共查询到20条相似文献,搜索用时 31 毫秒
1.
Comparison of the macroscopic and single channel conductance properties of colicin E1 and its COOH-terminal tryptic peptide 总被引:10,自引:0,他引:10
J O Bullock F S Cohen J R Dankert W A Cramer 《The Journal of biological chemistry》1983,258(16):9908-9912
A COOH-terminal tryptic fragment (Mr approximately equal to 20,000) of colicin E1 has been proposed to contain the membrane channel-forming domain of the colicin molecule. A comparison is made of the conductance properties of colicin E1 and its COOH-terminal fragment in planar bilayer membranes. The macroscopic and single channel properties of colicin E1 and its COOH-terminal tryptic fragment are very similar, if not indistinguishable, implying that the NH2-terminal, two-thirds of the colicin E1 molecule, does not significantly influence its channel properties. The channel-forming activity of both polypeptides is dependent upon the presence of a membrane potential, negative on the trans side of the membrane. The average single channel conductance of colicin E1 and the COOH-terminal fragment is 20.9 +/- 3.9 and 19.1 +/- 2.9 picosiemens, respectively. The rate at which both proteins form conducting channels increases as the pH is lowered from 7 to 5. Both molecules require negatively charged lipids for activity to be expressed, exhibit the same ion selectivity, and rectify the current to the same extent. Both polypeptides associate irreversibly with the membrane in the absence of voltage, but subsequent formation of conducting channels requires a negative membrane potential. 相似文献
2.
Dependence of the conformation of a colicin E1 channel-forming peptide on acidic pH and solvent polarity 总被引:4,自引:0,他引:4
The secondary structure content of the COOH-terminal tryptic peptide of colicin E1 has been measured by analysis of UV circular dichroism spectra as a function of pH in aqueous medium and in the presence of the nonionic detergents octyl glucoside and Triton X-100. The alpha-helical content of the peptide increased by approximately 10%, from 45-47% to 56-57%, in the presence of the nonionic detergents, but not in aqueous medium, as the pH was decreased from 4.5 to 3.5. This pH dependence of conformation is similar to that reported elsewhere for the in vitro activity and binding of this peptide. A smaller increase in helical content was observed for the peptide in aqueous medium or in Triton X-100 as the pH was decreased from 6.5 to 4.5. The letter change in helical content was not seen in octyl glucoside which was present at a detergent:peptide stoichiometry 100 times that of Triton. The mean residue ellipticity measured at 222 nm for peptide added to asolectin vesicles by a freeze-thaw treatment was slightly larger at pH 3.5, and substantially larger at pH 4.5, than found at these pH values in the detergent solutions. Changes in helical content at the former, but not the latter pH, could be attributed to peptide insertion. It appears that protonation of one or more acidic amino acid residues in the COOH-terminal region of the molecule causes a conformational change that can be attributed to an extra helical domain that is stabilized in a nonpolar environment. From the similar pH dependence of the conformational change and in vitro binding and activity, it is inferred that interaction of this domain with the membrane is essential for binding and insertion. 相似文献
3.
Isolation and molecular and functional properties of the amino-terminal domain of colicin A 总被引:3,自引:0,他引:3
M Knibiehler S P Howard D Baty V Geli R Lloubès P Sauve C Lazdunski 《European journal of biochemistry》1989,181(1):109-113
A plasmid was constructed which allowed easy and efficient production and purification of the NH2-terminal domain of colicin A. In only three steps, an homogenous 18-kDa polypeptide was obtained. The NH2- and COOH-terminal sequences of the protein were determined and showed that it corresponded to the NH2-terminal 171 amino acid residues of the 63-kDa colicin A. Although colicin A is a highly asymmetric protein, hydrodynamic studies indicated that the NH2-terminal domain (designated AT) has a globular structure. This fragment is not the receptor-binding domain of colicin A but is required for the transfer of colicin A across the outer membrane of sensitive cells. However, it has a low affinity for phospholipid films and this affinity is not pH-dependent, in contrast to that of colicin A. 相似文献
4.
H Suga K Shirabe T Yamamoto M Tasumi M Umeda C Nishimura A Nakazawa M Nakanishi Y Arata 《The Journal of biological chemistry》1991,266(21):13537-13543
Structural changes upon binding to the membrane of a COOH-terminal channel-forming thermolytic fragment of colicin E1 have been studied by means of a variety of spectroscopic techniques. Circular dichroism measurements show that the thermolytic fragment predominantly takes a helical structure in aqueous and detergent solutions. Fourier transform infrared spectroscopic measurements indicate that the content of the beta-structure is significantly increased when the thermolytic fragment is bound to vesicles. On the basis of the result of tryptophan fluorescence measurements, we have concluded that each of the three tryptophan residues of the thermolytic fragment exists in different environments, i.e. one is buried in the lipid bilayer, one exists on the cis side of the vesicles, and one exists near the surface of the lipid bilayer. The Fourier transform infrared and fluorescence data have been used along with the crystal structure of colicin A, which is highly homologous to colicin E1 in structure and function, to propose a model of the thermolytic fragment bound to the lipid vesicles. 相似文献
5.
Interactions of colicin A domains with phospholipid monolayers and liposomes: relevance to the mechanism of action 总被引:1,自引:0,他引:1
M Frenette M Knibiehler D Baty V Géli F Pattus R Verger C Lazdunski 《Biochemistry》1989,28(6):2509-2514
The colicin A polypeptide chain (592 amino acid residues) contains three domains which are linearly organized and participate in the sequential steps involved in colicin action. We have compared the penetrating ability in phospholipid monolayers and the ability to promote vesicle fusion at acidic pH of colicin A and of protein derivatives containing various combinations of its domains. The NH2-terminal domain (171 amino acid residues), required for translocation across the outer membrane, has little affinity for dilauroylphosphatidylglycerol (DLPG) monolayers at all pHs tested. The central domain has a pH-dependent affinity, although lower than that of the entire colicin A. The COOH-terminal domain contains a high-affinity lipid binding site, but in addition an electrostatic interaction is required as a first step in the process of penetration into negatively charged DLPG films. In contrast to the constructs containing the ionophoric domain, the NH2-terminal domain alone has no fusogenic activity for liposomes. These results are discussed with regard to the mechanism of entry and action of colicin A in sensitive cells. Our results suggest the existence of a pH-dependent interaction between the receptor binding domain (amino acid residues 172-388) and the pore-forming domain of colicin A (amino acid residues 389-592). 相似文献
6.
For structural studies of integral membrane proteins, including their 3D crystallization, the judicious use of detergent for solubilization and purification is required. Detergent binding by the solubilized protein is an important parameter to determine the hydrodynamic properties in terms of size and aggregational (monomeric/oligo(proto)meric) state of the protein. Detergent binding can be measured by gel filtration chromatography under equilibrium conditions and after separation from mixed micelles of solubilized lipid and detergent. Using sarcoplasmic reticulum Ca(2+)-ATPase as an example, we demonstrate in this protocol complete procedures for measurement of detergent binding using (i) radiolabeled n-dodecyl-beta-D-maltoside (DM) or (ii) from measurements of the increase in refractive index due to the presence of bound detergent on the protein. The latter measurement can also be performed by sedimentation velocity (SV) analysis in the analytical ultracentrifuge which in addition allows determination of the sedimentation coefficient. In combination with estimation of Stokes radius by gel filtration calibration, the molecular mass and asymmetry of the solubilized protein can be calculated. In the proposed protocols, the gel chromatographic procedures require 1 d; SV experiments are performed just after size exclusion. The whole time for these experiments is 24 h. Data analysis of analytical ultracentrifugation requires a couple of days. 相似文献
7.
The in vitro activity of many pore-forming toxins, in particular, the rate of increase in the membrane conductance induced by the channel-forming domain (P178) of colicin E1 is maximum at an acidic pH. However, after P178 binding at acidic conditions, a subsequent pH shift from 4 to 6 on both sides of the planar bilayer lipid membrane caused a large increase in the trans-membrane current which was solely due to an increase in the number of open channels. This effect required the presence of anionic lipid. Replacing the His440 residue of P178 by alanine eliminated the pH-shift effect thereby showing that it is associated with deprotonation of this histidine residue. It was concluded that alkalinization-induced weakening of the electrostatic interactions between colicin and the membrane surface facilitates conformational changes required for the transition of membrane-bound colicin molecules to an active channel state. 相似文献
8.
The dissolution and formation of egg phosphatidylcholine (PC) vesicles by the detergent octyl glucoside were examined systematically by using resonance energy transfer between fluorescent lipid probes, turbidity, and gel filtration chromatography. Resonance energy transfer was exquisitely sensitive to the intermolecular distance when the lipids were in the lamellar phase and to the transitions leading to mixed micelles. Turbidity measurements provided information about the aggregation of lipid and detergent. Several reversible discrete transitions between states of the PC-octyl glucoside system were observed by both methods during dissolution and vesicle formation. These states could be described as a series of equilibrium structures that took the forms of vesicles, open lamellar sheets, and mixed micelles. As detergent was added to an aqueous suspension of vesicles, the octyl glucoside partitioned into the vesicles with a partition coefficient of 63. This was accompanied by leakage of small molecules and vesicle swelling until the mole fraction of detergent in the vesicles was just under 50% (detergent:lipid ratio of 1:1). Near this point, a transition was observed by an increase in turbidity and release of large molecules like inulin, consistent with the opening of vesicles. Both a turbidity maximum and a sharp increase in fluorescence were observed at a detergent to lipid mole ratio of 2.1:1. This was interpreted as the lower boundary of a region where both lamellar sheets and micelles are at equilibrium. At a detergent:lipid ratio of 3.0:1, another sharp change in resonance energy transfer and clarification of the suspension were observed, demarcating the upper boundary of this two-phase region. This latter transition is commonly referred to as solubilization.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
9.
The exothermic thermal denaturation transition of band 3, the anion transporter of the human erythrocyte membranes, has been studied by differential scanning calorimetry, in ghost membranes and in nonionic detergent micelles. In detergent micelles the transmembrane domain of band 3 gave an irreversible denaturation transition (C transition). However, no thermal transition was observed for the N-terminal cytoplasmic domain when band 3 was solubilised in detergent micelles. A reduction in enthalpy (190-300 kcal mol-1) with an accompanying decrease in thermal denaturation temperatures (48-60 degrees C) for the C transition was observed in detergent solubilised band 3 when compared with ghost membranes. Unlike ghost membranes, two thermal transitions for band 3 in detergent micelles were observed for the C transition when in the presence of excess covalent inhibitor, 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS), which derive from the thermal unfolding of a single protein with two different thermal stabilities; DIDS-stabilised (75 degrees C) and DIDS-insensitive (62 degrees C). A reduction in the denaturation temperature for the transmembrane domain of band 3 was observed when compared with intact band 3 although no significant differences was observed in the corresponding enthalpy values. This indicates some cooperativity of the two domains of band 3 in maintaining the transmembrane conformation. The results presented in this study show that detergents of intermediate micelle size (e.g. Triton X-100 and C12E8) are required for optimal thermal stability of band 3. 相似文献
10.
2H and 31P NMR techniques were used to study the effects on acyl chain order and lipid organization of the well-characterized pore-forming domain of colicin A (20-kDa thermolytic fragment of colicin A) upon insertion in model membrane systems derived from the Escherichia coli fatty acid auxotrophic strain K 1059, which was grown in the presence of [11,11-2H2]-labeled oleic acid. Addition of the protein to dispersions of the E. coli total lipid extract, in a 1/70 molar ratio of peptide to lipids, resulted in a large pH-dependent decrease in quadrupolar splitting of the 2H NMR spectra. The decrease of the quadrupolar splitting obtained at the various pH values was correlated with the pH dependence of the insertion of the protein in monolayer films using the same E. coli lipid extracts. The pK governing the perturbing effects on the order of the fatty acyl chains was around 5, in agreement with the values of the pH-dependent conformational changes of the pore-forming domain of colicin A required for membrane insertion as reported by van der Goot et al. [(1991) Nature 354, 408-410]. 31P NMR measurements show that the bilayer organization remains intact upon addition of the protein to dispersions of lipid extract. Surprisingly, 31P NMR measurements as a function of temperature indicate that the pore-forming domain of colicin A even stabilizes bilayer lipid structure at pH 4. Both the large effect of the protein on acyl chain order and its bilayer-stabilizing activity are indicative of a surface localization of the protein.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
11.
Equilibrium measurements of the binding of central nervous system myelin basic protein to sodium dodecyl sulphate, sodium deoxycholate and lysophosphatidylcholine have been obtained by gel permeation chromatography and dialysis. This protein associates with large amounts of each of these surfactants: the apparent saturation weight ratios (surfactant/protein) being 3.58 +/- 0.12 and 2.30 +/- 0.15 for dodecyl sulphate at ionic strengths 0.30 and 0.10, respectively 1.34 +/- 0.10 for deoxycholate (at 0.12 ionic strength) and 4.0 +/- 0.5 for lysophosphatidylcholine. Binding to the ionic surfactants increases markedly close to their critical micelle concentrations. Sedimentation analysis shows that at 0.30 ionic strenght in excess dodecyl sulphate the protein is monomeric. It becomes dimeric when the binding ratio falls below 1 at a free detergent concentration of approximately 0.25 mM: below this concentration much of the protein and deterent forms an insoluble complex. The amount of dodecyl sulphate bound at high concentrations and at both above-mentioned ionic strengths corresponds closely to that expected for interaction of a single poly-peptide with two micelles. Variability of deoxycholate micelle size on interaction with other molecules precludes a similar analysis for this surfactant. Association was observed only with single micelles of lysophosphatidylcholine. The results provide strong evidence for dual lipid-binding sites on basic protein and indicate that lipid bilayer cross-linking by this protein may be effected by single molecules. 相似文献
12.
Functional domains of colicin A 总被引:16,自引:3,他引:13
D. Baty M. Frenette R. Lioubès V. Geli S. P. Howard F. Pattus C. Lazdunski 《Molecular microbiology》1988,2(6):807-811
A large number of mutations which introduce deletions in colicin A have been constructed. The partially deleted colicin A proteins were purified and their activity in vivo (on sensitive cells) and in vitro (in planar lipid bilayers) was assayed. The receptor-binding properties of each protein were also analysed. From these results, we suggest that the NH2-terminal region of colicin A (residues 1 to 172) is involved in the translocation step through the outer membrane. The central region of colicin A (residues 173 to 336) contains the receptor-binding domain. The COOH-terminal domain (residues 389 to 592) carries the pore-forming activity. 相似文献
13.
On the nature of the structural change of the colicin E1 channel peptide necessary for its translocation-competent state 总被引:5,自引:0,他引:5
Acidic pH conditions required in vitro for membrane binding and activity of the channel-forming colicin E1 resulted in an increased susceptibility to proteases of the 178-residue thermolytic channel peptide, an increased accessibility to acrylamide of a fluorescence probe linked to cysteine-505 of the peptide, and an increased partition into nonionic detergent. The structural change in the peptide sensed by the fluorescence probe caused by a transition from pH 6.0 to 3.5 occurred in less than 1 s. The presence of low concentrations of detergents (0.001% SDS or 0.44% octyl beta-D-glucoside) or urea (0.2 M) at pH 6 or 4 also increased the susceptibility of the channel peptide to proteases. The increase in protease susceptibility and acrylamide accessibility at low pH, as well as partition of the peptide into nonionic detergent, suggested that acidic pH or the detergents might cause peptide unfolding. However, the hydrodynamic radius of the channel peptide at pH 6, 21-23 A, was not changed at pH 3.5 or by detergents or urea under conditions that increased the susceptibility of the peptide to protease. The activity of the channel peptide at pH 6 measured with liposomes and planar bilayers, which was a factor of 10(3)-10(4) smaller than that at pH 4, was increased by 2-4 orders of magnitude by 0.001% SDS or 0.44% octyl beta-D-glucoside, with an additional small increment of activity on planar bilayers caused by 0.01% SDS. A small increase in Stokes radius of the peptide in the presence of SDS could be detected that was approximately correlated with increased activity. 相似文献
14.
DNA regions encoding the various domains of a protein can be expressed as separate entities by inserting at appropriate sites a 'STOP-Shine-Dalgarno-sequence-ATG' cassette encoding a termination codon, a Shine-Dalgano sequence and an initiation codon within the structural gene. This technique has been used to obtain a 137-amino-acid-residue pore-forming protein designated DA70C comprising the final 136-amino-acid-residue COOH-terminal of colicin A preceded by an NH2-terminal methionine. Da70C was correctly expressed but poorly released to the extracellular medium. Its purification involved, as a final step, a partition in Triton X-114 thus demonstrating that hydrophobic regions are exposed in this protein. The ability of DA70C to form ion channels in planar lipid bilayers was investigated and pore properties were analyzed. The results indicate that helices 1-3 of the 204-amino-acid-residue colicin pore-forming domain (containing 10 alpha-helices) are not involved in ion conduction through the channel. However, they are important in maintaining the stability of the soluble state of the COOH-terminal domain. 相似文献
15.
A model approach is developed to study intermediate steps and transientstructures in a course of the membrane self-assembly. The approach isbased on investigation of mixed lipid/protein-detergent systems capable ofthe temperature induced transformation from a solubilized micellar stateto closed membrane vesicles. We performed a theoretical analysis ofself-assembling molecular structures formed in binary mixtures ofdimyristoylphosphatidylcholine (DMPC) and sodium cholate (NaC). Thetheoretical model is based on the Helfrich theory of curvature elasticity,which relates geometrical shapes of the structures to their free energy inthe Ginzburg-Landau approximation. The driving force for the shapetransformation is spontaneous curvature of amphiphilic aggregates which isnonlinearly dependent on the lipid/detergent composition. An analysis ofthe free energy in the regular solution approximation shows that theformation of mixed structures of different shapes (discoidal micelles,rod-like micelles, multilayer membrane structures and vesicles) ispossible in a certain range of detergent/lipid ratios. A transition fromthe flat discoidal micelles to the rod-like cylindrical micelles isinduced by curvature instabilities resulting from acyl chain melting andinsertion of detergent molecules into the lipid phase. Nonideal mixing ofthe NaC and DMPC molecules results in formation of nonideal cylindricalaggregates with elliptical cross section. Further dissolution of NaCmolecules in DMPC may be accompanied with a change of their orientation inthe lipid phase and leads to temperature-induced curvature instabilitiesin the highly curved cylindrical geometry. As a result the rod-likemicelles fuse into less curved bilayer structures which transformeventually to the unilamellar and multilamellar membrane vesicles. Thetheoretical analysis performed shows that a sequence of shapetransformations in the DMPC/NaC mixed systems is determined by thesynergism of four major factors: detergent/lipid ratio, temperature (acylchain melting), DMPC and NaC mixing, and reorientation of NaC molecules inmixed aggregates. 相似文献
16.
The effects of pH and temperature on the stability of interdomain interactions of colicin B have been studied by differential-scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The calorimetric properties were compared with those of the isolated pore-forming fragment. The unfolding profile of the full-length toxin is consistent with two endothermic transitions. Whereas peak A (T(m) = 55 degrees C) most likely corresponds to the receptor/translocation domain, peak B (T(m) = 59 degrees C) is associated with the pore-forming domain. By lowering the pH from 7 to 3.5, the transition temperature of peaks A and B are reduced by 25 and 18 degrees C, respectively, due to proton exchange upon denaturation. The isolated pore-forming fragment unfolds at much higher temperatures (T(m) = 65 degrees C) and is stable throughout a wide pH range, indicating that intramolecular interactions between the different colicin B domains result in a less stable protein conformation. In aqueous solution circular dichroism spectra have been used to estimate the content of helical secondary structure of colicin B ( approximately 40%) or its pore-forming fragment ( approximately 80%). Upon heating, the ellipticities at 222 nm strongly decrease at the transition temperature. In the presence of lipid vesicles the differential-scanning calorimetry profiles of the pore-forming fragment exhibit a low heat of transition multicomponent structure. The heat of transition of membrane-associated colicin B (T(m) = 54 degrees C at pH 3.5) is reduced and its secondary structure is conserved even at intermediate temperatures indicating incomplete unfolding due to strong protein-lipid interactions. 相似文献
17.
A spin-labeled pentadecapeptide was synthesized containing 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (TOAC) as the N-terminal amino acid and residues 253-266 (EYWSTFGNLHHISL) of the mass oncogene receptor, a membrane-bound protein from the G-protein coupled receptors family. According to predictions, this protein folds into seven transmembrane helices connected by three extra- and three intracellular loops, and the peptide encompasses part of the third extracellular loop and part of the seventh helix. Electron paramagnetic resonance (EPR) spectra of the spin-labeled peptide (TOAC-14) were obtained in aqueous solution as a function of pH and temperature, in a secondary structure-inducing solvent [trifluoroethanol (TFE)], and in the presence of detergent micelles and phospholipid bilayers. The charged and uncharged amino groups of TOAC and TOAC-14 yielded spectra with different isotropic hyperfine splittings (aN). The slow exchange between protonated and unprotonated forms in the EPR time scale gave rise to composite spectra weighted by the Henderson-Hasselbalch equation. Plots of aN vs pH allowed the determination of the amino group pK values (8.4 and 4.5, for TOAC and TOAC-14, respectively). A small change in aN centered at pH 6.5 was ascribed to the titration of the histidines. Values of calculated rotational correlation times were indicative of a pH-induced conformational change. A conformational change was also observed in TFE. TOAC-14 bound to micelles irrespective of peptide and detergent head group charge. In contrast, the peptide bound to phospholipid bilayers only when both carried opposite charges. The slow exchange (in the EPR time scale) between membrane-bound and free TOAC-14 allowed the calculation of the peptide's partition coefficient. The spectral line shapes were affected by aggregate size and degree of packing of the constituent molecules. It is proposed that pH, polarity, and lipid environment can affect the conformation of water-exposed regions of membrane-bound receptors, thereby playing a role in the mechanism of signal transduction. 相似文献
18.
Amphiphilic,glycophosphatidylinositol-specific phospholipase C (PI-PLC)-insensitive monomers and dimers of acetylcholinesterase 总被引:5,自引:0,他引:5
Suzanne Bon Terrone L. Rosenberry Jean Massoulié 《Cellular and molecular neurobiology》1991,11(1):157-172
1. In a recent study, we distinguished two classes of amphiphilic AChE3 dimers in Torpedo tissues: class I corresponds to glycolipid-anchored dimers and class II molecules are characterized by their lack of sensitivity to PI-PLC and PI-PLD, relatively small shift in sedimentation with detergent, and absence of aggregation without detergent. 2. In the present report, we analyze the amphiphlic or nonamphiphilic properties of globular AChE forms in T28 murine neural cells, rabbit muscle, and chicken muscle. The molecular forms were identified by sucrose gradient sedimentation in the presence and absence of detergent and analyzed by nondenaturing charge-shift electrophoresis. Some amphiphilic forms showed an abnormal electrophoretic migration in the absence of detergent, because of the retention of detergent micelles. 3. We show that the amphiphilic monomers (G1a) from these tissues, as well as the amphiphilic dimers (G2a) from chicken muscle, resemble the class II dimers of Torpedo AChE. We cannot exclude that these molecules possess a glycolipidic anchor but suggest that their hydrophobic domain may be of a different nature. We discuss their relationship with other cholinesterase molecular forms. 相似文献
19.
The possible involvement of topogenic export sequences within the colicin A polypeptide chain has been investigated. Different constructs have been made using various techniques to introduce deletions in the central and NH2-terminal regions of colicin A. Together, these deletions span the region from amino acid 15 to the end of the protein. None of these regions was found to be required for extracellular release or had any effect on the efficiency of this process. By inserting a termination codon, a Shine-Dalgarno sequence and an initiation codon into the gene for colicin A, the NH2-terminal and central plus COOH-terminal domains could be demonstrated to be released to the same extent when produced as separate polypeptides as when produced as linked ones. The introduction into the COOH-terminal domain of mutations promoting cytoplasmic aggregation had no effect on the secretion of the NH2-terminal polypeptide. These results demonstrated that no specific interaction between the NH2- and COOH-terminal regions of the colicin A polypeptide chain is involved in the release of colicin A. We are led to conclude that there is no topogenic export signal in the polypeptide chain of colicin A involved in the release mechanism. Thus the process is non-specific with respect to the colicin itself and depends solely on the expression of the colicin A lysis protein (Cavard et al., 1985, 1987). The expression of the protein causes the release of not only the colicin but also many other cellular proteins, including beta-lactamase, EF-Tu, and chloramphenicol acetyltransferase. 相似文献
20.
Rat lung membrane vasoactive intestinal peptide (VIP) receptors were covalently labeled with 125I-VIP, extracted in Triton X-100 and n-octyl-beta-D-glucopyranoside, and analyzed by gel filtration and sucrose density gradient sedimentation. The fractions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, and the identity of the 125I-VIP.receptor complex was demonstrated by its co-migration with the covalently labeled 55-kDa receptor unit identified previously. Furthermore, the radioactivity in the peak corresponding to the 125I-VIP.receptor complex was displaced in the presence of unlabeled VIP in a dose-dependent manner. The following hydrodynamic properties were determined for VIP receptors in each detergent solution: in Triton X-100, Stokes radius of 6.1 +/- 0.4 nm, sedimentation coefficient (S20,w) of 7.35 +/- 0.45 S, and partial specific volume (v) of 0.809 +/- 0.015 ml/g; in n-octyl-beta-D-glucopyranoside, Stokes radius of 5.6 +/- 0.00 nm, S20,w of 10.87 +/- 0.22 S, and partial specific volume of 0.783 +/- 0.020 ml/g. The apparent molecular weight of the 125I-VIP.receptor.detergent complex was calculated as 270,000 +/- 36,000 in Triton X-100 and 320,000 +/- 32,000 in n-octyl-beta-D-glucopyranoside. The amount of detergent bound to the receptor was estimated by using the two sets of hydrodynamic data and the significantly different partial specific volumes of the two detergents. Thus, the molecular weight of the receptor alone was calculated as 54,600 daltons, indicating that approximately 3.9 g of Triton X-100 and 4.9 g of n-octyl-beta-D-glucopyranoside were bound per g of receptor. This species contained the 55-kDa binding unit and appeared to be glycosylated as evidenced by its specific binding to wheat germ agglutinin-Sepharose. These results indicate that the rat lung VIP receptor is a glycoprotein with a single polypeptide chain of 55 kDa. The large amount of detergent bound suggests that the receptor is extensively embedded in the membrane. 相似文献