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1.
Studies on human pregnancy-associated plasma protein A. Purification by affinity chromatography and structural comparisons with alpha 2-macroglobulin. 总被引:1,自引:0,他引:1
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R G Sutcliffe B M Kukulska-Langlands J R Coggins J B Hunter C H Gore 《The Biochemical journal》1980,191(3):799-809
Pregnancy-associated plasma protein-A (PAPP-A) has been purified by a combination of methods including antibody-affinity chromatography. The resultant protein, obtained in 16% yield from maternal serum, appeared as a single major component on non-denaturing polyacrylamide and SDS/polyacrylamide gel electrophoresis. The protein showed a single component when analysed by isoelectric focusing under denaturing conditions in the presence and absence of reduction and had a pI of 4.34 and 4.42 respectively. These pI values were indistinguishable from those of alpha 2-macroglobulin (alpha 2M). The molecular weight of the PAPP-A polypeptide as shown by SDS/polyacrylamide-gel electrophoresis was 187000, with a minor component of mol.wt. 82500 that was attributed to proteolysis. Since native PAPP-A had a molecular weight on gel chromatography very similar to that of alpha 2M (620000--820000), it was concluded that PAPP-A was a homotetramer. In the absence of reduction, a high-molecular-weight (420000) protomer of PAPP-A was found. It was deduced that PAPP-A, like alpha 2M, is a dinner, whose protomers are composed of disulphide-linked polypeptide chains. It was found that the molecular weight of the PAPP-A polypeptide exceeded that of alpha 2M by 3.3%, but that the total carbohydrate content of PAPP-A exceeded that of alpha 2M by 10% and that its neutral carbohydrate content exceeded that of alpha 2M by between 7.4 and 9.0%. The significance of the estimated molecular weights of alpha 2M (181000) and its major tryptic fragments is discussed in the light of published values. A tryptic fragment alpha 2M (82500 mol.wt.) was apparently the same size as the major tryptic fragment of PAPP-A. 相似文献
2.
Comparative studies of pregnancy associated plasma protein-A and alpha 2-macroglobulin using metal chelate chromatography 总被引:1,自引:0,他引:1
Pregnancy Associated Plasma Protein-A (PAPP-A), shares many physicochemical and functional similarities with alpha-2-macroglobulin (alpha 2M). Comparative studies between these two proteins and various metal chelate Sepharoses have shown that both PAPP-A and alpha 2M bind to the copper and zinc, but not to the calcium, manganese or magnesium chelate gels. In addition to PAPP-A and alpha 2M, fibronectin and Pregnancy Zone Protein (PZP) were also found to bind to the copper and zinc chelate gels. Analysis of proteins bound to the zinc chelate Sepharose showed alpha 2M to have the highest affinity for the matrix, followed by PAPP-A and fibronectin, which coeluted, and then PZP. Following the zinc chelate chromatography minor qualitative changes were detected only in PAPP-A, but the eluted proteins retained in-vitro functional activity. By atomic absorption, PAPP-A, alpha 2M and fibronectin were found to contain zinc. 相似文献
3.
Characterization of trimming Man9-mannosidase from pig liver. Purification of a catalytically active fragment and evidence for the transmembrane nature of the intact 65 kDa enzyme. 总被引:2,自引:0,他引:2
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An alpha 1,2-mannosidase (Man9-mannosidase) involved in N-linked oligosaccharide processing has been purified about 16,000-fold from pig liver crude microsomes (microsomal fractions) by CM-Sepharose and DEAE-Sephacel chromatography, concanavalin A (Con A)-Sepharose chromatography and, as the key step of the procedure, affinity chromatography on immobilized N-5-carboxypentyl-l-deoxymannojirimycin (CP-dMM). On SDS/polyacrylamide-gel electrophoresis under reducing conditions, the isolated enzyme migrated as a single protein band with a molecular mass of 49 kDa. The enzyme does not bind Con A and is not susceptible to glycopeptidase F, indicating that it lacks N-linked oligosaccharides of the high-mannose or complex type. Purified Man9-mannosidase has a pH optimum close to 6.0 and requires bivalent cations for activity, with Ca2+ being most effective. The enzyme is inhibited strongly by basic sugar analogues of mannose such as 1-deoxymannojirimycin (dMM, Ki approximately 5 microM), N-methyl-dMM (Ki approximately 55 microM) and CP-dMM (Ki approximately 150 microM), whereas NN-dimethyl-dMM and the mannosidase II inhibitor swainsonine were hardly or not at all inhibitory. A homogeneous preparation of the 49 kDa enzyme cleaves specifically three of the four alpha 1,2-mannosidic linkages in the natural Man9-GlcNAc2 (M9) substrate. The relative rates by which the parent and intermediate structures are hydrolysed were found to be about 3:2:5 for M9, M8 and M7 respectively. The enzyme displays only marginal activity toward the remaining alpha 1,2-mannosidic linkages in the Man9-GlcNAc2 oligosaccharide (relative rate of M6 hydrolysis approximately 0.02) and is not active against nitrophenyl and methylumbelliferyl alpha-mannosides. This unique substrate specificity suggests that Man9-mannosidase processing differs from that catalysed by other trimming alpha 1,2-mannosidases hitherto reported. A polyclonal antibody raised against the denatured 49 kDa polypeptide not only recognizes a protein band of similar size in Western blots of crude microsomes, but also reacts strongly with a 65 kDa protein species. On trypsin treatment of detergent-solubilized microsomes, the 65 kDa protein is converted specifically into a stable 49 kDa fragment, indicating a precursor-product relationship between the two proteins. We conclude from this observation that the 65 kDa protein represents the intact form of Man9-mannosidase from which the 49 kDa enzyme which we have isolated has been generated, with retention of catalytic activity, by proteolysis during purification. Proteolytic studies with sealed microsomes suggest that the intact 65 kDa enzyme is a protein with a membrane-spanning domain, as well as a cytosolic polypeptide domain of size at least 3 kDa. 相似文献
4.
Three high molecular weight protease inhibitors of rat plasma. Isolation, characterization, and acute phase changes 总被引:1,自引:0,他引:1
K Lonberg-Holm D L Reed R C Roberts R R Hebert M C Hillman R M Kutney 《The Journal of biological chemistry》1987,262(1):438-445
Rat blood plasma contains three high molecular weight thiol ester-containing proteinase inhibitors, alpha 1-macroglobulin (alpha 1M), alpha 1-inhibitor III (alpha 1I3), and alpha 2-macroglobulin (alpha 2M). Rat serums have been analyzed using a two-dimensional gel electrophoretic technique which optimizes recovery of high molecular weight proteins. alpha 1M, and (alpha beta)4-tetramer in native solution, separated in the second sodium dodecyl sulfate-containing electrophoretic dimension as a disulfide-linked (alpha beta)2-dimer with an approximate Mr of 360 kDa. alpha 1I3 separated in the gels as a single 190-kDa polypeptide. It is also a monomer in native solution by ultracentrifugation criteria. Native rat alpha 2M is a tetramer, but it separates in the gels as a disulfide-linked dimer with an Mr of approximately 360 kDa. The kinetics of changes in concentration of these proteins during the induction of polyarthritis was also measured by quantitative immunoelectrophoresis. In rats with adjuvant-induced polyarthritis, the concentration of alpha 1I3 dramatically decreases and alpha 2M appears and continues to increase in a biphasic manner for 2 weeks. The alpha 1M concentration remains relatively constant. All three macroglobulins were purified utilizing modern rapid chromatographic techniques, and parallel comparisons of their native physicochemical properties were carried out. The N-terminal sequence of the alpha-chain of rat alpha 1M was also shown to share sequence homology with that of alpha 2M. In agreement, Esnard et al. (Esnard, F., Gutman, N., El Moujahed, A., and Gauthier, F. (1985) FEBS Lett. 182, 125-129) recently reported that alpha 1I3 also contains a thiol ester bond, as do alpha 1M and alpha 2M, since it reacts covalently with [14C]methylamine and is cleaved autolytically at 80 degrees C. We have examined negatively stained preparations of native, trypsin-treated, and methylamine-treated human alpha 2M, rat alpha 2M, and rat alpha 1M in the electron microscope. Trypsin appears to convert globular ring-shaped native molecules to rectangular box-like structures, in agreement with the conclusions of a recent report on human alpha 2M (Tapon-Bretaudiere, J., Bros, A., Couture-Tosi, E., and Delain, E. (1985) EMBO J. 4, 85-89). 相似文献
5.
Two-domain structure of the native and reactive centre cleaved forms of C1 inhibitor of human complement by neutron scattering 总被引:6,自引:0,他引:6
S J Perkins K F Smith S Amatayakul D Ashford T W Rademacher R A Dwek P J Lachmann R A Harrison 《Journal of molecular biology》1990,214(3):751-763
The C1 inhibitor component of human complement is a member of the serpin superfamily, and controls C1 activation. Carbohydrate analyses showed that there are seven O-linked oligosaccharides in C1 inhibitor. Together with six N-linked complex-type oligosaccharides, the carbohydrate content is therefore 26% by weight and the molecular weight (Mr) is calculated as 71,100. Neutron scattering gives an Mr of 76,000 (+/- 4000) and a matchpoint of 41.8 to 42.3% 2H2O, in agreement with this carbohydrate and amino acid composition. Guinier plots to determine the radius of gyration RG were biphasic. Neutron contrast variation of C1 inhibitor in H2O-2H2O mixtures gave an overall radius of gyration RG at infinite contrast of 4.85 nm, from analyses at low Q, and a cross-sectional RG of 1.43 nm. The reactive centre cleaved form of C1 inhibitor has the same Mr and structure as the native molecule. The length of C1 inhibitor, 16 to 19 nm, is far greater than that of the putative serpin domain. This is attributed to an elongated structure for the carbohydrate-rich 113-residue N-terminal domain. The radial inhomogeneity of scattering density, alpha, is large at 59 x 10(-5) from the RG data and 28 x 10(-5) from the cross-sectional analysis, and this is accounted for by the high oligosaccharide content of C1 inhibitor. The scattering data were modelled using small spheres. A two-domain structure of length 18 nm based on two distinct scattering densities accounted for all the contrast variation data. One domain is based on the crystal structure of alpha 1 antitrypsin (7 nm x 3 nm x 3 nm). The other corresponds to an extended heavily glycosylated N-terminal domain of length 15 nm, whose long axis is close to the longest axis of the serpin domain. Calculation of the sedimentation coefficient s0(20),w for C1 inhibitor using the hydrodynamic sphere approach showed that a two-domain head-and-tail structure with an Mr of 71,000 and longest axis of 16 to 19 nm successfully reproduced the s0(20),w of 3.7 S. Possible roles of the N-terminal domain in the function of C1 inhibitor are discussed. 相似文献
6.
G. A. Rivas J. A. Aznárez P. Usobiaga J. L. Saiz J. González-Rodríguez 《European biophysics journal : EBJ》1991,19(6):335-345
Human platelet plasma membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa) form a Ca(2+)-dependent heterodimer, the integrin GPIIb/IIIa, which serves as the receptor for fibrinogen and other adhesive proteins at the surface of activated platelets. Below the critical micellar concentration of Triton X100 (TtX), the three glycoproteins do not bind appreciably to TtX and form association products of large size. The size-exclusion chromatographic patterns of GPIIb, GPIIIa and GPIIb/IIIa have been obtained at 0.2% TtX, and the molecular properties of the association products and monomer fractions have been determined by analysis of the detergent bound to the glycoproteins, laser-light scattering, sedimentation velocity, and electron microscopy (TEM). The monomer of the GPIIb-TtX complex was identified by the molecular mass (M) of the glycoprotein moiety (125 +/- 15 kDa), the molecular size (9.5 +/- 1.5 nm x 11 +/- 1.5 nm) and globular shape observed by TEM. It has a molecular mass (M*) of 197 +/- 20 kDa, a sedimentation coefficient s degrees 20* of 5.8 +/- 0.1 S, a Stokes radius R s* of 6.8 +/- 0.4 nm, and a frictional ratio f*/fmin* of 1.7 +/- 0.14. The (GPIIb)n-TtX complexes are disulphide-bonded size-heterogeneous association products of GPIIb, tetramers being the smallest species found. GPIIIa has a greater propensity to self-associate than GPIIb, this tendency being lower below 1 mg GPIIIa/ml, 0.1 mM Ca2+, pH 9.0. The (GPIIIa)n-TtX complexes are noncovalent size-heterogeneous association products of GPIIIa, tetramers being the smallest form observed. The monomer of the GPIIIa-TtX complex was identified by the 103 +/- 15 kDa M determined for the glycoprotein moiety, and the 9 +/- 1.5 nm x 10 +/- 1.5 nm size and globular shape observed by TEM. It has a M* of 136 +/- 15 kDa, a s degrees 20* of 3.9 +/- 0.3 S, a Rs* of 6.4 +/- 0.5 nm, a f*/fmin* of 1.9 +/- 0.3, and, when stored at pH 7.4, has a certain tendency to form filamentous association products (20-70 nm x 2-5 nm), as observed by TEM. The GPIIb/IIIa-TtX complex in 0.2% TtX/0.1 mM Ca2+ elutes as a single monomeric fraction, as deduced from the 210 +/- 15 kDa M determined for its glycoprotein moiety and the 12 +/- 1.5 nm x 14 +/- 1.5 nm size of the globular forms observed by TEM.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
7.
M Hayakawa T Hori S Shibamoto M Tsujimoto N Oku F Ito 《Archives of biochemistry and biophysics》1991,286(2):323-329
Human placental membranes exhibited high-affinity receptors for tumor necrosis factor (TNF) (Kd = 5.6 x 10(-10) M) with a density of 1.2-1.7 x 10(10) sites/mg protein. The receptors were solubilized from these membranes with 1% Nonidet P-40, and the solubilized receptor was adsorbed to Con A-Sepharose and wheat germ agglutinin agarose columns, indicating that the TNF receptor derived from human placenta contains carbohydrate chains recognized by these lectins. TNF binding activity was eluted from a column of Sephacryl S-300 as a single peak of Mr 300 kDa. The solubilized receptor was further purified by TNF-Sepharose prepared by coupling of TNF to tresyl-activated Sepharose 4B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified sample resolved five major bands of Mr 90, 78, 41, 35, and 11 kDa, suggesting that these polypeptides constitute a multimeric complex with a molecular mass of 300 kDa, as observed in gel filtration study. Furthermore, the TNF-Sepharose-bound fraction demonstrated GTP gamma S binding and GTPase activity. Immunoblot analysis showed that the 41- and 35-kDa polypeptides were recognized by antisera against alpha subunits and beta subunit of GTP-binding proteins, respectively. These results suggest that the native TNF receptor couples to a guanine nucleotide-binding protein to form a large complex structure in human placental membranes. 相似文献
8.
A series of non-steroidal anti-inflammatory drugs (NSAIDs) and some of their metabolites were evaluated for their ability to inhibit the proteolytic activity of human granulocyte elastase (HGE) and cathepsin G (HGC-G). The enzyme activity was monitored using specific synthetic chromogenic substrates. The results obtained indicated that phenylbutazone, sulindac, piroxicam and gold sodium thiomalate significantly inhibited HGE (Ki less than 0.5 mM), while only sulindac, diflunisal and gold sodium thiomalate were effective inhibitors of HGC-G (Ki less than 0.4 mM). Studies on metabolites of some of the NSAID tested were found to be superior inhibitors of both HGE and HGC-G than the parent molecules. Moreover, of the 18 compounds examined, the major metabolite of sulindac, sulindac sulphide was the most potent inhibitor of HGE (Ki = 0.01 mM) and HGC-G (Ki = 0.15 mM). 相似文献
9.
Overgaard MT Sorensen ES Stachowiak D Boldt HB Kristensen L Sottrup-Jensen L Oxvig C 《The Journal of biological chemistry》2003,278(4):2106-2117
Pregnancy-associated plasma protein-A (PAPP-A) is a metzincin superfamily metalloproteinase responsible for cleavage of insulin-like growth factor-binding protein-4, thus causing release of bound insulin-like growth factor. PAPP-A is secreted as a dimer of 400 kDa but circulates in pregnancy as a disulfide-bound 500-kDa 2:2 complex with the proform of eosinophil major basic protein (pro-MBP), recently shown to function as a proteinase inhibitor of PAPP-A. Except for PAPP-A2, PAPP-A does not share global similarity with other proteins. Three lin-notch (LNR or LIN-12) modules and five complement control protein modules (also known as SCR modules) have been identified in PAPP-A by sequence similarity with other proteins, but no data are available that allow unambiguous prediction of disulfide bonds of these modules. To establish the connectivities of cysteine residues of the PAPP-A.pro-MBP complex, biochemical analyses of peptides derived from purified protein were performed. The PAPP-A subunit contains a total of 82 cysteine residues, of which 81 have been accounted for. The pro-MBP subunit contains 12 cysteine residues, of which 10 have been accounted for. Within the 2:2 complex, PAPP-A is dimerized by a single disulfide bond; pro-MBP is dimerized by two disulfides, and each PAPP-A subunit is connected to a pro-MBP subunit by two disulfide bonds. All other disulfides are intrachain bridges. We also show that of 13 potential sites for N-linked carbohydrate substitution of the PAPP-A subunit, 11 are occupied. The large number of disulfide bonds of the PAPP-A.pro-MBP complex imposes many restraints on polypeptide folding, and knowledge of the disulfide pattern of PAPP-A will facilitate structural studies based on recombinant expression of individual, putative PAPP-A domains. Furthermore, it will allow rational experimental design of functional studies aimed at understanding the formation of the PAPP-A.pro-MBP complex, as well as the inhibitory mechanism of pro-MBP. 相似文献
10.
Three guinea pig testicular, low-molecular-weight, acid-stable inhibitors specific for trypsin-like proteinases were isolated, purified, and characterized. The procedure comprised acid extraction of testicular acetone powder, pH precipitation of the extract, gel filtration of the supernatant on Sephadex G-100 and G-50, ion-exchange chromatography on SP-Sephadex, followed by QAE-Sephadex. Final purification was by rechromatography on Sephadex G-50 superfine gel. The three proteinase inhibitors were labeled A, B, and Cnb, the latter to denote nonbinding of Cnb to the QAE-Sephadex. Components A and Cnb showed competitive, whereas B showed noncompetitive, inhibition against trypsin. All three inhibitors were active against trypsin but were ineffective against chymotrypsin. The inhibition constants, Ki, were obtained using trypsin-catalyzed hydrolysis of the N-benzyloxycarbonyl-L-arginyl amide of 7-amino-4-trifluoro-methylcoumarin (CBZ-Arg-AFC) at pH 8.0. The values were calculated to be, for A, 1.5 x 10(-8) M; for B, 1.5 x 10(-8) M; and, for Cnb, 2.2 x 10(-7) M. The Ki values calculated from inhibition of trypsin-catalyzed hydrolysis of the active site titrant 4-methylumbelliferyl-p-guanidinobenzoate (MUGB) using Easson-Stedman plots were, for A, 7.7 x 10(-9) M; for B, 6.7 x 10(-9) M; and, for Cnb, 1.4 x 10(-7) M. The Mrs as determined by active site titration with MUGB were A, 11.2 kDa; B, 10.5 kDa; Cnb, 17.0 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave Mr values for A of 11 kDa, for B of 4 kDa, and for Cnb of 19 kDa. The discrepancy in Mr values for B indicates that it may function as a dimer or trimer in the active state. 相似文献
11.
Inhibitors of trypsin and alpha-chymotrypsin with Mr of about 7000 Da and isoelectric points of greater than 10 and 9.9, respectively, were isolated from the venom of the common viper Vipera berus berus, using gel filtration and ion exchange chromatography. The inhibitor I prefers alpha-chymotrypsin (Ki = 4.6 X 10(-10) M) for the formation of an enzymeinhibitor complex at a molar ratio of 1:1. The inhibitor II prefers trypsin (Ki = 6.7 X 10(-11) M), forms an EI-complex at a molar ratio of 1:2, but also inhibits alpha-chymotrypsin (Ki = 1.4 X 10(-9) M) and hog pancreatic kallikrein (Ki = 1.6 X 10(-8) M). The inhibitor II contains no valine or methionine. 相似文献
12.
Polydisulfides of urea (PDSU), thiourea (PDSTU), biuret (PDSB), and gallic acid (PDSG) and their monomer analogues (urea, biuret, and gallic acid) inhibited (in a competitive manner) tetramethylbenzidine (TMB) peroxidation catalyzed by ferritin in 0.1 M acetate buffer, pH 4.2, containing 10% dimethylformamide. Their efficiency characterized in terms of inhibition constants, Ki, increased in the following order PDSU < PDSB approximately PDSTU < PDSG. This order is determined by the reactivity of monomers with respect to HO* radicals which are the main oxidizing agents in the system ferritin--H2O2. Polydisulfide antioxidants exhibit the intramolecular synergism of the inhibiting action (non-additivity of antiradical activity relative to their monomers) that was quantitatively characterized by alpha = (Ki)pol/(Ki)mon x n, where n is the number of monomers in the polymeric inhibitors. The alpha values increased from 1.5 up to 5.18 in the following order: PDSG < PDSU < PDSB. Significantly higher inhibiting efficiency of polydisulfide antioxidants as compared to monomer forms and synergism of the inhibitory action offer promising opportunities of their use as quenchers of free radical processes in biochemical systems. 相似文献
13.
Kunitz-type protease inhibitor found in rat mast cells. Purification, properties, and amino acid sequence 总被引:2,自引:0,他引:2
A low molecular weight serine protease inhibitor, named trypstatin, was purified from rat peritoneal mast cells. It is a single polypeptide with 61 amino acid residues and an Mr of 6610. Trypstatin markedly inhibits blood coagulation factor Xa (Ki = 1.2 x 10(-10) M) and tryptase (Ki = 3.6 x 10(-10) M) from rat mast cells, which have activities that convert prothrombin to thrombin. It also inhibits porcine pancreatic trypsin (Ki = 1.4 x 10(-8) M) and chymase (Ki = 2.4 x 10(-8) M) from rat mast cells, but not papain, alpha-thrombin, or porcine pancreatic elastase. Trypstatin forms a complex in a molar ratio of 1:1 with trypsin and one subunit of tryptase. The complete amino acid sequence of this inhibitor was determined and compared with those of Kunitz-type inhibitors. Trypstatin has a high degree of sequence homology with human and bovine inter-alpha-trypsin inhibitors, A4(751) Alzheimer's disease amyloid protein precursor, and basic pancreatic trypsin inhibitor. However, unlike other known Kunitz-type protease inhibitors, it inhibits factor Xa most strongly. 相似文献
14.
W Y Almawi P J Dolphin R L Thies W R McMaster J G Levy B L Pope 《Journal of immunology (Baltimore, Md. : 1950)》1988,141(8):2529-2535
The lymphokine suppressor cell-inducing factor (SIF), obtained from 15 liters of serum-free culture supernatants of the natural suppressor cell line, M1-A5, has been purified to apparent homogeneity by a combination of gel filtration, ion exchange chromatography, and reverse-phase-HPLC. Purity of SIF was assessed by the migration of the factor as a single band on SDS-PAGE, and the elution from reverse-phase-HPLC column as a single and sharp peak. SIF activity was retained after both procedures. Two protein factors with SIF activity were isolated from M1-A5 culture supernatants. The first protein factor (SIF alpha) had a Mr of 43 kDa, and the second protein factor (SIF beta) had a Mr of 6 kDa. Final purification of SIF alpha yielded 5 micrograms protein with specific activity of 4 x 10(6) U/mg protein. Final purification of SIF beta yielded 40 micrograms protein with specific activity of 7.5 x 10(7) U/mg protein. The relationship between SIF alpha and SIF beta, as well as the relationship with other suppressor factors, will be addressed. 相似文献
15.
Baculovirus-directed expression of the human insulin receptor and an insulin-binding ectodomain 总被引:4,自引:0,他引:4
J I Paul J Tavaré R M Denton D F Steiner 《The Journal of biological chemistry》1990,265(22):13074-13083
In this report we describe the use of the baculovirus expression system to overproduce the human insulin holoreceptor (HIR) and a truncated, secretory version of the HIR cDNA (HIRsec) consisting of the alpha subunit and the extracellular portion of the beta subunit (beta'). Sf9 cells infected with the full-length HIR viruses synthesize recombinant HIR (rHIR) with an insulin-binding alpha subunit of apparent Mr = 110,000 and a beta subunit of apparent Mr = 80,000. Uncleaved alpha beta proreceptor accumulates in infected cells. Both of these forms assemble into higher order disulfide-linked dimers or heterotetramers of apparent Mr greater than 350,000. Insulin-binding activity in cells infected with rHIR viruses is present predominantly on the extracellular aspect of the plasma membrane (greater than 80%). Insulin binding to the full-length rHIR occurs with typical complex kinetics with Kd1 = 0.5-1 x 10(-9) M and Kd2 = 10(-7) M and receptors are present in large amounts in infected cells (1 x 10(6) receptors/cell; 1-2 mg HIR/10(9) cells). The full-length rHIR undergoes insulin-dependent autophosphorylation; half-maximal activation of beta subunit autophosphorylation occurs at 1-2 x 10(-8) M. The alpha beta proreceptor also becomes phosphorylated in vitro. Analysis of tryptic phosphopeptides derived from in vitro autophosphorylated beta subunit and alpha beta proreceptor reveals a pattern of phosphorylation that is indistinguishable from that of authentic placental HIR. Sf9 cells infected with rHIRsec viruses synthesize and secrete an (alpha beta')2 heterotetrameric complex having an insulin-binding alpha subunit of apparent Mr = 110,000 and a truncated beta' subunit of apparent Mr = 45,000 that lacks kinase activity. The rHIRsec complex purified from the conditioned medium of infected cells binds insulin with high affinity (Kd = 10(-9) M). 相似文献
16.
Alpha 2-macroglobulin used to isolate intracellular endopeptidases from mammalian cells in culture. 总被引:1,自引:0,他引:1
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Extracts of cell cultures labelled with [3H]leucine were incubated with human alpha 2-macroglobulin (alpha 2M), a plasma proteinase inhibitor. The proteinase-alpha 2M complexes were then precipitated with immobilized monoclonal antibodies to alpha 2M and analysed by SDS/polyacrylamide-gel electrophoresis. Parallel experiments were done with methylamine-inactivated alpha 2M to check for unspecific binding of cell proteins to alpha 2M. Several 3H-labelled cell proteins bound to active, but not to inactivated, alpha 2M. Such proteins are likely to be proteinases. Putative endopeptidases of subunit Mr 112000, 78,000, 53,000, and in some experiments 88,000 and 16,000, were trapped by alpha 2M in supernatant fractions from IMR90 human fibroblasts, EBTr bovine fibroblasts and HeLa human carcinoma cells. No additional proteins were trapped in the presence of ATP. The Mr-78,000 endopeptidase was identified as calpain II by immunoblotting. At pH 5.3 putative endopeptidases of subunit Mr 80,000, 53,000 and 28,000-32,000 were trapped from IMR90-fibroblast extracts. Immunoblotting showed that both cathepsin B and cathepsin D were present in the Mr-28,000-32,000 electrophoretic bands. The use of alpha 2M and immobilized antibody to alpha 2M thus allows a rapid enrichment of endopeptidases from cell extracts. Some potentials and limitations of the method are discussed. 相似文献
17.
Recombinant eglin c is a potent reversible inhibitor of human pancreatic elastase. At pH 7.4 and 25 degrees C, kass. = 7.3 x 10(5) M-1.s-1, kdiss. = 2.7 x 10(-4) s-1 and Ki = 3.7 x 10(-10) M. Stopped-flow kinetic indicate that the formation of the stable enzyme-inhibitor complex is not preceded by a fast pre-equilibrium complex or that the latter has a dissociation constant greater than 0.3 microM. The elastase-eglin c complex is much less stable at pH 5.0 and 25 degrees C, where kdiss. = 1.1 x 10(-2) s-1 and Ki = 7.3 x 10(-8) M. At pH 7.4 the activation energy for kass. is 43.9 kJ.mol-1 (10.5 kcal.mol-1). The kass. increases between pH 5.0 and 8.0 and remains essentially constant up to pH 9.0. This pH-dependence could not be described by a simple ionization curve. Both alpha 2-macroglobulin and alpha 1-proteinase inhibitor are able to dissociate the elastase-eglin c complex, as evidenced by measurement of the enzymic activity of alpha 2-macroglobulin-bound elastase or by polyacrylamide-gel electrophoresis of mixtures of alpha 1-proteinase inhibitor and elastase-eglin c complex. The rough estimate of kdiss. obtained with the alpha 2-macroglobulin dissociation experiment (1.6 x 10(-4) s-1) was of the same order of magnitude as the constant measured with the progress curve method. Eglin c strongly inhibits the solubilization of human aorta elastin by human pancreatic elastase. The extent of inhibition is the same whether elastase is added to a suspension of elastin and eglin c or whether elastase is preincubated with elastin for 3 min before addition of eglin c. However, the efficiency of the inhibitor sharply decreases if elastase is reacted with elastin for more prolonged periods. 相似文献
18.
The subunit stoichiometry of a large, multisubunit protein can be determined from the molar amino acid compositions (i amino acids) of the protein and its subunits. The number of copies of the subunits (1, 2, ... j) is calculated by solving all possible combinations of simultaneous equations in j unknowns (i!/j!(i - j)!). Calculations carried out using the published amino acid compositions determined by analysis and the compositions calculated from the sequences for two proteins of known stoichiometry provided the following results: Escherichia coli aspartate transcarbamoylase (R6C6, Mr = 307.5 kDa), R = 5.6 to 6.6 and C = 5.8 to 6.3, and spinach ribulose-bisphosphate carboxylase (L8S8, Mr = 535 kDa), L = 7.3 to 9.1 and S = 5.6 to 10.6. Calculations were also carried out with the amino acid compositions of two much larger proteins, the E. coli pyruvate dehydrogenase complex, Mr = 5280 kDa, subunits E1 (99.5 kDa), E2 (66 kDa), and E3 (50.6 kDa), and the extracellular hemoglobin of Lumbricus terrestris, Mr = 3760 kDa, subunits M (17 kDa), D1 (31 kDa), D2 (37 kDa), and T (51 kDa); the results for PDHase were E1 = 20 to 24, E2 = 18 to 31, E3 = 21 to 33 and those for Lumbricus hemoglobin were M = 34 to 46, D1 = 13 to 19, D2 = 13 to 18, and T = 34 to 36. Although the sample standard deviations of the mean values are generally high, the proposed method works surprisingly well for the two smaller proteins and provides physically reasonable results for the two larger proteins. 相似文献
19.
Covalent crosslinking analysis of angiotensin receptors on differentiated NG108-15 cells 总被引:1,自引:0,他引:1
M D Carrithers K A Koide V K Raman S Masuda J A Weyhenmeyer 《Biochemical and biophysical research communications》1990,170(3):1096-1101
Neuroblastoma x glioma hybrid cells (NG108-15) were used as a model system to characterize neuronal-glial type angiotensin (ANG) receptors by covalent crosslinking analysis. After differentiation with 1.5% DMSO and 0.5% fetal bovine serum for four to five days, saturation analysis revealed a single high affinity site with a Kd = 1.35 +/- 0.42 nM and a Bmax = 468 +/- 106 fmol/mg protein. Using the homobifunctional crosslinking reagent bis(sulfosuccinimidyl) suberate (BS3), a site with an estimated Mr of 78 kDa was specifically labeled with 125I-ANG II as determined by SDS-polyacrylamide gel electrophoresis. Both ANG II and ANG III (10(-6) M) inhibited specific labeling. The Ki for ANG III binding was similar by both pharmacologic (Ki = 3.33 +/- 0.98 nM) and gel densitometric (Ki = 2.65 +/- 0.32 nM) analyses. We conclude that the 78 kDa protein represents a high affinity ANG binding site with similar affinities for both ANG II and ANG III. 相似文献
20.
Erythrocyte Mr 28,000 transmembrane protein exists as a multisubunit oligomer similar to channel proteins 总被引:33,自引:0,他引:33
A novel Mr 28,000 erythrocyte transmembrane protein was recently purified and found to exist in two forms, "28kDa" and "gly28kDa," the latter containing N-linked carbohydrate (Denker, B. M., Smith, B. L., Kuhajda, F. P., and Agre, P. (1988) J. Biol. Chem. 263, 15634-15642). Although 28kDa protein resembles the Rh polypeptides biochemically, structural homologies were not identified by immunoblot or two-dimensional iodopeptide maps. The NH2-terminal amino acid sequence for the first 35 residues of purified 28kDa protein is 37% identical to the 26-kDa major intrinsic protein of lens (Gorin, M. B., Yancey, S. B., Cline, J., Revel, J.-P., and Horwitz, J. Cell 39, 49-59). Antisera to a synthetic peptide corresponding to the NH2-terminus of 28kDa protein gave a single reaction of molecular mass 28kDa on immunoblots of erythrocyte membranes. Selective digestions of intact erythrocytes and inside-out membrane vesicles with carboxypeptidase Y indicated the existence of a 5-kDa COOH-terminal cytoplasmic domain. Multiple studies indicated that 28kDa and gly28kDa proteins exist together as a multisubunit oligomer: 1) similar partial solubilizations in Triton X-100; 2) co-purification during ion exchange and lectin affinity chromatography; 3) cross-linking in low concentrations of glutaraldehyde; and 4) physical analyses of purified proteins and solubilized membranes in 1% (v/v) Triton X-100 showed 28kDa and gly28kDa proteins behave as a large single unit with Stokes radius of 61 A and sedimentation coefficient of 5.7 S. These studies indicate that the 28kDa and gly28kDa proteins are distinct from the Rh polypeptides and exist as a multisubunit oligomer. The 28kDa protein has NH2-terminal amino acid sequence homology and membrane organization similar to major intrinsic protein and other members of a newly recognized family of transmembrane channel proteins. 相似文献