Inositol is a constituent of detergent-solubilized immunoaffinity-purified rat liver 5'-nucleotidase.
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ABSTRACT:
SUBMITTER: Bailyes EM
PROVIDER: S-EPMC1133719 | biostudies-other | 1990 Feb
REPOSITORIES: biostudies-other
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Project description:Patched (Ptc) is a twelve-pass transmembrane protein that functions as a receptor for the Hedgehog (Hh) family of morphogens. In addition to Ptc, several accessory proteins including the CDO/Ihog family of co-receptors are necessary for proper Hh signaling. Structures of Hh proteins bound to members of the CDO/Ihog family are known, but the nature of the full Hh receptor complex is not well understood. We have expressed the Drosophila Patched and Mouse Patched-1 proteins in Sf9 cells and find that Sonic Hedgehog will bind to Mouse Patched-1 in isolated Sf9 cell membranes but that purified, detergent-solubilized Ptc proteins do not interact strongly with cognate Hh and CDO/Ihog homologs. These results may reflect a nonnative conformation of detergent-solubilized Ptc or that an additional factor or factors lost during purification are required for high-affinity Ptc binding to Hh.
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Project description:Lactogenic receptors from rat liver microsomal fraction ('microsomes') were extracted by treatment with 1% (w/v) Triton X-100. Triton X-100 exerts an inhibitory effect on both the binding reaction and the separation of the free hormone from the complex. The association and dissociation of 125I-labelled human somatotropin are time- and temperature-dependent processes. The association rate constant, k1, is 6.7 x 10(6) mol . litre-1 . min-1 at 25 decrees C, and the dissociation rate constant, k-1, is 1.1 x 10(-3) min-1 at 25 degrees C. Scatchard analysis of saturation data reveals the existence of a single class of receptors and that solubilization leads to a slight decrease in affinity and a sharp increase in binding capacity. The dissociation constant, Kd, of the solubilized preparation is 0.22 nM and the binding capacity 2900 fmol/mg of protein. Similar results were obtained from competition experiments. Binding of 125I-labelled human somatotropin to the solubilized receptors is specifically inhibited by hormones with lactogenic activity. Incubation of the solubilized preparation with trypsin resulted in an 80% decrease in binding activity. The solubilized form of the receptor has a slightly increased sensitivity to the inactivation by trypsin, heat and extremes of pH, with respect to the membrane-bound form.
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Project description:The H(+),K(+)-ATPase pumps protons or hydronium ions and is responsible for the acidification of the gastric fluid. It is made up of an ?-catalytic and a ?-glycosylated subunit. The relation between cation translocation and the organization of the protein in the membrane are not well understood. We describe here how pure and functionally active pig gastric H(+),K(+)-ATPase with an apparent Stokes radius of 6.3 nm can be obtained after solubilization with the non-ionic detergent C(12)E(8), followed by exchange of C(12)E(8) with Tween 20 on a Superose 6 column. Mass spectroscopy indicates that the ?-subunit bears an excess mass of 9 kDa attributable to glycosylation. From chemical analysis, there are 0.25 g of phospholipids and around 0.024 g of cholesterol bound per g of protein. Analytical ultracentrifugation shows one main complex, sedimenting at s(20,)(w) = 7.2 ± 0.1 S, together with minor amounts of irreversibly aggregated material. From these data, a buoyant molecular mass is calculated, corresponding to an H(+),K(+)-ATPase ?,?-protomer of 147.3 kDa. Complementary sedimentation velocity with deuterated water gives a picture of an ?,?-protomer with 0.9-1.4 g/g of bound detergent and lipids and a reasonable frictional ratio of 1.5, corresponding to a Stokes radius of 7.1 nm. An ?(2),?(2) dimer is rejected by the data. Light scattering coupled to gel filtration confirms the monomeric state of solubilized H(+),K(+)-ATPase. Thus, ?,? H(+),K(+)-ATPase is active at least in detergent and may plausibly function as a monomer, as has been established for other P-type ATPases, Ca(2+)-ATPase and Na(+),K(+)-ATPase.
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Project description:Protein stability in detergent or membrane-like environments is the bottleneck for structural studies on integral membrane proteins (IMP). Irrespective of the method to study the structure of an IMP, detergent solubilization from the membrane is usually the first step in the workflow. Here, we establish a simple, high-throughput screening method to identify optimal detergent conditions for membrane protein stabilization. We apply differential scanning fluorimetry in combination with scattering upon thermal denaturation to study the unfolding of integral membrane proteins. Nine different prokaryotic and eukaryotic membrane proteins were used as test cases to benchmark our detergent screening method. Our results show that it is possible to measure the stability and solubility of IMPs by diluting them from their initial solubilization condition into different detergents. We were able to identify groups of detergents with characteristic stabilization and destabilization effects for selected targets. We further show that fos-choline and PEG family detergents may lead to membrane protein destabilization and unfolding. Finally, we determined thenmodynamic parameters that are important indicators of IMP stability. The described protocol allows the identification of conditions that are suitable for downstream handling of membrane proteins during purification.
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Project description:A soluble 'low-Km' 5'-nucleotidase has been described previously in several organs. It has been presumed to be of cytosolic origin and thus to play a role in the intracellular production of adenosine. Its catalytic properties are similar to those of the ecto-5'-nucleotidase of cell membranes. In the present study we compared molecular properties of the two enzymes in the kidney of the rat. The Mr of the main peak of soluble 'low-Km' 5'-nucleotidase in gel-filtration chromatography was similar to that of the ecto-5'-nucleotidase solubilized by a phosphatidylinositol-specific phospholipase C from renal brush-border membranes. In phase-partition experiments using Triton X-114, the soluble enzyme appeared to be hydrophobic. Its hydrophobicity was decreased on treatment with a phosphatidylinositol-specific phospholipase C, suggesting that the soluble 'low-Km' 5'-nucleotidase contains the phosphatidylinositol anchor which is characteristic for the ecto-enzyme. An anti-ecto-5'-nucleotidase antiserum provoked an almost complete inhibition of the soluble enzyme. Immunoblotting using anti-ecto-5'-nucleotidase antiserum revealed in the high-speed supernatants a polypeptide with a similar Mr to the subunit of the ecto-5'-nucleotidase. The soluble 'low-Km' 5'-nucleotidase, like the ecto-5'-nucleotidase, bound specifically to concanavalin A. We conclude that the soluble 'low-Km' 5'-nucleotidase is not a cytosolic enzyme, but that it most probably originates from the solubilization of the ecto-5'-nucleotidase, and that it therefore cannot participate in the intracellular production of adenosine.
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Project description:The presence of lactogenic and somatogenic binding sites in intact microsomal membranes and in detergent-solubilized microsomal membrane preparations of female rat liver has been studied by affinity cross-linking-SDS/polyacrylamide-gel electrophoresis. In microsomal membrane preparations an Mr 40,000 lactogenic binder is present which is not disulphide-linked to another protein. Triton X-100 solubilization of membranes results in the appearance of three lactogenic 125I-human growth hormone (125I-hGH) binders with Mr values of 87,000, 40,000 and 35,000, and one somatogenic 125I-hGH binder with Mr 32,000. Treatment of rats with oestrogen increased the amount of lactogenic and somatogenic binding species in liver. The lactogenic binding sites are present as one entity in Triton X-100-solubilized preparations, clearly separated from the somatogenic binder as analysed by gel chromatography. Furthermore, 125I-hGH interacts with an Mr 95,000 somatogenic binder in membrane preparations to which the hormone can be cross-linked only following Triton X-100 solubilization.
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Project description:High-throughput generation of antibodies against cellular components is currently a challenge in proteomics, therapeutic development and other biological applications. It is particularly challenging to raise antibodies that target membrane proteins due to their insolubility in aqueous solutions. To address these issues, a yeast display library of human single-chain antibody fragments (scFvs) was efficiently screened directly against detergent-solubilized and biotinylated lysates of a target cell line, thereby avoiding issues with membrane protein insolubility and eliminating the need for heterologous expression or purification of antigens. Antibody clones that specifically bind plasma membrane proteins or intracellular proteins were identified, depending on the biotinylation method applied. Antibodies against a predetermined target could also be identified using cell lysate as an antigen source as demonstrated by selecting an scFv against the transferrin receptor (TfR). When secreted from yeast and purified, the selected scFvs are active under physiological conditions in the absence of detergents. In addition, this method allows facile characterization of target antigens because it is compatible with yeast display immunoprecipitation. We expect that this method will prove useful for multiplex affinity reagent generation and in targeted antibody screens.
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Project description:Despite the ever-increasing usage of small-angle scattering as a valuable complementary method in the field of structural biology, applications concerning membrane proteins remain elusive mainly due to experimental challenges and the relative lack of theoretical tools for the treatment of scattering data. This fact adds up to general difficulties encountered also by other established methods (crystallography, NMR) for the study of membrane proteins. Following the general paradigm of ab initio methods for low-resolution restoration of soluble protein structure from small-angle scattering data, we construct a general multiphase model with a set of physical constraints, which, together with an appropriate minimization procedure, gives direct structural information concerning the different components (protein, detergent molecules) of detergent-solubilized membrane protein complexes. Assessment of the method's precision and robustness is evaluated by performing shape restorations from simulated data of a tetrameric α-helical membrane channel (Aquaporin-0) solubilized by n-Dodecyl β-D-Maltoside and from previously published small-angle neutron scattering experimental data of the filamentous hemagglutinin adhesin β-barrel protein transporter solubilized by n-Octyl β-D-glucopyranoside. It is shown that the acquisition of small-angle neutron scattering data at two different solvent contrasts, together with an estimation of detergent aggregation number around the protein, permits the reliable reconstruction of the shape of membrane proteins without the need for any prior structural information.
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Project description:Data, both for and against the presence of a mitochondrial nitric-oxide synthase (NOS) isoform, is in the refereed literature. However, irrefutable evidence has not been forthcoming. In light of this controversy, we designed studies to investigate the existence of the putative mitochondrial NOS. Using repeated differential centrifugation followed by Percoll gradient fractionation, ultrapure, never frozen rat liver mitochondria and submitochondrial particles were obtained. Following trypsin digestion and desalting, the mitochondrial samples were analyzed by nano-HPLC-coupled linear ion trap-mass spectrometry. Linear ion trap-mass spectrometry analyses of rat liver mitochondria as well as submitochondrial particles were negative for any peptide from any NOS isoform. However, recombinant neuronal NOS-derived peptides from spiked mitochondrial samples were easily detected, down to 50 fmol on column. The protein calmodulin (CaM), absolutely required for NOS activity, was absent, whereas peptides from CaM-spiked samples were detected. Also, l-[(14)C]arginine to l-[(14)C]citrulline conversion assays were negative for NOS activity. Finally, Western blot analyses of rat liver mitochondria, using NOS (neuronal or endothelial) and CaM antibodies, were negative for any NOS isoform or CaM. In conclusion, and in light of our present limits of detection, data from carefully conducted, properly controlled experiments for NOS detection, utilizing three independent yet complementary methodologies, independently as well as collectively, refute the claim that a NOS isoform exists within rat liver mitochondria.
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Project description:1. A variety of detergents were used to solubilize 5'-nucleotidase from rat liver plasma membranes. 2. The zwitterionic detergent Sulphobetaine 14 gave optimal solubilization by the criteria of release into a high-speed-centrifugation supernatant and the formation of the smallest and least polydisperse active enzyme observed on polyacrylamide-gel electrophoresis. 3. The Sulphobetaine 14-solubilized enzyme from rat liver was purified by using the conventional techniques of ion-exchange chromatography and gel filtration, or by an immunoaffinity step with a monoclonal antibody immunoadsorbent. 4. 5'-Nucleotidase was purified at least 12 000-fold relative to liver homogenate by the immunoaffinity purification scheme and had a specific activity in the range 285-340 mumol/min per mg of protein. The yield was in the range 9-16%. 5. The purified enzyme shows a major polypeptide band of apparent Mr 70 000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and a minor band of apparent Mr 38 000. 6. A rational approach to the general problem of the purification of minor intrinsic membrane proteins is discussed, with the use of polyacrylamide-gel electrophoresis to determine the most appropriate detergent and monoclonal antibodies in subsequent immunoaffinity purification.
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