Band-selective 3D NOESY-TOCSY: Measurement of through-space correlations between aliphatic protons of membrane peptides and proteins in non-deuterated detergents

Summary Recently the use of band-selective excitation to obtain ¹H 2D NMR spectra of membrane peptides and proteins in non-deuterated detergents has been demonstrated [Seigneuret, M. and Levy, D. (1995) J. Biomol. NMR, 5, 345–352]. A limitation of the method was the inability to obtain through-space correlation between aliphatic protons. Here, a 3D F3-band-selective NOESY-TOCSY experiment is described that allows such correlations to be observed in the presence of an excess of non-deuterated detergent. Application to the measurement of proximities between aliphatic protons of the membrane peptide mastoparan X solubilized in non-deuterated n-octylglucoside is presented. With this additional experiment, it is now possible to obtain the same amount of structural constraints on membrane peptides and protein in non-deuterated detergent as in deuterated detergent and therefore to perform complete structural studies.     

Enrichment of hydrophobic membrane proteins using Triton X-114 and subsequent analysis of their N-glycosylation

BackgroundNumerous proteins depend on correct glycosylation for their proper function and nearly all membrane, as well as secreted, proteins are glycosylated. Glycosylation of membrane proteins plays a crucial role in many processes including the intercellular recognition and intermolecular interactions on the cell surface. The composition of N-glycans attached to membrane proteins has not been sufficiently studied due to the lack of efficient and reproducible analytical methods.MethodsThe aim of this study was to optimise cloud-point extraction (CPE) of membrane proteins with the non-ionic detergent Triton X-114 and analyse their N-glycosylation using hydrophilic interaction liquid chromatography (HILIC-UPLC). Purification of isolated proteins from the excess of detergent proved to be the key step. Therefore, several purification procedures were tested to efficiently remove detergent, while retaining maximum protein recoveries.ResultsCPE showed to be an efficient method to simultaneously extract membrane and soluble proteins, which subsequently resulted in different N-glycan profiles of the aforementioned protein groups. The resulting protocol showed satisfactory reproducibility and potential for N-glycan analysis of both membrane and intracellular (soluble) proteins from different kinds of biological material.ConclusionsThis method can be used as a new analytical tool for reliable detection and quantification of oligomannose and complex type N-glycans attached to membrane proteins, thus serving to distinguish between differences in cell types and states.General significanceThe simple method was successfully optimised to generate reliable HILIC-UPLC profiles of N-glycans released from membrane proteins. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.     

The vasoactive intestinal peptide receptor on intact human colonic adenocarcinoma cells (HT29-D4). Evidence for its glycoprotein nature.

We have previously shown that the mono [125I]iodinated vasoactive intestinal peptide (125I-VIP) could be covalently cross-linked on intact colonic adenocarcinoma cells (HT29). A major Mr 67,000 and a minor Mr 120,000 cross-linked polypeptides have been characterized [Muller, Luis, Fantini, Abadie, Giannellini, Marvaldi & Pichon (1985) Eur. J. Biochem. 151, 411-417]. The glycoprotein nature of these species was investigated using endo-beta-acetylglucosaminidase F (Endo F) treatment, enzymic and chemical desialylation and wheat germ agglutinin (WGA)-Sepharose affinity chromatography. Affinity-labelled VIP-binding proteins solubilized by Nonidet P-40 bound to WGA-Sepharose and could be eluted specifically with N-acetyl-D-glucosamine. Treatment with Endo F resulted in an increased electrophoretic mobility of both polypeptides. The major and the minor VIP-binding proteins were converted respectively into Mr 47,000 and 100,000 species, indicating removal of 20 kDa of N-linked oligosaccharides. Deglycosylation with trifluoromethanesulphonic acid also led to a 20 kDa loss in mass of the Mr 67,000 component, indicating the absence of additional O-linked sugars on this polypeptide. The presence of sialic acid on the major VIP-binding protein was demonstrated after treatment of intact cells with neuraminidase or by chemical desialylation with hydrochloric acid. We conclude from this study that the VIP receptor from intact HT29-D4 cells is a glycoprotein with N-linked oligosaccharide side chains containing sialic acid.     

GUANYLATE CYCLASES IN CNS: ENZYMATIC CHARACTERISTICS OF SOLUBLE AND PARTICULATE ENZYMES FROM MOUSE CEREBELLUM AND RETINA

Guanylate cyclase activity is present in both soluble and particulate fractions of homogenates of mouse cerebellum and retina. Soluble guanylate cyclases in cerebellum and retina have an apparent K_m for GTP of approx 40 and 70 μM, respectively; are stimulated by Ca²⁺ and Mg²⁺ in the presence of low Mn²⁺; and do not respond to NaN₃, NH₂OH or detergent. The particulate guanylate cyclase found in brain has an apparent K_m GTP of 237 7mu;M, is not stimulated by Ca²⁺ or Mg²⁺ in the presence of low Mn²⁺, but is stimulated by NaN₃, NH₂OH, and detergent. In particulate fractions of normal retina, guanylate cyclase has two apparent K_m GTP values (42 and 225 μM); has higher activity at low concentrations of Mn²⁺ (0.5 mM) than at high concentrations (5.0 mM); is inhibited by Ca²⁺; and does not respond to NaN₃, NH₂OH, or detergent. Retinas essentially devoid of photoreceptor cells (from mice with photoreceptor dystrophy) have soluble guanylate cyclase activity which is similar to that in normal retina, but have only 4% as much particulate guanylate cyclase activity. This residual particulate guanylate cyclase has an apparent K_m GTP value of 392 μM and other properties similar to particulate guanylate cyclase from brain. These data indicate the presence of three distinguishable guanylate cyclases in CNS: (1) a soluble enzyme present in both brain and retina: (2) a particulate enzyme which is also present in brain and in the inner or neural retina: and (3) another particulate enzyme which is apparently unique and confined to retinal photoreceptor cells.     

Lipid–protein nanodiscs for cell-free production of integral membrane proteins in a soluble and folded state: Comparison with detergent micelles,bicelles and liposomes

Production of integral membrane proteins (IMPs) in a folded state is a key prerequisite for their functional and structural studies. In cell-free (CF) expression systems membrane mimicking components could be added to the reaction mixture that promotes IMP production in a soluble form. Here lipid–protein nanodiscs (LPNs) of different lipid compositions (DMPC, DMPG, POPC, POPC/DOPG) have been compared with classical membrane mimicking media such as detergent micelles, lipid/detergent bicelles and liposomes by their ability to support CF synthesis of IMPs in a folded and soluble state. Three model membrane proteins of different topology were used: homodimeric transmembrane (TM) domain of human receptor tyrosine kinase ErbB3 (TM-ErbB3, 1TM); voltage-sensing domain of K⁺ channel KvAP (VSD, 4TM); and bacteriorhodopsin from Exiguobacterium sibiricum (ESR, 7TM). Structural and/or functional properties of the synthesized proteins were analyzed. LPNs significantly enhanced synthesis of the IMPs in a soluble form regardless of the lipid composition. A partial disintegration of LPNs composed of unsaturated lipids was observed upon co-translational IMP incorporation. Contrary to detergents the nanodiscs resulted in the synthesis of ~ 80% active ESR and promoted correct folding of the TM-ErbB3. None of the tested membrane mimetics supported CF synthesis of correctly folded VSD, and the protocol of the domain refolding was developed. The use of LPNs appears to be the most promising approach to CF production of IMPs in a folded state. NMR analysis of ¹⁵N-Ile-TM-ErbB3 co-translationally incorporated into LPNs shows the great prospects of this membrane mimetics for structural studies of IMPs produced by CF systems.     

Choice of reconstitution protocol modulates the aggregation state of full-length membrane-reconstituted synaptotagmin-1

Synaptotagmin-1 (Syt1) functions as the Ca²⁺ sensor in neuronal exocytosis, and it is routinely incorporated into lipid bilayers along with other components of the fusion machinery in order to reconstruct the in vivo fusion process. Here, we demonstrate that the detergent used to reconstitute full-length Syt1 has a significant effect on the state of the protein in bilayers. When octyl-β-d -glucopyranoside is used to reconstitute the protein, Syt1 is present in an aggregated state that is mediated by the long juxta-membrane linker. EPR spectra from spin labels in the two C2 domains of Syt1 no longer resemble those obtained from a soluble construct containing these domains, and the C2B domain no longer exhibits a Ca²⁺-dependent membrane insertion. In contrast, when reconstituted using 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, Syt1 is largely monomeric and the EPR spectra from C2A and C2B resemble those of the soluble construct. This result demonstrates that the choice of detergent used to reconstitute Syt1 can modulate the state of the neuronal Ca²⁺-sensor.     

Development of a generic approach to native metalloproteomics: application to the quantitative identification of soluble copper proteins in Escherichia coli

A combination of techniques to separate and quantify the native proteins associated with a particular transition metal ion from a cellular system has been developed. The procedure involves four steps: (1) labeling of the target proteins with a suitable short-lived radioisotope (suitable isotopes are ⁶⁴Cu, ⁶⁷Cu, ¹⁸⁷W, ⁹⁹Mo, ⁶⁹Zn, ⁵⁶Mn, ⁶⁵Ni); (2) separation of intact soluble holoproteins using native isoelectric focusing combined with blue native polyacrylamide gel electrophoresis into native–native 2D gel electrophoresis; (3) spot visualization and quantification using autoradiography; and (4) protein identification with tandem mass spectrometry. The method was applied to the identification of copper proteins from a soluble protein extract of wild-type Escherichia coli K12 using the radioisotope ⁶⁴Cu. The E. coli protein CueO, which has previously been only identified as a multicopper oxidase following homologous overexpression, was now directly detected as a copper protein against a wild-type background at an expression level of 0.007% of total soluble protein. The retention of the radioisotope by the copper proteins throughout the separation process corroborates the method to be genuinely native. The procedure developed here can be applied to cells of any origin, and to any metal having suitable radioisotopes. The finding that the periplasmic protein CueO is the only major form of soluble protein bound copper in E. coli strengthens the view that the bacterial periplasm contains only a few periplasmic copper proteins, and that the cytosol is devoid of copper proteins. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

ON THE SPATIAL ARRANGEMENT OF PROTEINS IN MICROSOMAL MEMBRANES FROM RAT LIVER

Rat liver rough microsomes were labeled enzymatically with ¹²⁵I using lactoperoxidase and glucose oxidase. In intact microsomes only proteins exposed on the outside face of the microsomal membrane were iodinated. Low concentrations of detergent (0.049% deoxycholate) were used to allow entrance of the iodination system into the vesicles without disassembling the membranes. This led to iodination of the soluble content proteins and to an increased labeling of the membrane proteins. The distribution of radioactivity in microsomal proteins was analyzed after separation by sodium dodecyl sulfate acrylamide gel electrophoresis. Most membrane proteins were labeled when intact microsomes were iodinated. No major membrane proteins were exclusively labeled in the presence of low detergent concentrations or after complete membrane disassembly. Therefore it is unlikely that there are major membrane proteins, other than glycoproteins, present only on the inner membrane face or completely embedded within the microsomal membrane. Microsomal proteins were also labeled by incubating rough microsomes with [³H]-NaBH₄ after reaction with pyridoxal phosphate. Microsomal membranes were permeable to these small molecular weight reagents as shown by the fact that proteins in the vesicular cavity as well as membrane proteins were labeled with this system.     

Identity of a membrane-bound vasoactive intestinal peptide-binding protein with calmodulin.

A vasoactive intestinal peptide (VIP)-binding protein purified from guinea pig lung membranes (p18) was digested with trypsin, and the amino acid sequence of the peptide fragments was determined. The sequence of six tryptic fragments of p18 was identical with subsequences present in mammalian calmodulin. Authentic porcine brain calmodulin and p18 co-migrated on an sodium dodecyl sulfate-electrophoresis gel and displayed identical chromatographic behavior on a reverse phase high performance liquid chromatography column. The VIP-binding properties of p18 and calmodulin were indistinguishable. Both proteins displayed saturable and apparent high affinity binding of VIP, evidenced by potent inhibition of complexation with [Tyr10-125I]VIP by unlabeled VIP (IC50 = 6.0-8.1 nM). Rat growth hormone releasing factor and a C terminally extended form of VIP ([Leu17]VIP-GKR) also displayed potent inhibition of the binding (IC50 = 6.4 and 4 nM, respectively). These neuropeptides are potential modulators of calmodulin function.     

Sup35 methionine oxidation is a trigger for de novo [PSI+] prion formation

ABSTRACT. The molecular basis by which fungal and mammalian prions arise spontaneously is poorly understood. A number of different environmental stress conditions are known to increase the frequency of yeast [PSI⁺] prion formation in agreement with the idea that conditions which cause protein misfolding may promote the conversion of normally soluble proteins to their amyloid forms. A recent study from our laboratory has shown that the de novo formation of the [PSI⁺] prion is significantly increased in yeast mutants lacking key antioxidants suggesting that endogenous reactive oxygen species are sufficient to promote prion formation. Our findings strongly implicate oxidative damage of Sup35 as an important trigger for the formation of the heritable [PSI⁺] prion in yeast. This review discusses the mechanisms by which the direct oxidation of Sup35 might lead to structural transitions favoring conversion to the transmissible amyloid-like form. This is analogous to various environmental factors which have been proposed to trigger misfolding of the mammalian prion protein (PrP^C) into the aggregated scrapie form (PrP^Sc).     

Respirable powder formulation of a shortened vasoactive intestinal peptide analog for treatment of airway inflammatory diseases

The aim of present study was to develop a respirable powder (RP) of a shortened vasoactive intestinal peptide (VIP) analog for inhalation. VIP and C‐terminally truncated VIP analogs were synthesized with a solid‐phase method. A structure‐activity relationship (SAR) study was carried out in terms with binding and relaxant activities of the peptides. Prepared RP formulation of a shortened VIP analog was physicochemically characterized by morphological, in vitro aerodynamic, and pharmacological assessments. The SAR study demonstrated that the N‐terminal 23 amino acid residues were required for biological activity of VIP. Upon chemical modification of VIP(1–23), [R^{15, 20, 21}, L¹⁷]‐VIP(1–23) was newly developed, which had higher binding activity in rat lung and smooth muscle relaxant effect in mouse stomach than VIP(1–23). The [R^{15, 20, 21}, L¹⁷]‐VIP(1–23)‐based RP, [R^{15, 20, 21}, L¹⁷]‐VIP(1–23)/RP, exhibited fine in vitro inhalation performance. Airway inflammation evoked by sensitization of antigen in rats was attenuated by pre‐treatment with the [R^{15, 20, 21}, L¹⁷]‐VIP(1–23)/RP at a dose of 50 μg‐[R^{15, 20, 21}, L¹⁷]‐VIP(1–23)/rat as evidenced by a 70% reduction of recruited inflammatory cells in bronchoalveolar lavage fluid. On the basis of these results, [R^{15, 20, 21}, L¹⁷]‐VIP(1–23)/RP might be a promising agent for treatment of airway inflammatory diseases.     

Parameter optimization for thermostable lipase production and performance evaluation as prospective detergent additive

Abstract

Lipase based formulations has been a rising interest to laundry detergent industry for their eco-friendly property over phosphate-based counterparts and compatibility with chemical detergents ingredients. A thermo-stable Anoxybacillus sp. ARS-1 isolated from Taptapani Hotspring, India was characterized for optimum lipase production employing statistical model central composite design (CCD) under four independent variables (temperature, pH, % moisture and bio-surfactant) by solid substrate fermentation (SSF) using mustard cake. The output was utilized to find the effect of parameters and their interaction employing response surface methodology (RSM). A quadratic regression with R²?=?0.955 established the model to be statically best fitting and a predicted highest lipase production of 29.4?IU/g at an optimum temperature of 57.5?°C, pH 8.31, moisture 50% and 1.2?mg of bio-surfactant. Experimental production of 30.3?IU/g lipase at above conditions validated the fitness of model. Anoxybacillus sp. ARS-1 produced lipase was found to resist almost all chemical detergents as well as common laundry detergent, proving it to be a prospective additive for incorporation.     

Purification of vasoactive intestinal peptide receptor from porcine liver by a newly designed one-step affinity chromatography

Vasoactive intestinal peptide (VIP) receptors were solubilized from porcine liver membrane using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. The solubilized VIP receptor has been purified approximately 50,000-fold to apparent homogeneity by a one-step affinity chromatography using a newly designed VIP-polyacrylamide resin. The purified receptor bound 125I-VIP with a Kd of 22.3 +/- 0.7 nM and retained its peptide specificity toward VIP-related peptides. The specific activity of the purified receptor (16,400 pmol/mg of protein) was very close to the theoretical value (18,900 pmol/mg of protein) calculated assuming one binding site/protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified receptor revealed a single band with an Mr of 53,000 after either silver staining or radioiodination. Affinity labeling of the purified receptor with 125I-VIP using dithiobis(succinimidyl propionate) gave a single radioactive band, the labeling of which was completely inhibited by an excess of unlabeled VIP. In conclusion, an Mr 53,000 protein containing the VIP-binding site was purified to homogeneity by a one-step affinity chromatography using immobilized VIP.     

Cycloheximide induces accumulation of vasoactive intestinal peptide (VIP) binding sites at the cell surface of a human colonic adenocarcinoma cell line (HT29-D4). Evidence for the presence of an intracellular pool of VIP receptors

Incubation of monolayers of HT29-D4 cells (a clone of the human colonic adenocarcinoma cell line HT29) in the presence of 17.5 microM cycloheximide resulted in an increase in the number of vasoactive intestinal peptide (VIP) binding sites at the cell surface without any change in the affinity of receptor for its ligand. The increase in 125I-VIP-binding capacity was dose-dependent between 0.35 microM and 17.5 microM cycloheximide and was correlated with the inhibition of protein biosynthesis. At higher concentrations of drug (17.5-100 microM) a plateau corresponding to a twofold increase in VIP-binding capacity was reached independently of the extent of protein synthesis inhibition. We found that VIP receptors of HT29-D4 cells with such an enhanced binding capacity behaved like those of control cells with respect to receptor internalization and recycling (i.e. the cycle of occupied receptors was insensitive to cycloheximide). After inactivation of 90% of cell-surface VIP receptors by alpha-chymotrypsin, we observed a biphasic kinetic of reappearance of VIP-binding sites. 40% of VIP-binding sites reappeared very quickly (less than 5 min) and 100% within 17 h. The fast recovery of VIP receptors was probably due to the deployment of new binding sites from an intracellular pool. The rate and extent of recovery of these receptors were similar in control cells and in cycloheximide-treated cells. However, the slow recovery was inhibited in cycloheximide-treated cells probably because a pool of immature receptors was depleted by the drug before the alpha-chymotrypsin treatment. Our data are consistent with the existence of two different intracellular pathways of occupied and unoccupied VIP receptors.     

Associations of maize protein bodies with cytoskeleton, membranes, and ribosomes in the endosperm of wild type and opaque-2 mutant

Maize endosperm was homogenized in a cytoskeleton-stabilizing buffer, filtered and layered on gradients of 20–80% sucrose and analyzed by monitoring their UV absorbance. A major peak of UV-light absorbing material was detected on the gradient, at about 60–65% sucrose (density of approximately 1.3 g·ml⁻¹). Biochemical, fluorescence microscopic, and immunoblot analyses of this peak showed that it consisted of protein bodies associated with actin, membranes, and RNA (ribosomes). Seeds of wild type and opaque-2 mutant were then homogenized, the homogenate was modified using detergents and/or cytoskeleton-disrupting agents, and centrifuged on sucrose gradients. In wild type maize endosperm, detergent treatment caused the major peak (protein bodies) to increase in density so that they sediment further down the gradient. However, in opaque-2 the protein bodies formed a broader, but smaller peak which, upon treatment with detergent, generated protein bodies which pelleted to the bottom of the gradient. Analysis of gradient fractions by gel electrophoresis and immuno-blotting showed that both the wild type and the mutant had cytoskeleton proteins in the upper regions (soluble, non-polymerized microfilaments and microtubules) as well as in the peak regions. Comparisons of both the UV-absorbance profiles and the immunoblot data suggest that the protein bodies from the two maize types associate differently with the membranes and the cytoskeleton.     

Solubilization and Characterization of Striatal Dopamine Receptors

Abstract: Dopamine receptor binding proteins were sol-ubilized with the detergent 3–(3–cholamidopropyl) dimethylammonio - 2 - hydroxy - 1– propanesulfonate (CHAPSO) from bovine and rat striatal membranes. The binding of the dopamine antagonist [³H]spiroperidol ([³H]Spi) to the solubilized dopamine receptors was determined by the polyethyleneglycol method. The CHAPSO-solubilized dopamine receptor binding proteins remain in the supernatant fraction following centrifuga-tion at 100,000 ×g for 2 h. The CHAPSO-solubilized dopamine receptor proteins, as well as the prelabeled [³H]Spi-receptor protein complex, bind specifically to wheat germ agglutinin (WGA)-agarose columns, which is consistent with an identification as glycoproteins. HPLC analysis of the CHAPSO-solubilized, prelabeled [³H]Spi-receptor protein complex (CHAPSO preparation) reveals association with a high molecular weight form, indicating the formation of aggregates and/or micelles. Treatment of the WGA-agarose-bound [³H]Spi-receptor protein complex with digitonin (CHAPSO-digitonin preparation) results in dissociation of the high molecular weight form into lower molecular weight forms. The HPLC profile of the prelabeled [³H]Spi-receptor complex in the CHAPSO-digitonin preparation reveals two radioactive peaks. The major peak had a retention time of 16 min, corresponding to an apparent MW of 175,000, whereas the minor peak had a retention time of 21 min, corresponding to an apparent MW of 49,000. The CHAPSO-solubilized dopamine receptor binding proteins are sensitive to modulation by GTP, indicating that the association with the GTP binding component is preserved in the “soluble” state. The potencies of dopamine antagonists and agonists for inhibiting the binding of [³H]Spi to CHAPSO-solubilized dopamine receptor proteins are similar to those for membrane-bound proteins. Chronic treatment with haloperidol increases the B_max, and does not change the K_D for [³H]Spi in the CHAPSO-solubilized and in the membrane-bound preparations. Thus, the CHAPSO-solubilized dopamine receptor proteins retain the binding characteristics of the supersensitive membrane-bound dopamine receptors.     

利用蛋白质组学研究新德里金属b-内酰胺酶1对鲍曼不动杆菌的影响

【目的】比较临床分离的亲缘关系近的多药耐药鲍曼不动杆菌MDR-ZJ06(blaNDM-1–)和ABC3229(blaNDM-1+)的差异蛋白质组,以期发现新德里金属?-内酰胺酶1(New Delhimetallo-β-lactamase-1,NDM-1)对鲍曼不动杆菌生长代谢的影响。【方法】利用2-DE联合MALDI-TOF MS/MS技术鉴定差异表达蛋白,并在GO注析的基础上,对差异蛋白进行通路分析、功能分类和富集分析,并作出蛋白与蛋白相互作用网络。【结果】发现ABC3299相对于MDR-ZJ06有51个差异表达蛋白,其中11个蛋白表达上调,40个蛋白表达下调,并且这些差异蛋白主要涉及降低碳代谢、氨基酸代谢、脂肪酸代谢和细胞壁合成,增加铁离子转运系统形成。【结论】这个结果揭示了NDM-1可能是通过减缓细菌自身的代谢,增加自身铁的摄取使细菌机体系统地抵抗抗生素从而达到耐药。     

Alterations in the cell surface proteins of Drosophila during morphogenesis

Summary Unevaginated and evaginated Drosophila imaginal discs were surface-labeled with ¹²⁵I. Relative labeling was greater in eleven peptides and lower in three peptides of evaginated discs compared to unevaginated discs. These results are compared to the effects of 20-hydroxyecdysone (20-HOE) on metabolic labeling of membrane proteins fractionated from imaginal discs, and on cell surface labeling of a hormone-responsive Drosophila tissue culture line. A group of ³⁵S-methionine labeled membrane fraction peptides whose metabolic labeling is 20-HOE dependent have isoelectric points and apparent molecular weights very similar to those of a group of proteins only labeled in iodinated evaginated discs, supporting the conclusion that these are hormone-dependent, cell surface proteins (Rickoll and Fristrom 1983). Based upon two-dimensional gel electrophoretic and immunological criteria three of the proteins showing increased labeling in evaginated discs are related to three proteins induced by 20-HOE in tissue culture cells. Two different subsets of radiolabeled peptides were observed in the imaginal discs based upon detergent solubility. Some of the proteins which are soluble in NP-40 plus urea but insoluble in NP-40 alone may be localized in the basal lamina of the imaginal discs, a structure which labels heavily with ¹²⁵I and is lacking in tissue culture cells. In discs, the majority of hormone-dependent changes in radiolabeled peptides were seen in the fraction solubilized by NP-40 and urea with a sulfhydryl reducing agent, while in tissue culture cells, the majority of differences is seen in the fraction solubilized by NP-40 only. We speculate that these proteins may be involved in similar processes, e.g., cell rearrangement, that occur during both disc morphogenesis and 20-HOE induced aggregation in tissue culture cells.This work was supported by grants CD-205 from the American Cancer Society, RR08132 from NIH to C.A.P. and GM 19937 from NIH to J.W.F.     

THE CELL-FREE SYNTHESIS OF ALKALOID-BINDING PROTEINS IN IMMATURE RAT BRAIN1

Abstract— cell-free amino acid incorporating system from immature rat brain, consisting of ribosomal and soluble fractions, has been investigated for its capacity to incorporate [¹⁴C]amino acids into specific soluble proteins that interact with vinblastine sulfate and colchicine. The soluble ¹⁴C-labeled proteins formed in the cell-free system during incubation were compared with similar soluble proteins from immature rat brain which had been labeled in vivo by the incorporation of ¹⁴C-labeled amino acids. Criteria for the formation of vinblastine-binding, ¹⁴C-labeled proteins were: (1) aggregation of ¹⁴C-labeled soluble protein by one mm -vinblastine sulfate and (2) immunoprecipitation of ¹⁴C-labeled soluble protein by an antiserum against vinblastine sulfate-precipitable material. Criteria for the formation of [³H]colchicine-binding, ¹⁴C-labeled protein were based upon: (1) co-precipitation of the ³H-and ¹⁴C-labeled materials by vinblastine sulfate and (2) the coincidence of ³H- and ¹⁴C-labeled elution peaks from columns of Sephadex G-200, DEAE-Sephadex A-50 and isoelectric focusing. Both in the in vitro and in the in vivo system, ¹⁴C-labeled amino acids were incorporated into soluble proteins of the post-microsomal supernatant fraction. Proteins labeled with ¹⁴C-labeled amino acids in vitro and in vivo yielded comparable and qualitatively identical results by the criteria tested, including the formation of immunoprecipitates. In the in vitro system, ¹⁴C-labeled amino acids were incorporated into protein with a molecular weight of approx 120,000, an isoelectric point of 5.3 and with a chromatographic mobility on Sephadex G-200 which is identical to [³H]colchicine-binding protein. The above experimental results are presumptive evidence for the synthesis of vinblastine-binding and colchicine-binding proteins in the in vitro cell-free system.     

Deuterated detergents for structural and functional studies of membrane proteins: Properties,chemical synthesis and applications

Abstract

Detergents are amphiphilic compounds that have crucial roles in the extraction, purification and stabilization of integral membrane proteins and in experimental studies of their structure and function. One technique that is highly dependent on detergents for solubilization of membrane proteins is solution-state NMR spectroscopy, where detergent micelles often serve as the best membrane mimetic for achieving particle sizes that tumble fast enough to produce high-resolution and high-sensitivity spectra, although not necessarily the best mimetic for a biomembrane. For achieving the best quality NMR spectra, detergents with partial or complete deuteration can be used, which eliminate interfering proton signals coming from the detergent itself and also eliminate potential proton relaxation pathways and strong dipole-dipole interactions that contribute line broadening effects. Deuterated detergents have also been used to solubilize membrane proteins for other experimental techniques including small angle neutron scattering and single-crystal neutron diffraction and for studying membrane proteins immobilized on gold electrodes. This is a review of the properties, chemical synthesis and applications of detergents that are currently commercially available and/or that have been synthesized with partial or complete deuteration. Specifically, the detergents are sodium dodecyl sulphate (SDS), lauryldimethylamine-oxide (LDAO), n-octyl-β-D-glucoside (β-OG), n-dodecyl-β-D-maltoside (DDM) and fos-cholines including dodecylphosphocholine (DPC). The review also considers effects of deuteration, detergent screening and guidelines for detergent selection. Although deuterated detergents are relatively expensive and not always commercially available due to challenges associated with their chemical synthesis, they will continue to play important roles in structural and functional studies of membrane proteins, especially using solution-state NMR.