Structural analysis of the hepatic glucagon receptor. Identification of a guanine nucleotide-sensitive hormone-binding region

[125I-Tyr10]Monoiodoglucagon [( 125I]MIG) was cross-linked to liver membrane glucagon receptors with hydroxysuccinimidyl-p-azidobenzoate, and the products were analyzed by sodium dodecyl sulfate-gel electrophoresis. Autoradiograms of the gel obtained after a 24-h exposure showed one major band at Mr = 63,000 that was sensitive to GTP and excess unlabeled glucagon. Exposure for 7 days showed labeling of an additional Mr = 33,000 species that was also sensitive to excess unlabeled glucagon. The Mr = 33,000 peptide can be obtained by subtilisin, trypsin, elastase, or Staphylococcus aureus V8 protease treatment of the [125I]MIG-occupied receptor in the membrane or in Lubrol-PX solution. In contrast, limited proteolysis of membranes containing vacant receptors results in labeling of a Mr = 24,000 peptide. The Mr = 24,000 peptide specifically binds [125I]MIG in a GTP-sensitive manner. The Mr = 33,000 peptide also retains GTP sensitivity since it releases bound [125I]MIG upon addition of GTP. Elastase treatment of the electroeluted Mr = 33,000 peptide yields the Mr = 24,000 and 15,000 fragments. The Mr = 15,000 peptide is the smallest fragment of the receptor as yet identified. Treatment of the Mr = 63,000 receptor with [125I]MIG cross-linked to it with endo-beta-N-acetylglucosaminidase F results in four distinct fragments with Mr values of 61,000, 56,000, 51,000, and 45,000; prolonged treatment resulted in the accumulation of the last two. Neither the Mr = 33,000 nor the Mr = 24,000 fragment appeared to be substrates for endo-beta-N-acetylglucosaminidase F. These data indicate that glucagon receptor is a glycoprotein of approximately 60,000 daltons which contains at least four N-linked glycans accounting for 18,000 daltons of its mass. Both its glucagon binding function and its capacity to interact with the stimulatory regulator of adenylyl cyclase are contained within a fragment of only approximately 21,000 daltons that does not contain any N-linked glycans. Hormone occupancy of the receptor results in a conformational change so as to expose a region that is susceptible to proteolysis by proteases of varying specificities to yield a peptide of approximately 30,000 daltons that also does not contain N-linked glycans.     

Presence of highly selective receptors for PACAP (pituitary adenylate cyclase activating peptide) in membranes from the rat pancreatic acinar cell line AR 4-2J

We characterized highly selective receptors for PACAP, the pituitary adenylate cyclase activating peptide, in the tumoral acinar cell line AR 4-2J derived from the rat pancreas. PACAP, a novel hypothalamic peptide related to vasoactive intestinal peptide (VIP), was tested as the full natural 38-residue peptide (PACAP-38) and as an N-terminal amidated 27-residue derivative (PACAP-27). The binding sites showed considerable affinity for [125I]PACAP-27 (Kd = 0.4 nM) and PACAP-38, while their affinity for VIP and the parent peptide helodermin was 1000-fold lower. These receptors were coupled to adenylate cyclase, the potency of PACAP-38 and PACAP-27 (Kact = 0.2 nM) being much higher than that of VIP (Kact = 100 nM) and helodermin (Kact = 30 nM). Chemical cross-linking of [125I]PACAP-27 followed by SDS-PAGE and autoradiography revealed a specifically cross-linked peptide with an Mr of 68,000 (including 3000 for one PACAP-27 molecule).     

Subunit structure of the erythropoietin receptor

Chemical cross-linking of the red blood cell hormone, erythropoietin (Epo), to its receptor on erythroid cells has revealed the presence of two proteins closely associated with Epo, but the relationship between these two proteins is controversial. Using the cross-linking reagents disuccinimidyl suberate and dithiobissuccinimidyl propionate, we show that 125I-Epo can be specifically conjugated in a complex of 224kDa using mouse fetal liver cells, bone marrow cells, and Friend virus-induced splenic erythroblasts as demonstrated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions. Under reducing conditions, the 224-kDa complex appeared as two Epo conjugates of 136 kDa and 119 kDa, and these bands were also observed to a variable extent in some nonreducing gels. Disulfide linking of the 136-kDa and 119-kDa bands was confirmed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis run under nonreducing followed by reducing conditions. With increasing time of 125I-Epo binding to Friend virus erythroblasts in the presence of sodium azide to inhibit receptor internalization, the 136-kDa and 119-kDa bands seen under reducing conditions increased markedly in intensity, whereas the 224-kDa band seen under nonreducing conditions declined. These results suggest that the 224-kDa Epo conjugate is inefficiently solubilized under nonreducing conditions following prolonged periods of Epo binding. A single class of saturable, high affinity receptors for Epo on each of the cell types tested is demonstrated. It is concluded that the two disulfide-linked Epo-binding proteins which can be independently cross-linked to Epo form a single ligand binding site.     

Demonstration and structural comparison of receptors for insulin-like growth factor-I and -II (IGF-I and -II) in brain and blood-brain barrier

Specific receptors for insulin-like growth factors (IGF) I and II on microvessel-free rat brain cell membranes (RBCM) and in the microvessels that constitute the blood-brain barrier (BBB) were identified and characterized by means of affinity cross-linking techniques and specific anti-receptor antibodies. Two different models of BBB were examined: isolated rat brain capillaries and cultured bovine brain microvessel endothelial cells. Cross-linking with 125-I-IGF-I, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), revealed an alpha subunit of apparent Mr = 138,000 in both BBB preparations, compared to 120,000 in RBCM. Cross-linking was inhibited by unlabeled IGF and insulin, but not by antibody directed against the IGF-II receptor. When 125-I-IGF-II was cross-linked, followed by SDS-PAGE under reducing conditions, a major band of apparent Mr = 250,000 was identified in RBCM and both BBB preparations. This band, which migrated with an approximately equivalent Mr in both brain and BBB membranes, was inhibited by unlabeled IGF and by antibody specific for the IGF-II receptor. Thus, both rat and bovine brain microvessels possess classical Type I and II IGF receptors. While the alpha subunit of the Type I receptor of brain is smaller than that of the BBB, the Type II receptor of brain and BBB appear to be structurally and immunologically identical.     

Characterization of human red cell Rh (rhesus-)specific polypeptides by limited proteolysis

Human red cells of various Rh phenotypes were surface-labelled with 125I and the Rh-specific labelled polypeptides were isolated by preparative SDS-PAGE. The polypeptides were subjected to limited proteolysis and the resulting fragments were analysed by SDS-PAGE and autoradiography. Chymotryptic peptide maps of proteins obtained from Rh(D)-positive and -negative types appeared completely identical, whereas tryptic peptide maps revealed a difference: a fragment of Mr 17,500 was associated with the Rh(D) antigen, and one of Mr 19,000 with the Rh(C/c,E/e) antigens. Treatment of Rh polypeptides with carboxypeptidase Y prior to tryptic digestion resulted in a shift of nearly all tryptic fragments, including a fragment of Mr 8,000, indicating that the surface label was incorporated into the C-terminal part of the molecule.     

Expression of the erythropoietin receptor on a human myeloma cell line

We demonstrated the expression of the erythropoietin (EPO) receptor by a human myeloma cell line (MM-S1) which was established in our laboratory. EPO dose-dependently stimulated the proliferation of MM-S1 cells. Binding of radioiodinated EPO (125I-Epo) to MM-S1 cells was competitively inhibited by unlabeled EPO, but not by other recombinant cytokines. Specific binding of 125I-Epo to MM-S1 cells was saturable, and the Scatchard analysis revealed 330 EPO binding sites per cell with a Kd of 0.56 nmol/L. Bound EPO was internalized by MM-S1 cells during incubation at 37 degrees C. This is the first report describing the expression of the EPO receptor by human cells other than those of the erythroid lineage.     

Specific photoaffinity crosslinking of [125I]cholecystokinin to pancreatic plasma membranes. Evidence for a disulfide-linked Mr 76 000 peptide in cholecystokinin receptors

In rat pancreatic plasma membranes, preincubated with [125I]cholecystokinin-33 (CCK-33) and washed free of unbound tracer, the irradiation by UV light induced the irreversible binding of radioactivity to high molecular weight peptides as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) and autoradiography. This was not observed when the membranes were preincubated in the simultaneous presence of [125I]CCK-33 and of either an excess of unlabelled CCK-8 or of guanosine 5'-(beta, gamma-imido)-triphosphate. The radioactivity was mostly crosslinked with a Mr 96,000 peptide and peptide species of Mr greater than 200,000, after SDS solubilization in the absence of beta-mercaptoethanol. Peptide reduction with beta-mercaptoethanol converted the high molecular weight radioactive species into a Mr 76,000 peptide that contained as much as 65% of the radioactivity crosslinked. The Mr 76,000 peptide appears, therefore, to be a disulfide-linked constituent of rat pancreatic cholecystokinin receptors.     

Identification of the V1 vasopressin receptor by chemical cross-linking and ligand affinity blotting

Chemical and photoaffinity cross-linking experiments as well as ligand affinity blotting techniques were used to label the V1 vasopressin receptor. In order to determine the optimal reaction conditions, pig liver membranes were incubated with 5 nM [8-lysine]vasopressin (LVP) labeled with 125I and then cross-linked with the use of DMS (dimethyl suberimidate), EGS [ethylene glycol bis(succinimidyl succinate)] or HSAB (hydroxysuccinimidyl p-azidobenzoate) at different final concentrations. Consistently, EGS was found to label with high yield one band of Mr 60,000 in rat and pig liver membranes when used at a final concentration between 0.05 and 0.25 mM. The protein of Mr 60,000 is labeled in a concentration-dependent manner when pig liver membranes are incubated with increasing concentrations of 125I-LVP and then cross-linked with EGS. The label was displaced by increasing concentrations of unlabeled LVP or d(CH2)5 [Tyr2(Me),-Tyr9(NH2)]AVP (V1/V2 antagonist). A protein band of similar molecular mass was cross-linked with 125I-LVP in rat liver membranes. The reaction was specific since the incorporation of label into the protein of Mr 60,000 was inhibited by LVP, [8-arginine]vasopressin (AVP), the V1/V2-antagonist, and the specific V1-antagonist d(CH2)5 [Tyr2(Me)]AVP, only partially by [des-Gly9]AVP (V2-agonist) and by oxytocin, and not at all by angiotensin II. Incubation of nitrocellulose containing membrane proteins from pig liver with 125I-LVP showed the labeling of a band of Mr 58,000 that is inhibited by an excess of unlabeled LVP. This band of Mr 58,000 seems to correspond with the protein of Mr 60,000 revealed by the cross-linking experiment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding and processing of fibrinogen by rabbit hepatocytes

We describe a specific fibrinogen-hepatocyte interaction. Rabbit 125I-labeled fibrinogen (125I-FGN) was incubated at 4 degrees C with suspensions of rabbit hepatocytes (approximately 1 X 10(6) cells/ml). Bound ligand was separated from free by centrifugation of cells through oil and quantitated by gamma-scintillation counting. Specific binding, determined by subtraction of nonspecific binding in the presence of 8 mM EDTA from total binding in the presence of 2 mM CaCl2, required 3 h to plateau and represented approximately 70% of total binding. Specific binding was calcium-dependent and was negligible in buffer containing 2 mM MgCl2. Half-maximal saturation occurred at approximately 30 nM 125I-FGN with approximately 480,000 molecules/cell at saturation. Dilution experiments revealed comparable affinities for labeled and unlabeled fibrinogen. Total binding was irreversible as determined by addition of excess unlabeled fibrinogen or EDTA. Specific binding of 25 nM 125I-FGN was inhibited, in a concentration-dependent fashion, by unlabeled fibrinogen or fibrinogen fragment D95 (Mr = 95,000), but not by fibrinogen fragment E or Arg-Gly-Asp-containing peptides. Unlabeled fibrinogen (3.1 microM) completely abolished specific binding, whereas greater than 80% inhibition was achieved with 10 microM fragment D95. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography of 125I-FGN bound in the presence of calcium demonstrated disappearance of A alpha chains with formation of products of Mr greater than 200,000; EDTA or unlabeled fibrinogen prevented fibrinogen processing. These data describe a unique fibrinogen-hepatocyte interaction which differs considerably from the platelet-fibrinogen interaction, especially with regard to the processing of the fibrinogen molecule.     

Photoaffinity labeling of the canine renal receptor for parathyroid hormone

Studies were undertaken to identify and characterize components of the parathyroid hormone receptor. An analog of the bovine parathyroid hormone(1-34) sequence was derivatized at the 23-tryptophan position with the photoreactive reagent 2-nitro-5-azidophenylsulfenyl chloride. 2-Nitro-5-azidophenylsulfenyl-bovine parathyroid hormone (NAPS-bPTH) analog retained full biological activity with respect to receptor binding and activation of adenylate cyclase in canine renal cortical plasma membranes. 125I-bPTH(1-34) analog was also derivatized without loss of receptor-binding activity. When 125I-NAPS-bPTH(1-34) analog was incubated with canine renal plasma membranes and then photolyzed, at least two 125I-labeled membrane components were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The incorporation of 125I radioactivity into one of these components (Mr congruent to 60,000) was inhibited when incubation and photolysis were performed in the presence of excess, unlabeled bPTH(1-34). Furthermore, photolysis of membranes in the presence of NAPS-bPTH(1-34) analog led to activation of adenylate cyclase which persisted following washing to remove noncovalently bound peptide, suggesting a functional, covalent hormone-receptor complex had been formed. We conclude that the M r congruent to 60,000 membrane component may be a part of the renal receptor for parathyroid hormone.     

Characterization of a C5a receptor on human polymorphonuclear leukocytes (PMN)

Elucidation of the interactions between C5a and granulocytes is central to understanding the role of C5a in inflammation. In this study, interactions between C5a and PMN have been studied at two levels. Binding of human C5a to intact human cells has been characterized by using the radiolabeled ligand 125I-C5a. Binding is shown to be reversible, saturable, and to reach equilibrium in 60 to 90 min at 0 degrees C. Results show high affinity C5a binding sites with Kd = 2 X 10(-9) M and a range of 50,000 to 113,000 binding sites per PMN. These values for C5a receptors are comparable with the number of fMLP and LTB4 receptors on PMN. Binding of C5a to PMN fails to reach equilibrium at 37 degrees C because there is an irreversible loss of available surface receptors caused by an active internalization of the ligand-receptor complex. Interactions between C5a and human PMN were characterized further by cross-linking experiments, with the use of ethylene glycol bis succinimidylsuccinate (EGS). Cross-linking of 125I-C5a to intact PMN followed by subcellular fractionation revealed a single radioactive band present only in the plasma membrane fraction and visualized by autoradiography. Similar experiments resulted in a covalent linkage between 125I-C5a and a component in the isolated plasma membrane of PMN. The covalent complex containing C5a and a putative receptor has been visualized by autoradiography as a single 60,000 Mr complex on SDS-PAGE. The complex is not present when experiments are performed in the presence of excess unlabeled C5a or in the absence of EGS. Therefore, the putative receptor for C5a on human neutrophils is estimated to be approximately 48,000 Mr, assuming contribution of 12,000 to 13,000 daltons by the ligand 125I-C5a.     

Analysis of tegumental surface proteins of Schistosoma mansoni primary sporocysts

Axenically transformed primary sporocysts of Schistosoma mansoni (NMRI strain) were labeled with 125I in an effort to identify sporocyst proteins exposed at the tegumental surface. Using the 125I activating reagent, 1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenylglycoluril, in conjunction with SDS-PAGE and autoradiography, up to 12 bands were radiolabeled out of 60 components visualized by silver staining. Labeled proteins ranged in apparent Mr from greater than 200 to less than 12 kDa. Pronase treatment of living sporocysts after radioiodination removed all labeled material, suggesting that only surface proteins were being iodinated. Western blot analysis employing 5 monoclonal antibodies (MAB's) to sporocyst surface antigens revealed a wide range of reactivities which produced banding patterns closely reflecting autoradiograms of identical samples. The concomitant removal by Pronase of immunoreactive and radiolabeled surface proteins with identical Mr in the range of 90-130 kDa suggests that epitopes recognized by these antibodies are associated with these higher molecular weight surface proteins. However, although Pronase removes all labeled surface proteins, substantial nonradiolabeled, immunoreactive material with Mr less than 90 kDa still remains on enzyme-treated parasites. This indicates that MAB-reactive epitopes, in addition to their occurrence with surface proteins, are also associated with either unlabeled, protease-resistant surface components or internal antigens. The immunohistochemical localization of antibody-reactive material in gland-like structures within sporocysts supports an internal source for nonradiolabeled, immunoreactive components. Finally, the periodate sensitivity of the epitopes recognized by all tested MAB's suggests that carbohydrate moieties may represent a common and extremely immunogenic constituent of the sporocyst surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for vasoactive intestinal peptide receptors in apical membranes from tracheal epithelium

[125I]VIP (vasoactive intestinal peptide) bound to apical membranes isolated from the bovine tracheal epithelium with a half maximal inhibition by unlabeled VIP (IC50) of 0.6 x 10(-9)M and binding was reversible. Glucagon did not affect [125I]VIP binding to the membranes. [125I]VIP was covalently cross-linked to tracheal membrane proteins using disuccinimidyl suberate. SDS-polyacrylamide gel electrophoresis of labeled tracheal membranes revealed one major [125I]-receptor complex of Mr = 71,000 to which binding of [125I]VIP was inhibited by 10 microM unlabeled VIP. These results are consistent with the presence of a specific, high-affinity receptor for VIP, with a Mr = 71,000, in apical membrane vesicles isolated from the bovine tracheal epithelium.     

Detection of actin-binding proteins in human platelets by 125I-actin overlay of polyacrylamide gels

Actin-binding proteins have been identified in human platelets with a gel-overlay technique that uses 125I-G-actin. Platelet proteins were separated on SDS polyacrylamide gels using the buffer system of Laemmli (1970, Nature [Lond.] 227:680-685). The proteins were fixed in the gels with methanol-acetic acid, the SDS was washed out, and the proteins were renatured. The gels were incubated with 125I-G-actin from rabbit skeletal muscle that was radiolabeled with 125I according to the method of Bolton and Hunter (1973, Biochem. J. 133:529-538) and has been shown to retain biological activity. After nonspecifically bound radioactivity was washed out, gels were dried and processed for autoradiography. The 125I-G-actin binds to several proteins in human platelets, platelet extracts, and the particulate fraction. Control experiments demonstrate that the 125I-G-actin can be displaced by use of increasing amounts of unlabeled actin, that the binding is stable to 0.6 M NaCl, and that preheating the 125I-G-actin to 90 degrees C for 3 min eliminates all binding. Prominent 125I-G-actin-binding activities were present at Mr 90,000 and 40,000. The binding to the 90,000 Mr protein appears to be at least partially Ca++ sensitive, whereas the binding to the 40,000 Mr protein does not. 125I-G-actin bound to proteins in the SDS gels can be fixed in situ and compared directly with the stained gel. This technique should prove generally useful in identification and purification of some actin-binding proteins from cells and tissues.     

Differential binding of hemolymph proteins from schistosome-resistant and -susceptible Biomphalaria glabrata to Schistosoma mansoni sporocysts

Humoral factors have been associated with resistance of Biomphalaria glabrata to infection by Schistosoma mansoni. The goal of this study was to determine which serum (cell-free hemolymph) proteins bind to the surface of S. mansoni sporocysts. For this, 125I-labeled serum from schistosome-resistant (10-R2) and -susceptible (M-line) B. glabrata was incubated with sporocysts, washed, and then subjected to SDS-PAGE and autoradiography. Other samples examined included radiolabeled 10-R2 and M-line serum, sporocysts incubated with unlabeled serum followed by incubation with radiolabeled serum, and radiolabeled sporocysts. Results indicated that many polypeptides in the serum from both strains of B. glabrata were radiolabeled. Dominating both profiles were bands in the 90-210-kDa range. However, some differences between the serum of the 2 snail strains were observed with M-line serum having several radiolabeled polypeptides in the 31-40- and 66-85-kDa range that were absent in serum from 10-R2 B. glabrata. When sporocysts were incubated with radiolabeled serum, 3 polypeptides (116, 180, 210 kDa) from both snail strains bound to the surface of the parasite. Further, a 55-kDa polypeptide bound to sporocysts incubated with 10-R2 serum but did not bind to those parasites incubated with M-line serum. Preincubation of sporocysts with unlabeled serum prior to incubation with radiolabeled serum significantly inhibited the uptake of radiolabeled proteins. This differential binding of serum polypeptides from different strains of B. glabrata may be important in determining resistance or susceptibility of the snail to larval schistosome infection.     

Transferrin-binding and iron-binding proteins of rabbit reticulocyte plasma membranes. Three distinct moieties

1. Transferrin-membrane complexes and iron-binding membrane complexes were solubilized with sodium dodecyl sulfate from the plasma membranes of reticulocytes that had been incubated with (59Fe,125I)-labeled transferrin. Gel filtration of solubilized material demonstrated 125I-labeled transferrin complexed to two moieties, a minor component (Peak I) of apparent molecular weight 435,000 and a major component (Peak II) of apparent molecular weight 200,000. Most of the membrane 59Fe was located in Peak I. 2. Sepharose-bound anti-transferrin was used to purify the 125I-labeled transferrin-membrane complexes. The 59Fe/125I ratio in the transferrin complex purified from Peak I was the same as in the original transferrin and thus contained membrane-bound transferrin to which the 59Fe was still attached. The 59Fe/125I ratio in the purified Peak II transferrin complex was 0.33 times that of the original transferrin, indicating that more than 60% of its 59Fe had been delivered to the reticulocyte. 3. The purified transferrin complexes analyzed by SDS-polyacrylamide gel electrophoresis demonstrated a single band of apparent molecular weight 78,000 both by Coomassie blue stain for protein and by 125I radioactivity. The specific activity of this material was 0.27 and 0.56 times that of the original transferrin for Peak I and Peak II, respectively, indicating that transferrin in Peak I and II was bound to a membrane component with a molecular weight similar to that of transferrin. 4. The isoelectric focusing pattern of the Peak II transferrin complex showed isoelectric points of pH 6.7 and 6.2 compared to pH 5.4 for transferrin. 5. On the basis of these studies we propose that transferrin is first bound to a membrane protein and then delivers iron to a membrane component distinct and separate from the transferrin-binding moiety. Prior to its release, transferrin markedly depleted of iron is still bound to a component in the plasma membrane.     

Opiate receptor mediated internalization of 125I-beta-endorphin in human polymorphonuclear leucocytes

The early events in the interaction of (125I)-Tyr27-beta-endorphin with human polymorphonuclear leucocytes were investigated. Using ultrastructural autoradiography we found that the labeled peptide specifically bound to the plasma membrane and was internalized within two minutes of incubation at 37 degrees C. Both processes could be inhibited by unlabeled beta-endorphin or by the opiate antagonist diprenorphine. This finding was confirmed by radioreceptorassays. With longer incubation times the specific association of the labeled beta-endorphin with the cells decreased. About 10% of the tracer was degraded within 10 min of incubation as shown by gel chromatography. The morphological changes induced by 125I-beta-endorphin in the granulocytes were investigated under the microscope. The labeled peptide had the same biological effect as unlabeled beta-endorphin.     

Photoaffinity labeling of (Na+K+)-ATPase with [125I]iodoazidocymarin

A radioiodinated, photoactive cardiac glycoside derivative, 4'-(3-iodo-4-azidobenzene sulfonyl)cymarin (IAC) was synthesized and used to label (Na+K+)-ATPase in crude membrane fractions. In the dark, IAC inhibited the activity of (Na+K+)-ATPase in electroplax microsomes from Electrophorus electricus with the same I50 as cymarin. [125I]IAC binding, in the presence of Mg2+ and Pi, was specific, of high affinity (KD = 0.4 microM), and reversible (k-1 = 0.11 min-1) at 30 degrees C. At 0 degree C, the complex was stable for at least 3 h, thus permitting washing before photolysis. Analysis of [125]IAC photolabeled electroplax microsomes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (7-14%) showed that most of the incorporated radioactivity was associated with the alpha (Mr = 98,000) and beta (Mr = 44,000) subunits of the (Na+K+)-ATPase (ratio of alpha to beta labeling = 2.5). A higher molecular weight peptide (100,000), similar in molecular weight to the brain alpha(+) subunit, and two lower molecular weight peptides (12,000-15,000), which may be proteolipid, were also labeled. Two-dimensional gel electrophoresis (isoelectric focusing then SDS-PAGE, 10%) resolved the beta subunit into 12 labeled peptides ranging in pI from 4.3 to 5.5. When (Na+K+)-ATPase in synaptosomes from monkey brain cortex was photolabeled and analyzed by SDS-PAGE (7-14%), specific labeling of the alpha(+), alpha, and beta subunits could be detected (ratio of alpha(+) plus alpha to beta labeling = 35). The results show that [125I]IAC is a sensitive probe of the cardiac glycoside binding site of (Na+K+)-ATPase and can be used to detect the presence of the alpha(+) subunit in crude membrane fractions from various sources.     

The subunit structure of the follitropin receptor. Chemical cross-linking of the solubilized follitropin-receptor complex

Homobifunctional cross-linkers were utilized to characterize high affinity (Ka = 2.2 X 10(-10) M-1) follitropin (FSH) receptors in immature bovine testis. Following the formation of radioiodinated human FSH (125I-hFSH)-receptor complexes, the membranes were solubilized with Triton X-100 or beta-octyl glucoside and the supernatants from ultracentrifugation (220,000 X g) subjected to gel filtration (Sephadex G-200) to separate the labeled hormone-receptor complexes from the unbound 125I-hFSH. The appearance of a high molecular weight (greater than or equal to 200,000) radioactive component in the elution profile was abolished when an excess of unlabeled hFSH was included in the initial incubation. After concentration by ultrafiltration, the 125I-hFSH-receptor complex, as well as the free hormone, was treated with a variety of chemical cross-linkers and subjected to analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Bands of Mr = 65,000 and 83,000 observed in the autoradiograph of the hormone-receptor complex was not present in autoradiographs of free 125I-hFSH, nor were they present when an excess of unlabeled hFSH was included in the initial binding incubation mixtures. The 65,000 and 83,000 Mr bands were, therefore, considered to represent cross-linked complexes of labeled hFSH (Mr = 38,000) or its subunits (hFSH alpha, Mr = 16,000; hFSH beta, Mr = 21,000) and components of the FSH receptor. The bands were observed on autoradiographs when the extraction of the membranes was performed with either Triton X-100 or beta-octyl glucoside and when cross-linking was accomplished with disuccinimidyl suberate, ethylene glycol bis(succinimidyl succinate), or bis[2-(succinimido oxycarbonyl)oxyethyl]sulfone. The Mr of the native FSH receptor in the calf testis has been estimated at 146,000. Our studies demonstrate the multimeric nature of the FSH receptor. However, FSH is also composed of subunits, so that due to the complexity of the system, it was not possible to arrive at a precise assessment of the Mr or quaternary structure of the receptor subunits.