Phorbol ester binding and activation of protein kinase C on triton X-100 mixed micelles containing phosphatidylserine

A mixed micellar assay for the binding of phorbol-esters to protein kinase C was developed to investigate the specificity and stoichiometry of phospholipid cofactor dependence and oligomeric state of protein kinase C (Ca2+/phospholipid-dependent enzyme) required for phorbol ester binding. [3H]Phorbol dibutyrate was bound to protein kinase C in the presence of Triton X-100 mixed micelles containing 20 mol % phosphatidylserine (PS) in a calcium-dependent manner with a Kd of 5 X 10(-9) M. The [3H]phorbol dibutyrate X protein kinase C . Triton X-100 . PS mixed micellar complex eluted on a Sephacryl S-200 molecular sieve at an Mr of approximately 200,000; this demonstrates that monomeric protein kinase C binds phorbol dibutyrate. This conclusion was supported by molecular sieve chromatography of a similar complex where Triton X-100 was replaced with beta-octylglucoside. Phorbol dibutyrate activation of protein kinase C in Triton X-100/PS mixed micelles occurred and was dependent on calcium. The PS dependence of both phorbol ester activation and binding to protein kinase C lagged initially and then was highly cooperative. The minimal mole per cent PS required was strongly dependent on the concentration of phorbol dibutyrate or phorbol myristic acetate employed. Even at the highest concentration of phorbol ester tested, a minimum of 3 mol % PS was required; this indicates that approximately four molecules of PS are required. [3H]Phorbol dibutyrate binding was independent of micelle number at 20 mol % PS. The phospholipid dependencies of phorbol ester binding and activation were similar, with PS being the most effective; anionic phospholipids (cardiolipin, phosphatidic acid, and phosphatidylglycerol were less effective, whereas phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin did not support binding or activation. sn-1,2-Dioleoylglycerol displaced [3H]phorbol dibutyrate quantitatively and competitively. The data are discussed in relation to a molecular model of protein kinase C activation.     

The effect of triton X-100 on bovine brain synaptic membranes

Bovine brain synaptic membranes which were frozen and then extensively washed showed low affinity [³H]muscimol binding. These membranes contained GABA and calmodulin, apparently tightly bound within the membrane fraction. Membranes which were additionally treated with the detergent Triton X-100 showed high affinity [³H]muscimol binding. These membranes did not appear to contain GABA or calmodulin. Transmission electron microscopy studies demonstrated that the washed membrane fraction contained many synaptosomal and vesicular structures. Triton treatment led to the extensive rupture of these structures. These studies explain the well-reported findings of tightly bound GABA and calmodulin in brain membrane fractions, as being due to the entrapment of these compounds inside sealed membrane-bound structures which are still present after a freezethaw and extensive wash treatment, their complete removal requiring Triton-treatment to rupture the vesicles.     

Interactions of pig brain cytosolic sialidase with gangliosides. The formation of catalytically inactive enzyme-ganglioside complexes requires homogeneous ganglioside micelles and is a reversible phenomenon

Cytosolic sialidase A, obtained from pig brain and purified, interacts with ganglioside GT1b giving two catalytically inactive enzyme-ganglioside complexes. Treatment of these complexes with Triton X-100 under given conditions (1% detergent; 1 h at 37 degrees C; 0.1 M acetic acid-sodium acetate buffer, pH 4.8) leads to the liberation of part of the enzyme (about 47%) in a free and fully active form. Reversible inactivation of cytosolic sialidase requires the presence of homogeneous micelles of GT1b or of mixed micelles (for instance Triton X-100 and GT1b) with a high GT1b content. Triton X-100/ganglioside mixed micelles with a molar ratio above 50, as well as small unilamellar vesicles of egg yolk lecithin and GT1b (7-15 mol%), did not inactivate the enzyme at all; on the contrary these forms of ganglioside dispersion behaved as excellent substrates for the enzyme. It is to be concluded that under in vitro conditions the ability of ganglioside to interact with cytosolic sialidase, giving rise to catalytically inactive complexes or to Michaelis-Menten enzyme-substrate complexes, depends on the supramolecular organization of the ganglioside molecules. Arrangements of tightly packed molecules with strong side-side interactions facilitate the formation of complexes with the enzyme; arrangement with separated and loosely interacting molecules facilitates binding at the catalytically active site of the enzyme.     

Metals and activity of bovine heart cytochrome c oxidase are independent of polypeptide subunits III, VII, a, and b

Bovine heart cytochrome c oxidase, depleted of polypeptide subunits by alkaline detergent treatment, was characterized with respect to metal content, optical spectral properties, and oxidase activity. Treatment with 1.0% Triton X-100 at pH 9.5 followed by anion-exchange chromatography caused removal of subunit III, subunit VII, and polypeptides a and b. The metal atom stoichiometries of the control and the polypeptide-depleted enzyme were in both cases 2.5Cu/2Fe/1Zn/1Mg with metal-to-protein ratios significantly greater in the latter. The treated enzyme exhibited a red shifted oxidized Soret maximum and bound carbon monoxide upon reduction. Activity was markedly decreased by the treatment but was restored to control levels by incubation with 0.3% Tween 80 at pH 6.0. Therefore, subunit III, subunit VII, polypeptide a, and polypeptide b do not contain Cu, Fe, Zn, or Mg and are not essential for reduction of O2 by ferrocytochrome c.     

Convulsant/barbiturate activity on the soluble gamma-aminobutyric acid-benzodiazepine receptor complex

Cage convulsant t-butyl bicyclophosphoro[35S]thionate binding activity in rat brain membrane homogenates was solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]propane sulfonate (Chaps) and shown to co-purify with the benzodiazepine--gamma-aminobutyric acid (GABA) receptor complex on gel filtration and affinity chromatography. Whereas convulsant binding activity, but not GABA and benzodiazepine receptor binding, was eliminated by solubilization in other detergents like sodium deoxycholate or Triton X-100, or by addition of Triton X-100 to the extracts solubilized in the zwitterionic detergent, convulsant activity was not irreversibly lost or selectively unstable, but could be restored by exchanging the protein back into the detergent Chaps. The GABA-benzodiazepine receptor activity solubilized in Chaps alone, containing convulsant activity, and a sample in Chaps supplemented with Triton X-100 and lacking convulsant activity, did not differ in size as measured by gel filtration column chromatography or by radiation inactivation target size analysis. This suggests that convulsant binding activity does not require any additional protein subunits or other macromolecules nor any unique aggregation state relative to GABA and benzodiazepine receptor binding, and that all three activities reside on the same protein complex. As in intact brain, the target size for convulsant binding activity was 3-5 times that of benzodiazepine binding activity, suggesting that an oligomeric protein structure of the receptor complex with intact strong subunit interactions present in the native membrane environment is needed for convulsant activity, and that this and other properties are more preserved in Chaps than in other detergents.     

Interaction of detergents with cytochrome c oxidase.

The binding of ionic and nonionic, nondenaturing detergents to cytochrome c oxidase has been examined. All bind and displace part but not all of the phospholipid that is associated with the enzyme after isolation. From 6 to 10 phospholipid molecules, depending on the detergent used, do not exchange and these are mostly diphosphatidylglycerol molecules as first shown by Awasthi et al. ((1971) Biochim. Biophys. Acta 226, 42). The binding of Triton X-100 and deoxycholate to the cytochrome c oxidase complex has been studied in detail. Both bind to the enzyme above their critical micelle concentrations: Triton X-100 in the amount of 180 +/- 10 molecules per complex and deoxycholate in the amount of 80 +/- 4 molecules per complex. In nonionic detergents, cytochrome c oxidase exists as a dimer (4 heme complex). The enzyme is dissociated into the monomer or heme aa3 complex by delipidation in bile salts. Activity measurements in different detergents suggest that cytochrome c oxidase requires a flexible, hydrophobic environment for maximal activity and that the dimer or 4 heme complex may be the active species.     

Detergent dissociation of bovine liver phosphomannosyl binding protein

We have reported previously the isolation and partial characterization of a 215-kilodalton (Kd) phosphomannosyl binding protein from bovine liver membranes [3,9]. In the present studies evidence is presented that the binding protein is an aggregate. Four N-terminal amino acids were detected, and the complex could be dissociated into subunits. Bovine liver membranes were extracted with the detergent, Zwittergent, in the presence of protease inhibitors. The extract was subjected to affinity chromatography on phosphomannan-Sepharose 4B, and proteins with apparent Mr values of 215 and 57 Kd were eluted with mannose 6-phosphate. As reported previously, extraction with Triton X-100 yielded only the higher molecular weight material. When the binding protein was incubated at 4 degrees C in the presence of Zwittergent TM 3-14 the 215-Kd form slowly dissociated into smaller subunits; after two months, the major species had an apparent Mr of 57 Kd. The subunits derived from the binding protein were recognized by antiserum raised against purified binding protein. Dissociation of the binding protein by Zwittergent was enhanced by incubation at 37 degrees C, the presence of dithiothreitol, and low pH values. The subunit mixture enriched in the 57-Kd subunit had a lowered ability to bind ligands containing the phosphomannosyl recognition marker. Binding was partially restored (greater than 48% of the initial value) when dissociated receptor was back exchanged with Triton X-100.     

Solubilization of pituitary receptors for thyrotropin-releasing hormone.

Receptors for thyrotropin-releasing hormone were solubilized by Triton X-100. Membrane fractions from GH3 pituitary tumor cells were incubated with thyrotropin-releasing hormone in order to saturate specific receptor sites before the addition of detergent. The amount of protein-bound hormone solubilized by Triton X-100 was proportional to the fractional saturation of specific membrane receptors. Increasing detergent:protein ratios from 0.5 to 20 led to a progressive loss of hormone . receptor complex from membrane fractions with a concomitant increase in soluble protein-bound hormone. The soluble hormone . receptor complex was not retained by 0.22 micron filters and remained soluble after ultracentrifugation. Following incubation with high (2.5--10%) concentrations of Triton X-100 and other non-ionic detergents, or following repeated detergent extraction, at least 18% of specifically bound thyrotropin-releasing hormone remained associated with particulate material. Unlike the hormone receptor complex, the free hormone receptor was inactivated by Triton X-100. A 50% loss of binding activity was obtained with 0.01% Triton X-100, corresponding to a detergent:protein ratio of 0.033. The hormone . receptor complex was included in Sepharose 6B and exhibited an apparent Stoke radius of 46 A in buffers containing Triton X-100. The complex aggregated in detergent-free buffers. Soluble hormone receptors were separated from excess detergent and thyrotropin-releasing hormone by chromatography on DEAE-cellulose. Thyrotropin-releasing hormone dissociated from soluble receptors with a half-time of 120 min at 0 degrees C, while the membrane hormone . receptor complex was stable for up to 5 at 0 degrees C.     

Activation of the Sendai virus fusion protein (f) involves a conformational change with exposure of a new hydrophobic region

The F protein of paramyxoviruses is actively involved in the induction of membrane fusion. This fusion may be between viral and cellular membranes, as in the initiation of infection or in virus-induced lysis of erythrocytes, or between the plasma membranes of different cells. The F protein is activated by proteolytic cleavage to yield two disulfide-linked polypeptides (F1 and F2); however, its mechanism of action is not clear. In the present study, the conformations of the inactive, uncleaved precursor of glycoprotein (F0), and the active, cleaved form (F1,2) have been compared. The UV circular dichroism spectra of the two forms of the F protein indicate that cleavage results in a conformational change. Detergent-binding studies by velocity sedimentation analysis of Triton X-100-protein complexes revealed an increase in exposed hydrophobic surface of the protein on cleavage. The inactive F0 bound an estimated 27 molecules of Triton X-100/F polypeptide; these molecules are presumably bound to the hydrophobic region of the glycoprotein that anchors the spike-like protein in the virus membrane and that is common to both forms of F. The active form, F1,2, bound 67 molecules of Triton X-100. This increase in the number of detergent binding sites upon F protein activation indicates the presence of a hydrophobic region that is peculiar to the active form, and that may be of functional significance in the membrane fusion reaction.     

Solubilization of pituitary receptors for thyrotropin-releasing hormone

Receptors for thyrotropin-releasing hormone were solubilized by Triton X-100. Membrane fractions from GH₃ pituitary tumor cells were incubated with thyrotropin-releasing hormone in order to saturate specific receptor sites before the addition of detergent. The amount of protein-bound hormone solubilized by Triton X-100 was proportional to the fractional saturation of specific membrane receptors. Increasing detergent: protein ratios from 0.5 to 20 led to a progressive loss of hormone · receptor complex from membrane fractions with a concomitant increase in soluble protein-bound hormone. The soluble hormone · receptor complex was not retained by 0.22 μm filters and remained soluble after ultracentrifugation. Following incubation with high (2.5–10%) concentration of Triton X-100 and other non-ionic detergents, or following repeated detergent extraction, at least 18% of specifically bound thyrotropin-releasing hormone remained associated with particulate material. Unlike the hormone receptor complex, the free hormone receptor was inactivated by Triton X-100. A 50% loss of binding activity was obtained with 0.01% Triton X-100, corresponding to a detergent: protein ratio of 0.033.The hormone · receptor complex was included in Sepharose 6B and exhibited an apparent Stokes radius of 46 Å in buffers containing Triton X-100. The complex aggregated in detergent-free buffers. Soluble hormone receptors were separated from excess detergent and thyrotropin-releasing hormone by chromatography on DEAE-cellulose. Thyrotropin-releasing hormone dissociated from soluble receptors with a half-time of 120 min at 0°c, while the membrane hormone · receptor complex was stable for up to 5 h at 0°C.     

Effects of triton X-100 treatments on the composition and activities of membrane vesicles from pigeon erythrocytes

Membrane vesicles were prepared from pigeon erythrocytes. The effect of various treatments on the ability of these vesicles to trap and transport glycine was measured. Most of the work concerned the effects of the nonionic detergent, Triton X-100 (Triton).Triton inhibits the capacity of membrane vesicles to trap and transport glycine. The effects of Triton depend on temperaature and pH. At neutral pH and 41°C, the half-inactivating dose of Triton is 6 μl/g wet weight of membrane, while at neutral pH and O°C it is approx. 60 μl/g. At pH 8.5–8.8 and 0°C the half-inactivating dose is 15–20 μl/g. The Triton effect depends on the ratio of Triton to membrane, not the Triton ‘concentration’ in the aqueous phase.Protein is released from the membrane by Triton treatment at 0°C. At pH 8.5–8.8, the dose-response curve for protein release is very similar to the dose-response curve for inhibition of the capacities to trap and take up glycine. All three curves have steep and shallow regions, respectively below and above a Triton dose of 15 μl/g. The protein released by Triton treatment at 0°C is largely the lower molecular weight classes and the maximum protein release is 50–60% of the total membrane protein. The percent of this protein class released by a given Triton dose equals the percent inhibition of the capacity to trap glycine.Triton is bound by the membranes. In the dose range of 0–10 μl Triton/g wet membrane, about half of the added Triton is bound. This bound Triton is not readily removed by washing. Like protein release and inhibition of trapping and transport capacities, the binding process has two phases, one above and one below a dose of 15 μl/g. The membrane is saturated with Triton at a dose of 15 μl/g with 4.5 μl Triton bound/g wet weight, corresponding to 85–90 μl bound/g remaining membrane protein.Bovine serum high density lipoprotein efficiently removes bound Triton from the membrane. By removing Triton bound at 0°C with lipoprotein, the effects of Triton at 0 and 41°C could be distinguished and the time course of Triton action at 0°C could be measured. Triton action was nearly complete by 10 min at 0°C.Possible modes of action of Triton on these membranes are discussed. At least part of the action of Triton can be described as a process in which membrane proteins are partitioned between the membrane phase and an extramembranal Triton micelle phase.     

Optically Active Bis-β-keto Sulfonium Ylid

Particulate alcohol dehydrogenase of acetic acid bacteria that is mainly participated in vinegar fermentation was purified to homogeneous state from Gluconobacter suboxydans IFO 12528. Solubilization of enzyme from the bacterial membrane fraction by Triton X-100 and subsequent fractionation on DEAE-Sephadex A-50 and hydroxylapatite was successful in enzyme purification. A cytochrome c-like component was tightly bound to the dehydrogenase protein and existed as an enzyme-cytochrome complex. It was also confirmed that the alcohol dehydrogenase is not a cytochrome component itself. The molecular weight of the enzyme was determined to be 150,000, and gel electrophoresis showed the presence of three subunits having a molecular weight of 85,000, 49,000 and 14,400. The smallest subunit was corresponded to the cytochrome c-like component. Ethanol was oxidized in the presence of dyes in vitro but NAD or NADP were not required as hydrogen acceptor. Unlike NAD- linked alcohol dehydrogenase in yeast or liver and other primary alcohol dehydrogenases in methanol utilizing bacteria, the enzyme from the acetic acid bacteria showed its optimum pH at fairly acidic pH.     

Pseudomonas toxin binds triton X-114 at low pH.

Pseudomonas toxin was found to bind Triton X-114 at pH values below pH 5.0, whereas much less binding was observed at higher pH values. The toxin bound Triton X-114 at a higher pH value in the presence of 0.14 M-NaCl, -KCl or -NaNO3 than at low salt concentrations. The results suggest that low pH in an intracellular compartment facilitates the transport of Pseudomonas toxin across the membrane and into the cytosol by inducing a conformational change in the molecule.     

Gel chromatographic characterization of the hydrophobic interaction of glycosylphosphatidylinositol-alkaline phosphatase with detergents

The interaction of a glycosylphosphatidylinositol (GPI) protein with different detergents was studied for the first time with a purified protein. Four differently hydrophobic fractions of GPI-alkaline phosphatase (GPI-AP) from calf intestine were used as model proteins. The mode of interaction was determined by investigating (i) the self-aggregation behaviour of the GPI-AP fractions, (ii) the interference of detergents with GPI-AP binding to octyl-Sepharose, and (iii) the elution of GPI-AP bound to octyl-Sepharose. It was shown that polyoxyethylene-type detergents surprisingly interact much stronger than n-octylglucoside with GPI-AP, which is in contrast to the known behaviour of GPI-proteins in natural membranes. Gel filtration chromatography of Triton X-100 at concentrations above the critical micellar concentration yields three different micelle species with apparent molecular weights of about 166, 54, and 16 kDa. GPI-AP fraction II, which is shown to bear only one anchor per dimer, does not bind to any of these micelles. We demonstrate that a complex is formed containing about 150 Triton X-100 molecules and about 4700 molecules of water per molecule of GPI-AP dimer. The experimental findings are in accordance with a simple geometrical model based on the physical data of fatty acids and the arrangement, mean size, and shape of Triton X-100 molecules.     

Triton X-100 induced dissociation of beef heart cytochrome c oxidase into monomers

Purified beef heart cytochrome c oxidase, when solubilized with at least 5 mg of Triton X-100/mg of protein, was found to be a monodisperse complex containing 180 molecules of bound Triton X-100 with a protein molecular weight of 200 000, a Stokes radius of 66-72 A, and an s(0)20,w = 8.70 S. These values were determined by measurement of the protein molecular weight by sedimentation equilibrium in the presence of D2O, evaluation of the sedimentation coefficient, S(0)20,w, by sedimentation velocity with correction for its dependence upon the concentration of protein and detergent, and measurement of the effective radius by calibrated Sephacryl S-300 gel chromatography. The monomeric complex was judged to be homogeneous and monodisperse since the effective mass of the complex was independent of the protein concentration throughout the sedimentation equilibrium cell and a single protein schlieren peak was observed during sedimentation velocity. These results are interpreted in terms of a fully active monomeric complex that exhibits typical biphasic cytochrome c kinetics and contains 2 heme a groups and stoichiometric amounts of the 12 subunits normally associated with cytochrome c oxidase. With lower concentrations of Triton X-100, cytochrome c oxidase dimers and higher aggregates can be present together with the monomeric complex. Monomers and dimers can be separated by sedimentation velocity but cannot be separated by Sephacryl S-300 gel filtration, probably because the size of the Triton X-100 solubilized dimer is not more than 20% larger than the Triton X-100 solubilized monomer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization of ribulose-1,5-bisphosphate carboxylase from the membrane fraction of pea leaves

The solubilization of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from the membrane fraction was studied in whole leaf extracts and chloroplasts from pea. The amount of membrane-bound Rubisco was dependent on the pH of the chloroplastic lysate buffer. Maximum binding was found at pH 8.0, with about 8% of total leaf Rubisco being bound. The binding of Rubisco to the membranes was strong, and it was not released by repeated washing with hypotonic buffer or by changing ionic strength. Detergents such as Triton X-100, Tween 20, deoxycholate and dodecylsulfate were effective in solubilizing the membrane-bound Rubisco. Triton X-100 was most effective in the range of 0.04% to 0.2% and it solubilized Rubisco from the membrane without any decrease in enzyme activity.Abbreviations BSA bovine serum albumin - CABP carboxyarabinitol-1,5-bisphosphate - DTT dithiothreitol - LDS lithium dodecylsulfate - LHC light-harvesting chlorophyll protein complex - RuBP ribulose-1,5-bisphosphate - Rubisco RuBP carboxylase/oxygenase - SDS sodium dodecylsulfate - SDS-PAGE SDS-polyacrylamide gel electrophoresis     

Dimeric nature of apo-low density lipoprotein extracted with Triton X-100.

Human low density lipoprotein (LDL) was dissolved in 0.3 to 2.0% Triton X-100 at pH 7.5 and apo-LDL (B protein) was extracted from LDL to form B protein-Triton complex. Sedimentation equilibrium study of this complex in a solvent nearly isopycnic to Triton X-100 showed that the molecular weight of the protein in the complex was 570,000. The complex eluted almost at the void volume of a Sepharose 6B column, as would be expected for a complex with a total molecular weight of roughly 900,000, on the assumption that 0.52 g of Triton was bound to 1 g of protein (Helenius, A. and Simons, K. (1972) J. Biol. Chem. 247, 3656-3661). The sedimentation coefficient of the complex gave f/fmin = 2.2, indicating that the complex was either as asymmetric as a fibrinogen molecule or not compact. These results show that B protein exists in its complex with Triton X-100 as an elongated or a loosely expanded dimer based on the molecular weight of monomeric B protein of 270,000. B protein may also exist in LDL as a dimer.     

Action of detergents and pre- and postsynaptic localization of 3H-naloxone binding in synaptosomal membranes. A structural approach

3H-naloxone specific binding was carried out on synaptosomal membranes isolated from basal ganglia of the cat brain. A high- and a low-affinity site with Kd1 = 3.7 nM and Kd2 = 35 nM having B max 1 = 79 pmole/g protein and B max 2 = 224 pmole/g protein were found. The Hill number for the high- and low-affinity sites were, respectively, 1.01 and 0.86. Digitonin and Triton X-100 had an inhibitory effect on the binding at concentrations between 10(-5) and 10(-1)% (w/v). Deoxycholate and Nonidet P-40 also inhibited the binding of 3H-naloxone, but at 10(-4)% produced a 50% enhancement. After the binding to membranes, the 3H-naloxone receptor complex is stable to the action of Triton X-100 and dissociates slowly. In membranes bound with 10 nM 3H-naloxone and then submitted to 0.1-0.2% Triton X-100, in which only the presynaptic membrane disintegrates, the specific radioactivity is decreased. With a more drastic treatment that disintegrates the postsynaptic membrane, the 3H-naloxone binding to synaptosomal membranes is almost completely abolished. These results suggest that opiate receptors may be localized both pre- and postsynaptically in central synapses.     

Dithiothreitol activation of the insulin receptor/kinase does not involve subunit dissociation of the native alpha 2 beta 2 insulin receptor subunit complex

The subunit composition of the dithiothreitol- (DTT) activated insulin receptor/kinase was examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and gel filtration chromatography under denaturing (0.1% SDS) or nondenaturing (0.1% Triton X-100) conditions. Pretreatment of 32P-labeled insulin receptors with 50 mM DTT followed by gel filtration chromatography in 0.1% SDS demonstrated the dissociation of the alpha 2 beta 2 insulin receptor complex (Mr 400,000) into the monomeric 95,000 beta subunit. In contrast, pretreatment of the insulin receptors with 1-50 mM DTT followed by gel filtration chromatography in 0.1% Triton X-100 resulted in no apparent alteration in mobility compared to the untreated insulin receptors. Resolution of this complex by nonreducing SDS-polyacrylamide gel electrophoresis and autoradiography demonstrated the existence of the alpha 2 beta 2 heterotetrameric complex with essentially no alpha beta heterodimeric or free monomeric beta subunit species present. This suggests that the insulin receptor can reoxidize into the Mr 400,000 complex after the removal of DTT by gel filtration chromatography. Surprisingly, these apparently reoxidized insulin receptors were also observed to be functional with respect to insulin binding, albeit with a 50% decrease in affinity for insulin and insulin stimulation of the beta subunit autophosphorylation. To prevent reoxidation, the insulin receptors were pretreated with 50 mM DTT followed by incubation with excess N-ethylmaleimide prior to gel filtration chromatography in 0.1% Triton X-100. Under these conditions the insulin receptors migrated as the Mr 400,000 alpha 2 beta 2 complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Homogeneous functional insulin receptor from 3T3-L1 adipocytes. Purification using N alpha B1-(biotinyl-epsilon-aminocaproyl)insulin and avidin-sepharose

Insulin receptor was purified 10,000-fold from cultured mouse 3T3-L1 adipocytes in 35% overall yield. The specific activities of 125I-insulin binding and autophosphorylation increased in parallel, following the initial Triton X-100 extraction of membranes. The isolation protocol, performed entirely at pH 8.45, entailed adsorption by avidin-Sepharose CL-4B of a complex formed between Triton X-100-solubilized insulin receptor and N alpha B1-(biotinyl-epsilon-aminocaproyl)insulin, and the specific elution of the complex with biotin. The avidin-Sepharose CL-4B was a partially denatured preparation, showing estimated dissociation constants of 0.2 microM for biotin and approximately 1 microM for the bifunctional ligand at, pH 7, 4 degrees C. The bifunctional ligand was characterized by 70% competency in binding to avidin, 100% competency in binding to solubilized insulin receptor, full stimulation of autophosphorylation of the isolated receptor, and maximal stimulation of hexose uptake by intact 3T3-L1 adipocytes. The insulin binding properties of the insulin receptor were uniform throughout this purification procedure. At pH 8.45, 4 degrees C, an average Kd = 0.72 nM was determined for a single class of noninteracting insulin binding sites. The apparent autophosphorylation of the beta-subunit was also unchanged following affinity chromatography. A single oligomeric structure was established for the purified receptor, composed only of 135,000- and 95,000-Da subunits, whose association was lost by denaturation in the presence of reducing agent. This single structure occurred in the initial Triton X-100 extract. The purified insulin receptor was capable of autophosphorylating the beta-subunit and catalyzed phosphorylation of protein substrates.