Purification of the human placental alpha 2-macroglobulin receptor

The alpha 2-macroglobulin receptor was solubilized from human placental membranes, purified and characterized. Affinity cross-linking of labelled ligand to intact membranes showed a receptor size compatible with 400-500 kDa. The membranes were solubilized in 3-[(3-cholamidopropyl)dimethylammonio]propane sulfonate (CHAPS) and affinity chromatography was performed using Sepharose-immobilized alpha 2-macroglobulin-methylamine with elution in buffer containing 2 mM EDTA, pH 6.0. SDS-PAGE of the resulting receptor preparation showed a predominant approx. 440 kDa band (reducing conditions) and some minor accompanying proteins of 70-90 kDa and 40 kDa. The yield was 400-800 micrograms receptor preparation per placenta. The receptor preparation immobilized on nitrocellulose bound the alpha 2-macroglobulin-trypsin complex with a dissociation constant of about 400 pM. 125I-iodinated receptor preparation bound almost quantitatively to Sepharose-immobilized alpha 2-macroglobulin-methylamine in the presence of CHAPS alone, and bound 70-80% in the presence of 0.2% SDS. The labelled proteins were separated in the presence of 0.2% SDS by gel filtration or SDS-PAGE (unboiled samples). The 440 kDa protein accounted for the major part of the binding, although some approx. 80 kDa proteins, perhaps proteolytic degradation products, also showed binding activity.     

Characterization, size estimation and solubilization of alpha-macroglobulin complex receptors in liver membranes

Receptors for alpha 2-macroglobulin-proteinase complexes have been characterized in rat and human liver membranes. The affinity for binding of 125I-labelled alpha 2-macroglobulin.trypsin to rat liver membranes was markedly pH-dependent in the physiological range with maximum binding at pH 7.8-9.0. The half-time for association was about 5 min at 37 degrees C in contrast to about 5 h at 4 degrees C. The half-saturation constant was about 100 pM at 4 degrees C and 1 nM at 37 degrees C (pH 7.8). The binding capacity was approx. 300 pmol per g protein for rat liver membranes and about 100 pmol per g for human membranes. Radiation inactivation studies showed a target size of 466 +/- 71 kDa (S.D., n = 7) for alpha 2-macroglobulin.trypsin binding activity. Affinity cross-linking to rat and human membranes of 125I-labelled rat alpha 1-inhibitor-3.chymotrypsin, a 210 kDa analogue which binds to the alpha 2-macroglobulin receptors in hepatocytes (Gliemann, J. and Sottrup-Jensen, L. (1987) FEBS Lett. 221, 55-60), followed by SDS-polyacrylamide gel electrophoresis, revealed radioactivity in a band not distinguishable from that of cross-linked alpha 2-macroglobulin (720 kDa). This radioactivity was absent when membranes with bound 125I-alpha 1-inhibitor-3 complex were treated with EDTA before cross-linking and when incubation and cross-linking were carried out in the presence of a saturating concentration of unlabelled complex. The saturable binding activity was maintained when membranes were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]propane sulfonate (CHAPS) and the size of the receptor as estimated by cross-linking experiments was shown to be similar to that determined in the membranes. It is concluded that liver membranes contain high concentrations of an approx. 400-500 kDa alpha 2-macroglobulin receptor soluble in CHAPS. The soluble preparation should provide a suitable material for purification and further characterization of the receptor.     

Photoaffinity labeling of alpha 1-adrenergic receptors of rat heart

The photoaffinity probe [125I]aryl azidoprazosin was used to examine structural aspects of rat left ventricular alpha 1-adrenergic receptor. Autoradiography of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins from photoaffinity-labeled membranes revealed a specifically labeled protein of mass 77 kDa. Adrenergic drugs competed with the photoaffinity probe for binding to the receptor in a manner expected of an alpha 1-adrenergic antagonist. Because the autoradiographic pattern was unaltered by incubating labeled membranes in gel sample buffer containing high concentrations of reducing agents, the binding component of the cardiac alpha 1-adrenergic receptor appears to be a single polypeptide chain. The photoaffinity probe specifically labeled a single protein of approximately 68 kDa in membranes of cardiac myocytes prepared from rat left ventricles. The role played by sulfhydryls in receptor structure and function was also studied. Dithiothreitol (DTT) inhibited [3H]prazosin binding to left ventricular membranes and altered both the equilibrium dissociation constant and maximal number of [3H]prazosin-binding sites but not the ability of the guanine nucleotide guanyl-5'-yl imidodiphosphate to decrease agonist affinity for the receptors. When photoaffinity-labeled membranes were incubated with 40 mM DTT for 30 min at room temperature, two specifically labeled proteins of 77 and 68 kDa were identified. The DTT-induced conversion of the 77-kDa protein to 68 kDa was irreversible with washing, but the effect of DTT on [3H]prazosin binding was reversible. Both 77- and 68-kDa proteins were observed with liver membranes even in the absence of reducing agent. We suggest that the DTT-induced conversion of the 77-kDa protein to 68 kDa is due to enhancement in protease activity by the reductant. These results document that the cardiac alpha 1-adrenergic receptor is a 77-kDa protein, similar in mass to the receptor in liver and other sites. Proteolysis likely accounts for lower Mr forms of this receptor found in cardiac myocytes and in previous publications on hepatic alpha 1-receptors.     

Rat liver glucocorticoid receptor isolated by affinity chromatography is not a Mg2+- or Ca2+-dependent protein kinase

The glucocorticoid hormone receptor (92 kDa), purified 9000-fold from rat liver cytosol by steroid affinity chromatography and DEAE-Sephacel chromatography, was assayed for the presence of protein kinase activity by incubations with [gamma-32P]ATP and the photoaffinity label 8-azido-[gamma-32P]ATP. Control preparations isolated by affinity chromatography in the presence of excess steroid to prevent the receptor from binding to the affinity matrix were assayed for kinase activity in parallel. The receptor was not labeled by the photoaffinity label under photoactivation conditions in the presence of Ca2+ or Mg2+. A Mg2+-dependent protein kinase (48 kDa) that could be photoaffinity labeled with 8-azido-ATP copurified with the receptor. This kinase was also present in control preparations. The kinase could phosphorylate several minor contaminants present in the receptor preparation, including a protein (or proteins) of similar molecular weight to the receptor. The phosphorylation of 90-92-kDa proteins was independent of the state of transformation or steroid-binding activity of the receptor. These experiments provide direct evidence that neither the glucocorticoid receptor nor the 90-92-kDa non-steroid-binding protein associated with the molybdate-stabilized glucocorticoid receptor possesses intrinsic Ca2+- or Mg2+-dependent protein kinase activity.     

A novel guanine nucleotide-binding protein coupled to the alpha 1-adrenergic receptor. I. Identification by photolabeling or membrane and ternary complex preparation

The G-protein involved in alpha 1-adrenergic receptor signaling was identified using two different approaches. First, purified rat liver membranes were incubated with [alpha-32P]GTP in the absence or presence of the adrenergic agonist (-)-epinephrine, or in the presence of GTP. After UV irradiation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography, covalent labeling of a number of proteins was apparent and could be blocked by unlabeled GTP. In the preparation treated with (-)-epinephrine alone, labeling of a 74-kDa species was markedly enhanced. Enhanced labeling of 40-50-kDa species was also observed. Labeling of the 74-kDa protein was also evident in similarly treated membranes prepared from FRTL-5 thyroid cells, which contain abundant alpha 1-adrenergic receptors, but not in those prepared from turkey erythrocytes or NIH 3T3 fibroblasts, which are essentially devoid of alpha 1-receptors. Second, alpha 1-agonist-receptor-G-protein ternary complex formation was induced by incubating purified rat liver membranes with (-)-epinephrine. Rauwolscine (10(-7) M) and (+/-)-propranolol (10(-6) M) were included to prevent activation of alpha 2- and beta-adrenergic receptors by (-)-epinephrine. The ternary complex of hormone, receptor, and G-protein was solubilized, partially purified using heparin- and wheat germ agglutinin-agarose, and reconstituted into phospholipid vesicles. The vesicles displayed agonist-stimulated guanosine 5'-O-3-thiotriphosphate (GTP gamma S) binding that was blocked by phentolamine (10(-4) M). By contrast, stimulation of GTP gamma S binding was not evident when the vesicles were incubated with the beta-agonist, isoproterenol. Incubation of the vesicles with [alpha-32P]GTP or [alpha-32P]azido-GTP in the presence of (-)-epinephrine, followed by photolysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography, resulted in the covalent labeling of a 74-kDa protein. Labeling of this protein could be blocked by preincubation with phentolamine or unlabeled GTP. These findings provide direct evidence for the coupling of the alpha 1-adrenergic receptor to a previously uncharacterized G-protein (termed Gh), which has an apparent molecular mass of approximately 74 kDa.     

An endogenous Ca2+-sensitive proteinase converts the hepatic alpha 1-adrenergic receptor to guanine nucleotide-insensitive forms

An iodoazido[125I]prazosin analogue was employed to photoaffinity label alpha 1-adrenergic receptors in rat liver plasma membranes. Labeled proteins were separated by gradient polyacrylamide gel electrophoresis in sodium dodecyl sulfate, and (-)-epinephrine displacement of [3H]prazosin binding was concurrently measured in the presence or absence of guanosine 5'-O-(gamma-thiotriphosphate) (GTP[gamma S]). Inclusion of EGTA and/or proteinase inhibitors during membrane preparation and incubation increased the effect of GTP[gamma S] on alpha 1-adrenergic agonist binding and this could be correlated with increased concentrations of a 78 kDa photoaffinity labeled protein. In contrast, omission of EGTA or addition of exogenous Ca2+ diminished or abolished the effect of GTP[gamma S] on binding and caused loss of the 78 kDa form and the appearance of lower molecular weight labeled proteins. Age-dependent differences in GTP[gamma S] effects on alpha 1-adrenergic agonist binding were abolished when membranes were prepared and incubated in the presence of EGTA and proteinase inhibitors. However, the 78 kDa photoaffinity labeled protein observed in adult rats (over 225 g body weight) was not apparent in membranes from younger rats (50-75 g), even when the membranes were prepared and incubated in the presence of EGTA and proteinase inhibitors. Instead, a 68 kDa species was the major labeled protein. These data suggest that GTP effects on alpha 1-adrenergic agonist binding in rat liver membranes require the presence of either a 68 or 78 kDa alpha 1-adrenergic binding protein. Failure to inhibit proteolysis in the membranes leads to the generation of lower-molecular-weight binding proteins and the loss of GTP effects on alpha 1-adrenergic agonist binding, although [3H]prazosin binding characteristics are not changed. It is suggested that either the proteolyzed forms of the alpha 1-adrenergic receptor are unable to couple to a putative guanine nucleotide-binding regulatory protein, or that such a protein is concurrently proteolyzed and is thus unable to couple to the receptor.     

Ca2+-dependent proteolysis of the Ah receptor

We previously reported (J. Biol. Chem. (1986) 261, 6352-6465) that the photoaffinity ligand for the Ah receptor, [125I]-2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin, upon incubation with the liver cytosol fraction from C57BL/6 mice, labeled in a 1:1 ratio two peptides that had apparent molecular masses of 95 and 70 kDa and similar proteolytic fragmentation patterns. In the cytosolic fraction of Hepa 1 cells, a cloned murine hepatoma cell line, the product of photoaffinity labeling is almost exclusively a 95-kDa peptide which is rapidly hydrolyzed by a Ca2+-dependent proteinase to a 70-kDa peptide as well as other fragments. Thus, the ligand binding unit of the Ah receptor in C57BL/6 mouse liver and Hepa 1 cell is a 95-kDa peptide, and the 70-kDa fragment is a proteolytic artifact. The Ca2+-dependent proteinase which hydrolyzes the 95-kDa peptide has the properties of calpain II: (i) an absolute requirement for Ca2+, with maximal activity at 0.5 to 1.0 mM Ca2+; (ii) a pH optimum of 7.5 to 8.0; (iii) inhibition by EDTA, iodoacetamide, leupeptin and L-trans-epoxysuccinylleucylamido(4-guanidino)butane, but not by soybean trypsin inhibitor, aprotinin, or phenylmethanesufonyl fluoride. Upon chromatographic separation of the liver cytosol of C57BL/6 mice on DEAE-Sephacel, Ca2+-dependent proteinase activity (using casein or the labeled 95-kDa peptide as substrates) elutes with 0.25 M NaCl, and a specific proteinase inhibitor elutes with 0.15 M NaCl. Ca2+-dependent proteinase activity that hydrolyzes the 95-kDa peptide is found in the liver cytosols of several mammalian species.     

A novel guanine nucleotide-binding protein coupled to the alpha 1-adrenergic receptor. II. Purification, characterization, and reconstitution

In the previous paper, we reported the identification of a 74-kDa G-protein that co-purifies with the alpha 1-adrenergic receptor following ternary complex formation. We report here on the purification and characterization of this 74-kDa G-protein (termed Gh) isolated de novo from rat liver membranes. After solubilization of rat liver membranes with the detergent sucrose monolaurate, Gh was isolated by sequential chromatography using heparin-agarose, Ultrogel AcA 34, hydroxylapatite, and heptylamine-Sepharose columns. The protein, thus isolated, is not a substrate for cholera or pertussis toxin but displays GTPase activity (turnover number, 3-5 min-1) and high-affinity guanosine 5'-O-3-thiotriphosphate (GTP gamma S) binding (half-maximal binding = 0.25-0.3 microM), which is Mg2(+)-dependent and saturable. The relative order of nucleotide binding by Gh is GTP gamma S greater than GTP greater than GDP greater than ITP much much greater than ATP greater than or equal to adenyl-5'-yl imidodiphosphate, which is similar to that observed for other heterotrimeric G-proteins involved in receptor signaling. Moreover, specific alpha 1-agonist-stimulated GTPase (turnover number, 10-15 min-1) and GTP gamma S binding activity could be demonstrated after reconstitution of purified Gh with partially purified alpha 1-adrenergic receptor into phospholipid vesicles. The alpha 1-agonist stimulation of GTP gamma S binding and GTPase activity was inhibited by the alpha-antagonist phentolamine. A 50-kDa protein co-purifies with the 74-kDa G-protein. This protein does not bind guanine nucleotides and may be a subunit (beta-subunit) of Gh. These findings indicate that Gh is a G-protein that functionally couples to the alpha 1-adrenergic receptor.     

Binding of transforming growth factor-beta 1 to immobilized human alpha 2-macroglobulin.

Native alpha 2-macroglobulin (alpha 2M) and alpha 2M-methylamine were immobilized in 96-well microtiter plates. 125I-labeled transforming growth factor-beta 1 (TGF-beta 1) bound to both alpha 2M variants; however, greater binding was observed with alpha 2M-methylamine. Binding of 125I-TGF-beta 1 (0.2 nM) to immobilized alpha 2M-methylamine was inhibited by nonradiolabeled TGF-beta 1 (up to 74% with 0.4 microM TGF-beta 1). Approximately 10% of the TGF-beta 1-alpha 2M-methylamine complex was covalent. Treatment of alpha 2M-methylamine with iodoacetamide prior to immobilization completely eliminated covalent TGF-beta 1 binding; the total amount of 125I-TGF-beta 1-alpha 2M-methylamine complex detected was unchanged. The binding of 125I-TGF-beta 1 to immobilized alpha 2M-methylamine was not significantly inhibited by increasing the ionic strength to 1.0 M. Binding and complex dissociation were also unaffected by changes in pH within the range 6.9-8.9. Acidic pH dramatically decreased binding and promoted complex dissociation; no binding of 125I-TGF-beta 1 to immobilized alpha 2M-methylamine was detected at pH 3.5. The interaction of TGF-beta 1 with immobilized alpha 2M-methylamine was not significantly changed by 1.0 mM EDTA or 1.0 mM CaCl2. ZnCl2 (1.0 mM) completely eliminated binding. This result was not due to TGF-beta 1 precipitation or aggregation. Inhibition of 125I-TGF-beta 1 binding to alpha 2M-methylamine was 50% complete (IC50) with 30 microM ZnCl2. Native alpha 2M, thrombospondin, and alpha 2M-methylamine (in solution) decreased binding of 125I-TGF-beta 1 to immobilized alpha 2M-methylamine. The IC50 values for these three proteins were 520, 160, and 79 nM, respectively. The TGF-beta 1-binding activity of native alpha 2M may have reflected, at least in part, trace-contamination with alpha 2M-proteinase complex.     

Identification and characterization of calmodulin-binding proteins in mammalian sperm flagella

A calcium and calmodulin-regulated cyclic nucleotide phosphodiesterase has been shown to be an integral component of both rat and bovine sperm flagella. The calcium-activated enzyme was inhibited by both trifluoperazine (ID50 = 10 microM) and [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid (EGTA), and the basal activity measured in the presence of EGTA was stimulated by limited proteolysis to that observed in the presence of calcium/calmodulin. 125I-Calmodulin binding to purified rat sperm flagella has been characterized and the flagellar-associated calmodulin-binding proteins identified by a combination of gel and nitrocellulose overlay procedures and by chemical cross-linking experiments using dimethyl suberimidate. 125I-Calmodulin bound to demembranated rat sperm flagella in a time- and concentration-dependent manner. At equilibrium, 30-40% of the bound 125I-calmodulin remains associated with the flagella after treatment with EGTA or trifluoperazine. The majority of the bound 125I-calmodulin, both the Ca2+-dependent and -independent, was displaced by excess calmodulin. A 67-kDa calmodulin-binding protein was identified by both the gel and nitrocellulose overlay procedures. In both cases, binding was dependent on Ca2+ and was totally inhibited by trifluoperazine, EGTA, and excess calmodulin. On nitrocellulose overlays, the concentration of calmodulin required to decrease binding of 125I-calmodulin by 50% was between 10(-10) and 10(-11) M. Limited proteolysis resulted in the total loss of all Ca2+-dependent binding to the 67-kDa polypeptide. Chemical cross-linking experiments identified a major calcium-dependent 125I-calmodulin:polypeptide complex in the 84-90-kDa molecular mass range and a minor complex of approximately 200 kDa. Immunoblot analysis showed that the major 67-kDa calmodulin-binding protein did not cross-react with polyclonal antibodies raised against either the calcium/calmodulin-regulated cyclic nucleotide phosphodiesterase or phosphoprotein phosphatase (calcineurin) from bovine brain.     

Characterization of EGTA-washed synaptosomal membrane with emphasis on its calmodulin-binding proteins. Demonstration of possible reconstitution with added calcium/calmodulin.

Endogenous calmodulin (CaM) in the EGTA-washed cerebral-cortical synaptosomal membrane (SM) preparation was estimated below 3 micrograms/ml protein by the semiquantitative immunoblot analysis (Natsukari, N., Ohta, H. and Fujita, M. (1989) J. Immunol. Methods 125, 159-166). Membrane-bound CaM was immunoelectron-microscopically demonstrated in EGTA-washed, non-treated (control), and Ca(2+)-treated cerebral-cortical synaptosomal membranes (SM) as well as for the SM enriched with added CaM. The density of CaM increased in the above order. CaM-dependent adenylate cyclase and CaM-dependent protein kinase II (CaM-kinase II) activities were restored, whereas the phosphodiesterase (PDE) activity was not affected by exogenous CaM over all the Ca2+ concentrations tested. Adenylate cyclase at pCa 6.2 was synergistically activated either by GTP and CaM or by CaM and beta-adrenergic agonist, (+/-)-isoproterenol, reflecting the intactness of signal transduction pathway in the SM. Also demonstrated were the presence of protein kinase A, CaM-kinase II, and their endogenous substrates in the SM. Based on 32P-autoradiography and 125I-CaM overlay data certain CaM-binding proteins such as CaM-kinase II and synapsin I were identified on SDS-PAGE. Ca(2+)-dependent and -independent CaMBPs were distinguished by 125I-CaM gel overlay with and without Ca2+. The former had bigger molecular size (greater than or equal to 49 kDa) than the latter (less than or equal to 34 kDa). Yield of Ca(2+)-dependent CaMBPs was not affected by Ca2+ concentration during preparation of the SM while that of Ca(2+)-independent CaMBPs was reduced by exposure to 100 microM Ca2+. In contrast with the CaMBPs of brain SM, those of enterocyte and eyrthrocyte plasma membranes especially, microvillous membrane of the enterocyte, showed quite distinct CaMBP profiles. The present findings suggested that the EGTA-washed SM preparation made a useful system for studying the role of CaM in the brain SM.     

Insulin and phorbol ester stimulate phosphorylation of a 40-kDa protein in adipocyte plasma membranes

Several substrates of endogenous Ca2+- and phospholipid-sensitive protein kinase have been identified in plasma membranes and cytosol from rat adipocytes. Specifically, Ca2+ stimulates phosphorylation of a 40-kDa protein in isolated plasma membranes, an effect which is further enhanced by the addition of the phorbol ester tetradecanoylphorbol acetate and phospholipase C. The 40-kDa phosphoprotein is also present in the cytosol, and its phosphorylation is stimulated in a Ca2+-dependent manner by phosphatidylserine, diacylglycerol, and phorbol ester. Direct addition of insulin to adipocyte plasma membranes stimulates phosphorylation of the 40-kDa protein in a concentration-dependent manner. Maximal stimulation was observed at 10(-8) M insulin. At 6.7 X 10(-8) M insulin, phosphorylation of the 40-kDa protein was stimulated by 68 +/- 9% (n = 6). Addition of phorbol ester (1, 10, and 100 ng/ml) plus insulin further enhanced the phosphorylation (286 +/- 39, n = 3; 350 +/- 65, n = 4; and 323 +/- 42%, n = 5, stimulation, respectively). Analysis of the 40-kDa phosphoprotein by two-dimensional polyacrylamide gel electrophoresis revealed that incubations containing no additions, insulin, and/or phorbol ester all resulted in the generation of a single and apparently identical phosphorylated 40-kDa species. These studies indicate that insulin and Ca2+- and phospholipid-dependent protein kinase stimulate phosphorylation of a 40-kDa protein in adipocyte plasma membranes.     

Insulin-like growth factor II binding to the type I somatomedin receptor. Evidence for two high affinity binding sites

We have previously shown that the antireceptor antibody alpha IR-3 inhibits binding of 125I-somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) to the 130-kDa alpha subunit of the type I receptor in human placental membranes, but does not block 125I-insulin-like growth factor II (IGF-II) binding to a similar 130-kDa complex in these membranes. To determine whether the 130-kDa 125I-IGF-II binding complex represents a homologous receptor or whether 125I-IGF-II binds to the type I receptor at a site that is not blocked by alpha IR-3, type I receptors were purified by affinity chromatography on Sepharose linked alpha IR-3. The purified receptors bound both 125I-Sm-C/IGF-I and 125I-IGF-II avidly (KD = 2.0 X 10(-10) M and 3.0 X 10(-10) M, respectively). The maximal inhibition of 125I-Sm-C/IGF-I binding by the antibody, however, was 62% while only 15% of 125I-IGF-II binding was inhibited by alpha IR-3. In the presence of 500 nM alpha IR-3, Sm-C/IGF-I bound with lower affinity (KD = 6.5 X 10(-10) M) than IGF-II (KD = 4.5 X 10(-10) M) and IGF-II was the more potent inhibitor of 125I-Sm-C/IGF-I binding. These findings suggest that the type I receptor contains two different binding sites. The site designated IA has highest affinity for Sm-C/IGF-I and is blocked by alpha IR-3. Site IB has higher affinity for IGF-II than for Sm-C/IGF-I and is not blocked by alpha IR-3.     

alpha 2-Macroglobulin binding to cultured fibroblasts. Solubilization and partial purification of binding sites

Binding sites having the characteristics of receptors for "activated" alpha 2-macroglobulin (alpha 2M) have been solubilized with octyl-beta-D-glucoside from fibroblast membranes. When the detergent was removed by dialysis, the resulting insoluble extract was shown to bind 125I-alpha 2M specifically. Analysis of the binding data using a nonlinear curve-fitting program suggests that the solubilized preparation contains two classes of binding sites (KD = 0.34 nM and KD = 104 nM). Membranes or solubilized extracts from KB cells which lack alpha 2M binding sites did not specifically bind 125I-alpha 2M. The solubilized binding sites from fibroblasts were inactivated by boiling and trypsin treatment, and required Ca+2 for maximal binding. In addition, the high affinity binding of 125I-alpha 2M to the solubilized receptor was inhibited by bacitracin and by alpha-bromo-5-iodo-4-hydroxy-3-nitroacetophenone, two agents which interfere with the uptake of alpha 2M in cultured fibroblasts. Using a combination of ion exchange and gel permeation chromatography, we have purified the high affinity alpha 2M binding site approximately 100-fold from membrane derived from NIH-3T3 (spontaneously transformed) fibroblasts grown as tumors in mice. The receptor is apparently an acidic protein and the receptor octyl-beta-D-glucoside complex has a Stokes radius of 45-50 A as measured by gel filtration.     

Calcium/Calmodulin-Dependent Protein Kinase II in Squid Synaptosomes

The Ca2+/calmodulin (CaM)-dependent protein kinase II system in squid nervous tissue was investigated. The Ca2+/CaM-dependent protein kinase II was found to be very active in the synaptosome preparation from optic lobe, where it was associated with the high-speed particulate fraction. Incubation of the synaptosomal homogenate with calcium, calmodulin, magnesium, and ATP resulted in partial and reversible conversion of the Ca2+/CaM-dependent protein kinase II from its calcium-dependent form to a calcium-independent species. The magnitude of this conversion reaction could be increased by inclusion of the protein phosphatase inhibitor NaF or by substitution of adenosine 5'-O-(3-thiotriphosphate) for ATP. When [gamma-32P]ATP was used, proteins of 54 and 58 kilodaltons (kDa) as well as proteins greater than 100 kDa were rapidly 32P-labeled in a calcium-dependent manner. Major 125I-CaM binding proteins in the synaptosome membrane fraction were 38 and 54 kDa. The Ca2+/CaM-dependent protein kinase II was purified from the squid synaptosome and was shown to consist of 54- and 58-60-kDa subunits. The purified kinase, like Ca2+/CaM-dependent protein kinase II from rat brain, catalyzed autophosphorylation associated with formation of the calcium-independent form. These studies, characterizing the Ca2+/CaM-dependent protein kinase II in squid neural tissue, are supportive of the putative role of this kinase in regulating calcium-dependent synaptic functions.     

Identification of the hyaluronan receptor for endocytosis (HARE)

Rat liver sinusoidal endothelial cells (LECs) express two hyaluronan (HA) receptors, of 175 and 300 kDa, responsible for the endocytic clearance of HA. We have characterized eight monoclonal antibodies (mAbs) raised against the 175-kDa HA receptor partially purified from rat LECs. These mAbs also cross-react with the 300-kDa HA receptor. The 175-kDa HA receptor is a single protein, whereas the 300-kDa species contains three subunits, alpha, beta, and gamma at 260, 230, and 97 kDa, respectively (Zhou, B., Oka, J. A., and Weigel, P. H. (1999) J. Biol. Chem. 274, 33831-33834). The 97-kDa subunit was not recognized by any of the mAbs in Western blots. Based on their cross-reactivity with these mAbs, the 175-, 230-, and 260-kDa proteins appear to be related. Two of the mAbs inhibit (125)I-HA binding and endocytosis by LECs at 37 degrees C. All of these results confirm that the mAbs recognize the bone fide LEC HA receptor. Indirect immunofluoresence shows high protein expression in liver sinusoids, the venous sinuses of the red pulp in spleen, and the medullary sinuses of lymph nodes. Because the tissue distribution for this endocytic HA receptor is not unique to liver, we propose the name HARE (HA receptor for endocytosis).     

Affinity labelling of endothelin receptor and characterization of solubilized endothelin-endothelin-receptor complex

Chick cardiac membranes were affinity labelled by cross-linking to membrane-bound 125I-endothelin-1 with disuccinimidyl tartarate. SDS/PAGE and autoradiographic analysis of the 125I-endothelin-1-labelled material in the presence or absence of 2-mercaptoethanol revealed one major labelled band, corresponding to a molecular mass of 53 kDa, whose appearance was dose-dependently inhibited by the addition of unlabelled endothelin-1 (1-100 nM). Subtracting the molecular mass of 125I-endothelin-1 and disuccinimidyl tartarate, the binding protein appeared to have a molecular mass of 50 kDa. To investigate further the molecular properties of endothelin receptor, the 125I-endothelin-1-endothelin-receptor complex was solubilized from chick cardiac membranes using the detergent digitonin. Sucrose gradient sedimentation of the solubilized complex indicated a sedimentation coefficient of 13 S, whereas the complex of (+)-[3H]PN200-110, a dihydropyridine derivative, and dihydropyridine-sensitive Ca2+ channels sedimented at 22 S. A monoclonal antibody raised against dihydropyridine-sensitive Ca2+ channels from the chick brain did not immunoprecipitate the 125I-endothelin-1-endothelin-receptor complex. These data suggest that endothelin receptor is clearly distinct from dihydropyridine-sensitive Ca2+ channels and endothelin has its own specific 50-kDa receptor.     

Purification and characterization of the dihydropyridine-sensitive voltage-dependent calcium channel from cardiac tissue

The dihydropyridine-sensitive voltage-dependent Ca2+ channel from cardiac tissue was purified 900-fold using DEAE-Sephadex A-25, concanavalin A-Sepharose, and wheat germ agglutinin-Sepharose. The purified preparation was highly enriched in a peptide of 140,000 daltons when electrophoresed on sodium dodecyl sulfate gels in the presence of 2-mercaptoethanol, or 170,000 when electrophoresed in the presence of iodoacetamide. Polyclonal antibodies raised against the purified subunits of the rabbit skeletal muscle Ca2+ channel recognized the 170-kDa protein in preparations electrophoresed under nonreducing conditions, and the large peptide of 140 kDa and smaller peptides of 29-32 kDa in preparations analyzed under reducing conditions. Monoclonal antibodies, which were raised against the native Ca2+ channel from skeletal muscle, immunoprecipitated [3H]PN 200-110 binding activity from solubilized cardiac membranes and immunoprecipitated 125I-labeled peptides (from the purified cardiac Ca2+ channel preparation) which migrated as a single species of 170 kDa under nonreducing conditions, or as 140, 32, and 29 kDa under reducing conditions. The results show that the purified cardiac Ca2+ channel, like that previously purified from skeletal muscle, consists of a major component of 170 kDa which is comprised of a 140-kDa peptide linked by disulfide bonds to smaller peptides of 32-29 kDa. Peptide maps of the 140-kDa peptide purified from cardiac and skeletal muscle preparations were strikingly similar, suggesting a high degree of homology in their primary sequence.     

Homo- and heterotetrameric forms of the membrane-bound metalloendopeptidases meprin A and B

Meprin A and B are disulfide-linked, tetrameric metalloendopeptidases in renal brush border membranes. Meprin A contains 90-kDa subunits (alpha subunits) and is expressed in random-bred and some inbred strains of mice. Meprin B contains subunits of 110 kDa (beta subunits) in situ, and the enzyme from C3H mice, a strain that does not express alpha subunits, has been characterized. Evidence from this and previous studies indicate that beta subunits are expressed in all mouse strains. The tetrameric organization of these meprins was examined in brush border membrane fractions from a random-bred strain (ICR) and two inbred strains of mice (C57BL/6 and C3H/He). Lectin blotting using biotinylated concanavalin A revealed that membranes from the random-bred strain contained three oligomeric complexes of approximately 390, 440, and 490 kDa as determined after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of reducing agents. The subunits in all three oligomers were linked by disulfide bridges. Western blotting using an anti-alpha monoclonal antibody indicated that alpha subunits (90 kDa) were present in the 390- and 440-kDa complexes. Western blotting with a polyclonal antibody specific for beta subunits (110 kDa) indicated the presence of these subunits in the 440- and 490-kDa complexes. Electroelution of the individual oligomers followed by SDS-PAGE under reducing conditions confirmed that the 390- and 490-kDa molecules are homotetramers of alpha and beta subunits, respectively, and that the 440-kDa molecule is a heterotetramer consisting of disulfide-bridged alpha and beta subunits. C57BL/6 mice expressed both alpha and beta subunits and contained tetramers composed of alpha 4 and alpha 2 beta 2. C3H/He mice expressed only the 110-kDa beta subunits and the beta 4 oligomer. This type of multimeric organization of disulfide-linked subunits is unique for the known endopeptidases.     

Identification and characterization of glucose transport proteins in plasma membrane- and Golgi vesicle-enriched fractions prepared from lactating rat mammary gland.

Previous studies have indicated that turkey erythrocyte and rat liver membranes contain endogenous alpha beta heterodimeric insulin receptors in addition to the disulphide-linked alpha 2 beta 2 heterotetrameric complexes characteristic of most cell types. We utilized 125I-insulin affinity cross-linking to examine the structural properties of insulin receptors from rat liver and turkey erythrocyte membranes prepared in the absence and presence of sulphydryl alkylating agents. Rat liver membranes prepared in the absence of sulphydryl alkylating agents displayed specific labelling of Mr 400,000 and 200,000 bands, corresponding to the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric insulin receptor complexes respectively. In contrast, affinity cross-linking of membranes prepared with iodoacetamide (IAN) or N-ethylmaleimide identified predominantly the alpha 2 beta 2 heterotetrameric insulin receptor complex. Similarly, affinity cross-linking and solubilization of intact turkey erythrocytes in the presence of IAN resulted in exclusive labelling of the alpha 2 beta 2 heterotetrameric insulin receptor complex, whereas in the absence of IAN both alpha 2 beta 2 and alpha beta species were observed. Turkey erythrocyte alpha 2 beta 2 heterotetrameric insulin receptors from IAN-protected membranes displayed a 3-4-fold stimulation of beta subunit autophosphorylation and substrate phosphorylation by insulin, equivalent to that observed in intact human placenta insulin receptors. Turkey erythrocyte alpha beta heterodimeric insulin receptors, prepared by defined pH/dithiothreitol treatment of IAN-protected membranes, were also fully competent in insulin-stimulated protein kinase activity compared with alpha beta heterodimeric human placenta receptors. In contrast, endogenous turkey erythrocyte alpha beta heterodimeric insulin receptors displayed basal protein kinase activity which was insulin-insensitive. These data indicate that native turkey erythrocyte and rat liver insulin receptors are structurally and functionally similar to alpha 2 beta 2 heterotetrameric human placenta insulin receptors. The alpha beta heterodimeric insulin receptors previously identified in these tissues most likely resulted from disulphide bond reduction and denaturation of the alpha 2 beta 2 holoreceptor complexes during membrane preparation.