial cells have been postulated to contribute to the transport of albumin via a process involving transcytosis. In the present study, we have purified and characterized a 57- to 60-kDa (gp60) putative albumin-binding protein from bovine pulmonary microvessel endothelial cells. The endothelial cell membranes were isolated from cultured cells by differential centrifugation and solubilized with sodium cholate and urea. The solubilized extract was concentrated after dialysis by ethanol precipitation and reextracted with Triton X-100, and the resulting extract was subjected to DEAE-cellulose column chromatography. Proteins eluted from this column were further separated using preparative sodium dodecyl sulfate/polyacrylamide gel electrophoresis and used for immunizing rabbits. Fluorescence-activated cell sorter analysis using the anti-gp60 antibodies demonstrated the expression of gp60 on the endothelial cell surface. Affinity-purified anti-gp60 antibodies inhibited approximately 90% of the specific binding of 125I-labeled albumin to bovine pulmonary microvessel endothelial cell surface. The anti-gp60 antibodies reacted with gp60 from bovine pulmonary artery, bovine pulmonary microvessel, human umbilical vein, and rat lung endothelial cell membranes. Bovine anti-gp60 antibodies also reacted with bovine secreted protein, acidic and rich in cysteine (SPARC). However, bovine SPARC NH2-terminal sequence (1-56 residues) antibodies did not react with gp60, indicating that the endothelial cell-surface-associated albumin-binding protein gp60 was different from the secreted albumin-binding protein SPARC. We conclude that the endothelial cell-surface-associated gp60 mediates the specific binding of native albumin to endothelial cells and thus may regulate the uptake of albumin and its transcytosis.     

Characterization of the 3,3',5-triiodo-L-thyronine-binding site on plasma membranes from human placenta

The binding of [125I]T3 to sites on human placenta plasma membranes was characterized, and the binding site was solubilized after affinity labeling with N-bromoacetyl-[125I]T3 (BrAc[125I]T3). Two classes of T3-binding sites were detected. One class has a high affinity (Kd = 2.0nM) and a low capacity (approximately 320 fmol/mg protein); the other has a low affinity (Kd = 18.5 microM) and a high capacity (approximately 2.2 pmol/mg protein). The binding sites were found to be specific for T3 in that other thyroid hormone analogs (D-T3, rT3, D-T4, and L-T4) were less effective or ineffective in displacing the bound [125I]T3. The affinity labeling ligand BrAc[125I]T3 was found to specifically label a protein with an apparent mol wt of 65,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The BrAc[125I]T3-labeled protein was solubilized with 2 mM 3-[( 3-cholamidopropyl)dimethylammonio]1-propane sulfonate. The apparent mol wt of the labeled protein was between 140,000 and 150,000 by Sephadex-G-200 gel filtration. These data demonstrate that a high affinity binding site specific for T3 is present on plasma membranes from human placenta and that the binding site is a protein, most likely a dimer, with a native mol wt between 140,000 and 150,000.