The porcine LH/hCG receptor. Characterization and purification

Porcine luteal LH/hCG receptor (LH/hCG R) was solubilized with 70-80% recovery from the crude plasma membrane fraction by Triton X-100 in the presence of 25% glycerol and protease inhibitors. The solubilized receptor maintained 90% of original activity at -60 degrees C for 90 days. Equilibrium association constant (Ka) values of 1.92, 2.22, and 2.03 X 10(10) M-1 were observed for the whole homogenate, plasma membrane fraction, and solubilized LH/hCG R preparations, respectively. The specific binding capacity for the same fractions were 49, 70, 55 fmol/mg protein, respectively. Complexes of LH/hCG R and Triton X-100 were resolved into two components with approximate Mr = 2.7 X 10(5) and 5.4 X 10(5) by gel filtration on Sepharose 6B and two glycoprotein components by chromatography on concanavalin A-Sepharose. Solubilized porcine LH/hCG R was purified by two cycles of affinity chromatography on highly purified hCG-Sepharose with an overall recovery of 30-35% of the initial activity in the Triton extract. Purified porcine LH/hCG R had a specific binding capacity of 2300 pmol/mg protein and a Ka = 1.5 X 10(10) M-1. Silver staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels demonstrated that the major protein in porcine LH/hCG R preparations has Mr = 68,000. A weakly staining band at Mr = 45,000 was also observed in the purified receptor preparation. Analysis of iodinated purified LH/hCG R by autoradiography has confirmed these results. Porcine LH/hCG R was purified 40,000-fold by this method.     

Solubilization and partial purification of human placental cytochromes P-450

Human placental mitochondrial and microsomal cytochrome P-450 (P-450) was solubilized with sodium cholate in the presence of Emulgen 911 and the solubilized preparations purified by phenyl-Sepharose and DEAE-cellulose column chromatography. The final preparations exhibited specific activities between 3.0 and 7.0 nmol P-450/mg protein implying a 30–115-fold purification over the starting material. Androstenedione exhibited type I spectral interaction with the microsomal P-450 and pregnenolone a reverse type I spectrum with mitochondrial P-450. Hydrophobic column chromatography proved to be a rapid and efficient initial purification step for placental P-450s.     

Heterogeneity of the vacuolar pyrophosphatase protein fromChenopodium rubrum

Summary Activities of the tonoplast ATPase (V-ATPase EC 3.6.1.3) and PPase (V-PPase EC 3.6.1.1) provide the proton gradient driving the accumulation of various metabolites, organic and inorganic ions in the plant vacuole. We used anion exchange chromatography, liquid-phase isoelectric focusing (IEF), and continuous-elution native polyacrylamide gel electrophoresis (preparative PAGE) to enrich the V-PPase from solubilized tonoplast proteins from suspension cultured cells ofChenopodium rubrum L.The fractions were identified by their enzymatic activity, sensitivity towards the specific PPase inhibitor aminomethylenediphosphonate, apparent molecular weight, and immunological reactivity with an antibody raised against mung bean V-PPase. All these different methods used for the separation of solubilized tonoplast proteins revealed the existence of two physically separable V-PPase proteins exhibiting substrate specific enzymatic activity and 66 kDa apparent molecular weight after sodium dodecyl sulfate(SDS)-PAGE. The isoelectric points of the active V-PPase forms were 5.05 and 5.48 (V-ATPase 6.1). On the basis of the observation of high recoveries of enzymatic activity after different preparations we suggest that the V-PPase proteins separated may represent physiologically occurring forms of the enzyme which cannot be distinguished by SDS-PAGE and Western blot.     

Heterogeneity of the vacuolar pyrophosphatase protein fromChenopodium rubrum

Activities of the tonoplast ATPase (V-ATPase EC 3.6.1.3) and PPase (V-PPase EC 3.6.1.1) provide the proton gradient driving the accumulation of various metabolites, organic and inorganic ions in the plant vacuole. We used anion exchange chromatography, liquid-phase isoelectric focusing (IEF), and continuous-elution native polyacrylamide gel electrophoresis (preparative PAGE) to enrich the V-PPase from solubilized tonoplast proteins from suspension cultured cells of Chenopodium rubrum L.The fractions were identified by their enzymatic activity, sensitivity towards the specific PPase inhibitor aminomethylenediphosphonate, apparent molecular weight, and immunological reactivity with an antibody raised against mung bean V-PPase. All these different methods used for the separation of solubilized tonoplast proteins revealed the existence of two physically separable V-PPase proteins exhibiting substrate specific enzymatic activity and 66 kDa apparent molecular weight after sodium dodecyl sulfate(SDS)-PAGE. The isoelectric points of the active V-PPase forms were 5.05 and 5.48 (V-ATPase 6.1). On the basis of the observation of high recoveries of enzymatic activity after different preparations we suggest that the V-PPase proteins separated may represent physiologically occurring forms of the enzyme which cannot be distinguished by SDS-PAGE and Western blot.     

Effect of modification of Naja naja oxiana neurotoxin II on the stoichiometry of its complexes with acetylcholine receptor

It was discovered that illumination of the complex formed by the solubilized acetylcholine receptor from Torpedo marmorata and Lys25-p-azidobenzoyl derivative of neurotoxin II results in the appearance on the receptor of up to 4 additional binding sites. Acetylcholine and neurotoxin II, but not the long-chain neurotoxins bind specifically to these sites. The additional binding sites could be also detected after illuminating the receptor complex with other photoactivable derivatives, provided the latter were displaced from one of the two main binding sites by hexa(trifluoroacetyl)neurotoxin II. A similar, but less pronounced effect, was observed on binding Lys25 (Ac) derivative of neurotoxin II. The formation of the additional binding sites was found to depend on the activity of the receptor preparations as well as on the mutual influence of the two main toxin-binding sites.     

Purification of guanylyl cyclase from rod outer segments.

The particulate form of guanylyl cyclase from bovine rod outer segments has been solubilized and purified to near homogeneity by a combination of liquid chromatography and native gel electrophoresis. The procedure enriches enzyme activity 6700-fold from rod outer segment extracts to a final specific activity of 17.5 mumol/min per mg (when assayed with Mn-GTP as substrate). Purified preparations of guanylyl cyclase contain a single glycoprotein with an apparent molecular mass of 60,000 Da and a native isoelectric point of 7.6. Although crude or partially purified enzyme activity is modulated by sub-micromolar concentrations of Ca2+, the fully purified enzyme is insensitive to this cation. However, the purified enzyme remains sensitive to nitrovasodilators, being stimulated over 10-fold by sodium nitroprusside. These data suggest that retinal rods contain a unique isoform of guanylyl cyclase.     

Isolation and properties of the alpha-latrotoxin receptor.

The receptor protein of alpha-latrotoxin (alpha LTx, a neurotoxin with 'pure' presynaptic action isolated from black widow spider venom), was solubilized by Triton X-100 from bovine brain membranes and purified by affinity chromatography on alpha LTx-Sepharose. The purified receptor preparation contained four major polypeptides of molecular masses 200 (alpha), 160 (alpha'), 79 (beta) and 43 (gamma) kd according to SDS electrophoresis with molecular ratio alpha 1 alpha' 1 beta 2 gamma 2. The alpha- and alpha'-subunits are glycoproteins binding to wheat germ lectin and can be separated under non-denaturing conditions by anion exchange chromatography. Purified to homogeneity, both of them, though differing in the carbohydrate composition, retain the alpha LTx-binding activity and give closely related peptide maps. Anti-alpha antibodies recognize the alpha'-subunit as well. These results suggest that alpha LTx receptor is present in purified preparations in two very close forms containing the alpha- or alpha'-subunit. Beta and gamma proteins do not specifically bind alpha LTx and their physiological role is unclear. They form a complex with solubilized alpha- and alpha'-subunits independently of alpha LTx presence. The receptor proteins were purified to homogeneity by high performance gel filtration in the presence of SDS, their amino acid composition was determined.     

Solubilization and characterization of the beta-bungarotoxin-binding protein of chick brain membranes

The previously characterized ( Rehm , H., and Betz, H. (1982) J. Biol. Chem. 257, 10015-10022) neuronal binding protein for the presynaptic neurotoxin beta-bungarotoxin (beta-BuTx) was solubilized from synaptic membrane fractions of chick brain using the nonionic detergent Triton X-100. 125I-beta-BuTx bound to the solubilized protein with a dissociation constant (KD) of 1.9 +/- 0.1 nM. This binding of 125I-beta-BuTx was Ca2+-dependent and pharmacologically specific. From different basic proteins tested, only unlabeled beta-BuTx and its antagonist dendrotoxin inhibited 125I-beta-BuTx binding. Potassium ions were required during solubilization and binding in order to detect 125I-beta-BuTx-binding activity. Sedimentation in sucrose/H2O and sucrose/D2O gradients and gel exclusion chromatography on Sepharose 6B indicated a s20,w of 12.8 +/- 0.6 S and a Stokes radius of 8.6 +/- 0.2 nm for the solubilized beta-BuTx-binding component. From these data, the protein molecular weight of the beta-BuTx binding site was calculated to be 431,000 +/- 45,000.     

Purification and characterization of a protein associated with peripheral-type benzodiazepine binding sites

The photoaffinity ligand [3H]PK 14105 was utilized to modify covalently peripheral-type benzodiazepine binding sites in rat adrenal mitochondrial preparations. The photolabeled membrane preparations were then solubilized in 1% digitonin and the detergent-soluble extracts subjected to fractionation by ion-exchange chromatography and reversed-phase high performance liquid chromatography. This scheme resulted in the purification of the putative binding site protein for PK 14105 which we have entitled PKBS. Purified preparations of PKBS exhibited a single band with a Mr of approximately 17,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver-staining or autoradiographic detection. Additional criteria examining the purity of PKBS preparations were provided by radioiodination with Bolton-Hunter reagent, amino acid analysis, gas-phase sequencing, and reversed-phase chromatography suggesting that this protein was purified to apparent homogeneity. These results demonstrate that a protein associated with peripheral-type benzodiazepine recognition sites has been isolated thus facilitating more direct studies on the structure of this receptor and on the role of these binding sites in mediating responses elicited by benzodiazepines acting at these sites.     

Solubilization of Calcium Channel Antagonist Binding Sites from Rat Brain

Calcium antagonist binding sites were solubilized from rat brain membranes using the detergent 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulfonate (CHAPS). CHAPS-solubilized [3H]nitrendipine binding sites are saturable over a range of 0.05-4 nM and Scatchard analysis reveals a single, high-affinity (KD = 0.49 +/- 0.10 nM), low-capacity (Bmax = 56 +/- 4 fmol/mg of protein) binding site. Reversible ligand competition experiments using solubilized binding sites demonstrated appropriate pharmacologic specificity, with dihydropyridines (nifedipine = nitrendipine greater than Bay K 8644) completely displacing binding, verapamil partially displacing binding, and diltiazem enhancing binding, as previously described in membrane preparations. Lyophilized Crotalus atrox venom was purified by ion exchange chromatography followed by gel filtration to a single peptide band on sodium dodecyl sulfatepolyacrylamide gel electrophoresis. This fraction of molecular weight 60,000 competitively inhibits [3H]nitrendipine binding to both membrane and soluble preparations with an IC50 of 5 micrograms/ml. This polypeptide should serve as a useful ligand for future efforts in purifying the dihydropyridine calcium channel binding site in brain.     

Strategies for the purification of P-glycoprotein from multidrug-resistant Chinese hamster ovary cells.

P-glycoprotein, a hydrophobic 170-kDa integral protein overexpressed in the plasma membrane of multidrug-resistant cells, is proposed to function as an ATP-dependent drug efflux pump. Plasma membrane preparations highly enriched in P-glycoprotein were isolated from multidrug-resistant cells by discontinuous sucrose gradient and Ca2+ precipitation methods. Several strategies were used for P-glycoprotein purification, with the goal being to achieve both good yields and purity, while keeping experimental manipulation to a minimum. P-glycoprotein was solubilized from the plasma membrane using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Immunoaffinity chromatography using C219 monoclonal antibody produced low yields of moderately pure protein. Sequential lectin affinity chromatography on RCA-120 followed by lentil lectin resulted in a P-glycoprotein preparation that showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A fraction of P-glycoprotein did not bind to RCA-120, most likely as a result of heterogeneous glycosylation. A combination of chromatography on RCA-120 followed by immunoaffinity chromatography on C219 resulted in low yields of very pure P-glycoprotein.     

Isolation of human platelet glycoproteins.

Human platelet glycoproteins were isolated from whole platelets by two methods. The first method, that of affinity chromatography on wheat germ agglutinin, is based on the known affinity of lectins for cell surface glycoproteins. When solubilized whole platelets are used as starting material for this procedure, elution with N-acetylglucosamine yields primarily a glycoprotein of Mr approximately 150 000 as estimated by sodium dodecyl sulfate-acrylamide gel electrophoresis. The second method is based on the ability of the chaotropic salt lithium diiodosalicylate to extract glycoprotein from particulate cell fractions in water-soluble form. This method yields three major glycopeptides with apparent molecular weights after sulfhydryl reduction of 145 000, 125 000, and 95 000 as estimated on 5.6% sodium dodecyl sulfate-acrylamide gels. Carboxymethylation of these preparations in the presence of sulfhydryl-reducing agent further resolves a glycoprotein of Mr approximately 165 000. Treatment of whole platelets by periodate oxidation and sodium[3H]-borohydride reduction labels the three major glycoproteins extracted by lithium diiodosalicylate and the glycoprotein of Mr approximately 150 000 isolated on wheat germ agglutinin confirming their surface orientation. However, glycoprotein with Mr approximately 165 000 resolved by carboxymethylation of the lithium diiodosalicylate extracted glycoprotein mixture was not labelled by this method, suggesting that it represents the granule protein with similar electrophoretic characteristics described by others. Phosphorylation of intact platelets with 32Pi also results in labelling of glycoproteins isolated by both methods, suggesting that these molecules traverse the bilipid layer of the platelet membrane, bearing reactive groups on both outer and cytoplasmic surfaces.     

Relative localization of the bound acetylcholine receptor subunits and neurotoxin

A series of neurotoxin II (Naja naja oxiana) derivatives, each containing one p-azido-[14C]benzoyl group, have been prepared. Those labeled at Leu1, Lys15, Lys25, Lys26, or Lys46 associate specifically with the acetylcholine receptor from the Torpedo marmorata electric organs and form the crosslinks with it as a result of irradiation. Electrophoresis in polyacrylamide gel and gel chromatography revealed the contacts between the neurotoxins and alpha, beta, gamma and delta subunits of the receptor, modification of a particular subunit being governed by the photoactivable group position in the neurotoxin molecule. The differences of the two neurotoxin binding sites in the receptor were demonstrated by analysis of the photoinduced crosslinks under the conditions of one site being blocked by hexa (trifluoroacetyl) neurotoxin II. The mutual arrangement of the two bound neurotoxin molecules was established. On the basis of data obtained, two models for the acetylcholine receptor subunit topography were proposed.     

Solubilization and Partial Purification of α-Amino-3- Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding Sites from Rat Brain

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) binding sites were solubilized from rat brain membranes using 1% Triton X-100 in 0.5 M potassium phosphate buffer containing 20% glycerol. The solubilized binding sites were stable, permitting biochemical and pharmacological characterization as well as partial purification. Pharmacological and binding analyses indicated that the solubilized binding sites were similar to the membrane-bound sites. Both the solubilized and the membrane-bound preparations contained high- and low-affinity AMPA binding sites in the presence of potassium thiocyanate. A similar rank order for inhibition of [3H]AMPA binding by several excitatory amino acid analogs was obtained for the soluble and membrane-bound preparations. [3H]AMPA binding to both soluble and membrane-bound preparations was increased in the presence of potassium thiocyanate. The solubilized AMPA binding sites migrated as a single peak with gel filtration chromatography, with an Mr of 425,000. Beginning with the solubilized preparation, AMPA binding sites were purified 54-fold with ion-exchange chromatography and gel filtration. The characterization and purification of these soluble binding sites is potentially useful for the molecular characterization of this putative excitatory amino acid receptor subtype.     

High-Yield Purification of Glucokinase from Rat Liver

A rapid and reliable method for the purification of rat liver glucokinase was developed. The procedure consists of DEAE-cellulose ion-exchange chromatography, Phenyl-Sepharose hydrophobic interaction chromatography, DEAE-Affi Gel Blue dye-ligand chromatography, and duplicate steps of glucosamine-Sepharose affinity chromatography. Glucokinase was purified to a specific activity of 290 units/mg protein in a yield of 55% in 6 days. The final enzyme preparations were completely homogeneous in most experiments as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The estimated molecular weight (51.000) and sigmoidal saturation function for glucose of purified glucokinase were in good agreement with published data.     

High-yield purification of glucokinase from rat liver

A rapid and reliable method for the purification of rat liver glucokinase was developed. The procedure consists of DEAE-cellulose ion-exchange chromatography, Phenyl-Sepharose hydrophobic interaction chromatography, DEAE-Affi Gel Blue dye-ligand chromatography, and duplicate steps of glucosamine-Sepharose affinity chromatography. Glucokinase was purified to a specific activity of 290 units/mg protein in a yield of 55% in 6 days. The final enzyme preparations were completely homogeneous in most experiments as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The estimated molecular weight (51,000) and sigmoidal saturation function for glucose of purified glucokinase were in good agreement with published data.     

Effects of detergent on the binding of solubilized sodium channels to immobilized wheat germ agglutinin: structural implications

The binding of the solubilized voltage-dependent sodium channel from rat brain to immobilized wheat germ agglutinin (WGA) is detergent-dependent. When solubilized in sodium cholate, only 11% of total recovered channels bound to a WGA-Sepharose column. When solubilized in Triton X-100 or CHAPS, however, 80% and 60% could bind, respectively. The effect of cholate on sodium channel binding is relatively specific: the major rat brain glycoproteins which bind to immobilized WGA are roughly the same in either Triton or cholate, as analyzed by SDS gel electrophoresis. The structural implications for the channel are discussed.     

Isolation and partial purification of a Na+-dependent phlorizin receptor from dog kidney proximal tubule

This paper describes a new method for solubilization and partial purification of a Na+-dependent phlorizin receptor from dog kidney proximal convoluted tubule. Selective solubilization is carried out with 0.1% Na+-deoxycholate followed by complete solubilization with 0.5% deoxycholate. The 100,000 X g supernatant of the deoxycholate extract is then subjected to a combination of chromatofocusing and gel exclusion chromatography. Purification is monitored by a new column assay which permits detection of the Na+-dependent high affinity phlorizin receptor in solubilized preparations. Na+-dependent phlorizin binding exhibits the same characteristics on the column assay as in intact brush border vesicles. Binding is temperature-dependent, inhibited by proteolytic agents, Na+-dependent, and inhibited by excess cold phlorizin and D-glucose but not L-glucose. Quantitation of specific binding at different stages of the isolation procedure indicates a final purification of approximately 80-140-fold compared to intact brush border membrane fragments. Enrichment of specific phlorizin binding is paralleled by enrichment of a 61-66-kDa polypeptide on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. It is postulated that this polypeptide contains both the Na and the sugar specific binding site and represents a subunit of the intact Na+-dependent glucose transporter from dog kidney proximal tubule brush border membrane.     

Isolation of the bombesin/gastrin-releasing peptide receptor from human small cell lung carcinoma NCI-H345 cells.

Purification of the gastrin-releasing peptide (GRP) or bombesin receptor has proved elusive in part due to technical difficulties. In the present studies, the problem of oxidized radioligand was avoided by the use of 125I-GRP, which was verified to be not oxidized by high performance liquid chromatography. Specific 125I-GRP binding (at 0 degrees C) to intact human small cell lung carcinoma NCI-H345 cells which had been subjected to a dilute acid wash was 6 fmol/10(6) cells. Inhibition of GRP degradation by human H345 cell membranes through the use of phenanthroline or phosphoramidon permitted the development of binding assays for the GRP receptor in detergent-solubilized crude membrane preparations. The solubilized GRP receptor exhibited saturable, high affinity (KD = 1.3 nM), temperature-dependent specific binding averaging 402 +/- 65 fmol/mg protein (mean +/- S.E. for eight separate membrane preparations with 125I-GRP concentration = 3 nM), with a Bmax = 434 fmol/mg protein using a gel filtration binding assay. That the GRP receptor had been solubilized was demonstrated by its failure to pellet when centrifuged at 100,000 x g for 60 min, its passage through a 0.22-micron filter without loss of binding activity, and its elution in the void volume of a Sephadex G-50 gel filtration column, but within the inclusion volume of a Sephacryl S-200 column (Ve/V0 = 1.1). Isolation of the GRP receptor from human H345 cell-solubilized membranes was achieved by ligand affinity chromatography. A unique 70-kDa band on silver-stained reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis was reproducibly eluted from GRP14-27 affinity columns by an acidic high salt buffer, but binding activity was denatured by these conditions. The protein nature of the GRP receptor was demonstrated by its sensitivity to proteases after isolation. In addition, two unique bands of 65 and 70 kDa were eluted from the GRP14-27 affinity column with GRP14-27 in neutral buffer, and this eluate possessed specific 125I-GRP binding with a stoichiometry of approximately 1:1. Thus, reported here is the isolation of a functional membrane-associated, saturable, high affinity GRP receptor with temperature-dependent binding from the solubilized membranes of human H345 cells.     

Solubilization and partial purification of somatostatin-28 preferring receptors from hamster pancreatic beta cells.

Somatostatin-28 (SRIF-28) preferring receptors were solubilized from hamster beta cell insulinoma using the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate. The binding of the iodinated [Leu8-D-TRP22-Tyr25]SRIF-28 analog (referred to as 125I[LWY] SRIF-28) to the solubilized fraction was time-dependent, saturable, and reversible. Scatchard analysis of equilibrium binding data indicated that the solubilized extract contained two classes of SRIF-28-binding sites: a high affinity site (Kd = 0.3 nM and Bmax = 1 pmol/mg protein) and a low affinity site (Kd = 13 nM and Bmax = 4.7 pmol/mg protein). The binding of 125I[LWY]SRIF-28 to solubilized SRIF-28 receptors was sensitive to the GTP analog guanosine-5'-O-thiotriphosphate, suggesting that receptors are functionally linked to a G-protein. By anion-exchange chromatography of the solubilized extract followed by chromatography on wheat germ agglutinin, a 46-fold purification of SRIF-28 receptors was obtained. At this stage of purification, only high affinity sites were found (Kd = 1 nM) and the GTP effect was not maintained. A specific protein of 37 kDa was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after photoaffinity labeling. We suggest that this protein is the putative SRIF-28 receptor or a subunit thereof.