共查询到20条相似文献,搜索用时 31 毫秒
1.
The structure of crotapotin, a protein extracted, from the venom of the Crotalus durissus terrificus, in solution at pH = 1.5, was studied by SAXS. The experimental results yield structural parameter values of the molecular radius of gyration Rg = 13.6 A, volume v = 16.2 x 10(3) A3 A3 and maximal dimension Dmax = 46 A. The distance distribution function deduced from the scattering measurements is consistent with an overall molecular shape of an oblate ellipsoid of revolution with asymmetry parameter v = 0.45. 相似文献
2.
Single bilayer vesicles of dimyristoylphosphatidylcholine have been investigated by small-angle X-ray scattering at 28 degrees C. The results indicate that these vesicles are hollow spherical shell structures with an outer radius of approximately 12 nm and a molecular weight of (3.2 +/- 0.5) X 10(6). The shell was found to be 4.4 +/- 0.2 nm thick with a cross-sectional electron-density profile characteristic for a single phospholipid bilayer. Upon interaction of these vesicles with apolipoprotein C-III from human very low density lipoproteins at a protein/lipid ratio greater than 0.08 (g/g), a complex containing 0.25 g of protein/g of lipid, with molecular weight of (3.9 +/- 0.4) X 10(5), is formed. The shape analysis indicates a highly asymmetric particle with an internal partition of low and high electron density resembling that produced by a bilayer structure. Model calculations and curve-fitting procedures show good agreement between the experimental scattering curve and that computed for an oblate ellipsoidal structure with dimensions of 17 X 17 X 5 nm and a 1 nm thick shell of high electron density surrounding the core of low electron density. 相似文献
3.
Geometry of normal mammalian platelets by quantitative microscopic studies. 总被引:7,自引:3,他引:4
下载免费PDF全文
![点击此处可从《Biophysical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The shape distributions of normal and hardened human and rabbit erythrocytes and platelets were obtained for edge-on orientations of a few hundred freely rotating cells from analyses of microphotographs obtained similarly as by Ponder(1930, Q. J. Exp. Physiol. 20:29) by phase-contrast microscopy at 800 X magnification. Major average diameters (d) and thicknesses (t) were estimated for both normal and hardened cells, and were used to calculate an average geometric axis ratio, rp = t/d, which increases to unity as cells become more spherical. Our fixation procedure did not alter these shape parameters: rp was unchanged for erythrocytes, with d and t values similar to those reported by Ponder (1930); platelets had d X t = 3.6 +/- 0.7 mum X 0.9 +/- 0.3 mum and 3.1 +/- 0.4 mum X 0.6 +/- 0.3 mum, respectively, for human and rabbit cells, with rp = 0.26 and 0.20, respectively. Agreement in rp was found with data obtained by a novel rheo-optical method which allows for a direct statistical averaging for large populations (greater than 100 X 10(3) cells). Histograms and linear correlation studies were made of the above three parameters (d,t,rp), as well as volume (V), total surface area are (S), and sphericity index (S.I.) calculated for both "prolate ellipsoid" and "disc with rounded edges" models. Results indicate very high linear correlations between rp - t, rp - S. I., and d -S, with high correlations for t - V,d -V and S. Data are in agreement with the few reports in the literature determined by other methods, with the best model for platelets appearing to be an oblate spheroid. 相似文献
4.
Molecular characterisation of soybean polysaccharides: an approach by size exclusion chromatography, dynamic and static light scattering methods 总被引:2,自引:0,他引:2
Water soluble polysaccharides from soybean (SSPS) have a pectin-like structure and are used as stabilisers in acidified beverages. Physicochemical properties such as structure, molecular weight and shape or conformation are primary factors controlling their functional properties. Two soybean polysaccharides, a native SSPS and a modified SSPS treated with beta-(1-->4)-D-galactosidase (GPase/SSPS) were studied by dynamic and static light scattering (DLS, SLS) and size exclusion chromatography (SEC). Consecutive filtrations using a range of membrane pore size removed a small fraction of macromolecular aggregates from dilute polysaccharide solutions with relatively little effect on the major component molecules as monitored by DLS and SEC measurements. Access to aggregate-free dilute solutions of SSPS and GPase/SSPS allowed the direct measurement of molecular characteristics. SLS results showed that SSPS had a weight average molecular weight of (645+/-11)x 10(3)g/mol and a radius of gyration, Rg, of (23.5+/-2.8)nm. By comparing R(g) with the hydrodynamic radius, Rh (21.1+/-0.5 nm) obtained from DLS, the structural parameter rho (Rg/Rh) was found to be 1.1, suggesting that SSPS has an overall globular shape due to a highly branched structure. The modified SSPS had a significantly lower molecular weight (287+/-18)x 10(3)g/mol but a similar radius of gyration (23.2+/-1.7 nm). The structure parameter rho of GPase/SSPS was higher (rho=1.3) because of a smaller hydrodynamic radius (17.7+/-1.8 nm). This suggests that GPase/SSPS has a much less branched structure yet still differs significantly from a linear random coil conformation (rho=1.7-2.0). The results derived from SLS and DLS are in agreement with the conclusions obtained from a chemical analysis where the reduction of molecular weight of GPase/SSPS was caused by the cleavage of galactan side chains. 相似文献
5.
D W Goddette E C Uberbacher G J Bunick C Frieden 《The Journal of biological chemistry》1986,261(6):2605-2609
Small angle neutron scattering has been used to study the dimensions of G-actin and the formation of low molecular weight actin oligomers under conditions where rapid polymerization does not take place. In the presence of 200 microM Ca2+, actin in solution consists of a single component with a radius of gyration (Rg) of 19.9 +/- 0.4 A, consistent with the known molecular dimensions of the G-actin molecule. In the presence of 50 microM Mg2+, however, formation of an actin species with a larger Rg occurs over a 4-h period. Multicomponent fits were tried and the data were best fit assuming two components, the monomer and a species with an Rg of 29 +/- 1 A. This latter value is consistent with the dimensions expected for certain actin dimers. The apparent dissociation constant for dimer formation is approximately 150 microM with forward and reverse rate constants of 6.0 X 10(-7) microM-1 s-1 and 8.8 X 10(-5) s-1, respectively. Kinetic fluorescence experiments show that the dimer formed in the presence of low levels of Mg2+ is a nonproductive complex which does not participate in the polymerization process. However, the addition of cytochalasin D to actin in the presence of 50 microM Mg2+ rapidly induces the formation of dimers, presumably related to cytochalasin's ability to nucleate actin polymerization. 相似文献
6.
Expression of the transferrin receptor gene during the process of mononuclear phagocyte maturation 总被引:7,自引:0,他引:7
T Hirata P B Bitterman J F Mornex R G Crystal 《Journal of immunology (Baltimore, Md. : 1950)》1986,136(4):1339-1345
The expression of transferrin receptors by blood monocytes, human alveolar macrophages, and in vitro matured macrophages was evaluated by immunofluorescence, radioligand binding, and Northern analysis, using the monoclonal anti-human transferrin receptor antibody OKT9, [125I]-labeled human transferrin and a [32P]-labeled human transferrin receptor cDNA probe, respectively. By immunofluorescence, the majority of alveolar macrophages expressed transferrin receptors (86 +/- 3%). The radioligand binding assay demonstrated the affinity constant (Ka) of the alveolar macrophage transferrin receptor was 4.4 +/- 0.7 X 10(8) M-1, and the number of receptors per cell was 4.4 +/- 1.2 X 10(4). In marked contrast, transferrin receptors were not present on the surface or in the cytoplasm of blood monocytes, the precursors of the alveolar macrophages. However, when monocytes were cultured in vitro and allowed to mature, greater than 80% expressed transferrin receptors by day 6, and the receptors could be detected by day 3. Consistent with these observations, a transferrin receptor mRNA with a molecular size of 4.9 kb was demonstrated in alveolar macrophages and in vitro matured macrophages but not in blood monocytes. Thus, although blood monocytes do not express the transferrin receptor gene, it is expressed by mature macrophages, an event that probably occurs relatively early in the process of monocyte differentiation to macrophages. 相似文献
7.
Small angle neutron scattering (SANS) results revealed that contrary to most reports C9 is not a globular protein. Its radius of gyration (Rg) at pH 8 and an ionic strength of 0.5 is 32.2 +/- 1.4 A increasing to 35 A at physiologic ionic strength. In contrast, C8, which has a 2.2-fold larger mass, has a similar Rg value [34.6 +/- 1.6 A]. Calibration plots of Rg vs. M(r) indicate that native C8 is a spherical protein whereas native C9 is elongated. From previous reports it was known that native C8 and C9 associate in solutions of low ionic strength. SANS results confirmed this observation but also demonstrated that C8-C9 heterodimers are already formed at physiologic ionic strength. The dimeric complex is globular [Rg = 40 +/- 0.8 A] indicating that the proteins associate side-by-side rather than end-to-end. In contrast, in presence of the drug Suramin, a potent inhibitor of the assembly of the C5b-9 complex, C9 forms a complex with twice the molecular mass that is still elongated (Rg = 48.8 +/- 0.8 A), suggesting that in this case the protein dimerizes end-to-end via a bridging Suramin molecule. 相似文献
8.
9.
An analysis by low-angle neutron scattering of the structure of the acetylcholine receptor from Torpedo californica in detergent solution.
下载免费PDF全文
![点击此处可从《Biophysical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The acetylcholine receptor from the electric tissue of Torpedo californica is a large, integral membrane protein containing four different types of polypeptide chains. The structure of the purified receptor in detergent solution has previously been investigated by sedimentation analysis and gel filtration. Sedimentation analysis yielded a molecular weight of 250,000 for the protein moiety of the receptor monomer-detergent complex; hydrodynamic characteristics such as the Stokes radius, however, refer to the receptor-detergent complex. In this paper we report the results of our use of low-angle neutron scattering to investigate the shape of the receptor-detergent (Triton X-100 from Rohm & Haas Co., Philadelphia, Pa.) complex and separately of its protein and detergent moieties. By adjustment of the neutron-scattering density of the solvent with D2O to match that of one or the other of the moieties, its contribution to the scattering can be nearly, if not completely, eliminated. Neutron scattering from Triton X-100 micelles established that this detergent is contrast matched in approximately 18% D2O. Scattering measurements on the receptor-detergent complex in this solvent yielded a radius of gyration of the acetylcholine receptor monomer of 46 +/- 1A. The radius of gyration and molecular volume (305,000 A3) of the receptor are inconsistent with a compact spherical shape. These parameters are consistent with, for example, a prolate cylinder of dimensions (length x diameter) approximately 150 x approximately 50 A or an oblate cylinder, approximately 25 x approximately 130 A. More complex shapes are possible and in fact seem to be required to reconcile the present results with previous electron microscopic and x-ray analyses of receptor in membrane and with considerations of the function of the receptor in controlling ion permeability. The neutron-scattering data yield, in addition, an independent determination of the molecular weight of the receptor protein (240,000 +/- 40,000), the extent of Triton X-100 binding in the complex (approximately 0.4 g/g protein), and from the extended scattering curve, an approximation to the shape of the receptor-Triton X-100 complex, namely an oblate ellipsoid of axial ratio 1:4. 相似文献
10.
Staubli M.; Stauble U. P.; Waber U.; Straub P. W. 《Journal of applied physiology》1985,58(4):1170-1175
In a controlled study of 11 male volunteers the following changes (means +/- SD) were observed in venous blood during (D) and 75 min after (A) a period of 20 min of voluntary hyperventilation in comparison with before (B) hyperventilation (P values referring to the difference between D and B) erythrocyte count 5.18 +/- 0.17 X 10(6) (B), 5.70 +/- 0.21 X 10(6) (D) (P less than 0.001), and 5.18 +/- 0.16 X 10(6)/microliter (A); hemoglobin 15.7 +/- 0.6 (B), 17.2 +/- 0.7 (D) (P less than 0.001), and 15.8 +/- 0.6 g/dl (A); centrifuged hematocrit 46.6 +/- 1.0 (B), 50.4 +/- 1.7 (D) (P less than 0.001), and 47.0 +/- 1.8% (A). The platelets increased from 159 +/- 30 X 10(3) (B) to 205 +/- 40 X 10(3) (D) (P less than 0.001) and returned to 157 +/- 26 X 10(3)/microliter (A). The leukocytes (WBC) were 4,210 +/- 630 (B), 6,220 +/- 1,660 (D) (P less than 0.001), and 6,190 +/- 1,870/microliter (A) (P less than 0.002, as compared with B). The rise of WBC during hyperventilation was mainly due to a 83% increase of lymphocytes, whereas a 93% increase of neutrophil leukocytes accounted for the increased WBC 75 min posthyperventilation. The increase of the ratio of band forms to segmented neutrophils from 9 (B) to 19% (A) (P less than 0.01) indicates that band forms were released from the bone marrow. The results show that WBC and platelets can be mobilized by hyperventilation by as yet unidentified mechanisms. 相似文献
11.
Morphometric analysis of cell types in the ovine corpus luteum throughout the estrous cycle 总被引:5,自引:0,他引:5
C E Farin C L Moeller H R Sawyer F Gamboni G D Niswender 《Biology of reproduction》1986,35(5):1299-1308
The cellular composition of ovine corpora lutea obtained during the early (Day 4), mid (Days 8 and 12), and late (Day 16) stages of the estrous cycle was determined by morphometric analysis. Individual corpora lutea were collected via midventral laparotomy from a total of 19 ewes. A center slice from each corpus luteum was processed for electron microscopy and subsequent morphometric analysis of the numbers and sizes of steroidogenic and nonsteroidogenic cells. Luteal weight progressively increased throughout the estrous cycle (p less than 0.05). Corpora lutea collected on Day 16 were assigned to one of two subgroups on the basis of gross appearance and weight: nonregressed (NR, 542 +/- 25 mg) or regressed (R, 260 +/- 2 mg). There were no significant changes in the proportion of the corpus luteum occupied by small luteal cells (19 +/- 2%) or large luteal cells (36 +/- 1%) throughout the estrous cycle. The total number of steroidogenic cells per corpus luteum increased from 21.8 +/- 3.7 (X 10(6)) on Day 4 to 61.7 +/- 5.4 (X 10(6)) on Day 8 (p less than 0.05) and remained elevated thereafter. The number of small luteal cells was 10.0 +/- 2.7 (X 10(6)), 39.7 +/- 1.4 (X 10(6)), 46.1 +/- 5.8 (X 10(6)), 49.0 +/- 13.7 (X 10(6)), and 29.9 +/- 8.6 (X 10(6)) on Days 4, 8, 12, 16 (NR), and 16 (R), respectively (p less than 0.05, Day 4 vs. Days 8, 12, 16 NR). In contrast, the number of large luteal cells was 11.8 +/- 1.5 (X 10(6)) on Day 4 and did not vary significantly during the remainder of the estrous cycle. The numbers of nonsteroidogenic cell types increased (p less than 0.05) from Day 4 to Day 16 (NR) but were decreased in regressed corpora lutea (Day 16 R). Regression was characterized by a 50% decrease (p less than 0.05) in the total number of cells per corpus luteum from 243 +/- 57 ( X 10(6)) on Day 16 (NR) to 125 +/- 14 ( X 10(6)) on Day 16 (R) (p less than 0.05). Small luteal cells remained constant in volume throughout the entire estrous cycle (2520 +/- 270 microns 3), whereas large luteal cells increased in size from 5300 +/- 800 microns 3 on Day 4 to 16,900 +/- 3300 microns 3 on Day 16 (NR) (p less than 0.05). In summary, small luteal cells increased in number but not size throughout the estrous cycle, whereas large luteal cells increased in size but not number. 相似文献
12.
Hydrodynamic characterization of the size and shape of atropinesterase from Pseudomonas putida 总被引:2,自引:0,他引:2
Atropinesterase from Pseudomonas putida has been investigated by means of different ultracentrifugation methods under native and denaturing conditions. The following quantities were determined: sedimentation coefficient, translational diffusion and friction coefficient, partial specific volume and molecular weight. From these data the size, shape and hydration of the enzyme molecule in solution were estimated. The results suggest that atropinesterase is a globular protein which consists of a single polypeptide chain with a molecular weight of about 30,000. In solution under non-denaturing conditions, it occurs mainly as a dimer which hydrodynamically behaves as a rigid impenetrable particle. Calculations based on the spheroid model indicate that this particle resembles a hydrated sphere with a diameter of 6.1 +/- 0.2 nm and a hydration of 0.4 +/- 0.1 g of H2O/g of protein rather than a significantly less hydrated ellipsoid of revolution. Under denaturing conditions dissociation into monomers takes place. The effects of sodium dodecyl sulphate (SDS) on size and shape suggest that dimerization results from side-by-side association of two elongated monomers rather than from end-to-end association. Approximately 57 molecules of SDS are bound per dimer before dissociation occurs concomitant with the additional binding of about 19 molecules of detergent. 相似文献
13.
F Kilár I Simon S Lakatos F Vonderviszt G A Medgyesi P Závodszky 《European journal of biochemistry》1985,147(1):17-25
The structure of six human myeloma proteins: IgG1(Bal), IgG2(Klu), IgG3(Bak), IgG3(Het), IgG4(Kov) and IgG4(Pol), was studied in solution using small-angle X-ray scattering and hydrodynamic methods. For IgG1(Bal) and IgG3(Het) the experimental data, including radius of gyration (Rg degree), radii of gyration of the cross-section (Rq1, Rq2), intrinsic viscosity [eta], sedimentation coefficient (S degree 20,w) and molecular mass, were interpreted in terms of structural models based on the Fab and Fc conformations, observed in crystal, by varying the relative positions of the Fab and Fc parts, i.e. their relative angles and distances. The values Rg degree = (6.00 +/- 0.05) nm, S degree 20,w = (6.81 +/- 0.10) S and [eta] = 0.0062 +/- 0.0005 cm3/mg obtained for IgG1(Bal) are compatible with a planar model in which the angle between the Fab arms is about 120 degrees. For IgG3(Het) the following data were obtained: Rg degree = (4.90 +/- 0.05) nm, S degree 20,w = (6.32 +/- 0.01) S and [eta] = (0.0065 +/- 0.0005) cm3/mg. The apparent contradiction between the higher molecular mass and lower Rg degree and S degree 20,w values for IgG3(Het) in comparison to IgG1(Bal) can be resolved by proposing a 'non-planar' (tetrahedral) molecular shape, in which the long hinge peptide is in a folded conformation and the two Fab and Fc parts are in a closely packed arrangement. In this model the angle between the two Fab arms is about 90 degrees, in the average position. The X-ray scattering and hydrodynamic behaviour of the IgG2 and IgG4 types of antibodies appeared to be similar to IgG1(Bal). The parameters of the two IgG3 proteins are similar while they are different to the others. 相似文献
14.
Characterization of the extracellular haemoglobin of Haemopsis sanguisuga (L.). 总被引:4,自引:4,他引:0
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The haemoglobin from the blood of the horseleech, Haemopsis sanguisuga (L.), had a sedimentation coefficient, SO20, w, of 59.11 +/- 0.55 S, and a molecular weight as determined by sedimentation equilibrium of 3.71 X 10(6)+/-9904 X 10(6). In the electron microscope the molecule appeared to be made up of two hexagonal plates, as is found with other worm haemoglobins, with dimensions 24.4+/-2.0 nm (across the hexagon) and 15.2+/-1.4 nm (height). The amino acid composition and spectrum were closely similar to those of the haemoglobins of other annelids (e.g. Lumbricus). The alpha-helical content, calculated from circular-dichroism measurements in the far-u.v. region, was 56-63%. The haem content was 2.49%, corresponding to a minimum molecular weight per haem group of 24 800, but detergent-gel electrophoresis indicated the presence of polypeptide chains of mol.wts. 12 600, 14 800, 15 500 and 25 100. The pH-induced dissociation of the native molecule yielded compotosol of Soya-bean root nodules. 相似文献
15.
Interaction of single-chain urokinase-type plasminogen activator with human endothelial cells 总被引:6,自引:0,他引:6
E S Barnathan A Kuo L Rosenfeld K Karikó M Leski F Robbiati M L Nolli J Henkin D B Cines 《The Journal of biological chemistry》1990,265(5):2865-2872
The interaction of urokinase-type plasminogen activators with receptors on the surface of endothelial cells may play an important role in the regulation of fibrinolysis and cell migration. Therefore, we investigated whether human umbilical vein endothelial cells (HUVEC) express receptors for single-chain urokinase (scu-PA) on the cell surface and examined the effect of such binding on plasminogen activator activity. Binding of 125I-labeled scu-PA to HUVEC, performed at 4 degrees C, was saturable, reversible, and specific (k+1 4 +/- 1 X 10(6) min-1 M-1, k-1 6.2 +/- 1.4 X 10(-3) min-1, Kd 2.8 +/- 0.1 nM; Bmax 2.2 +/- 0.1 X 10(5) sites/cell; mean +/- S.E.). Binding of radiolabeled scu-PA was inhibited by both natural and recombinant wild-type scu-PA, high molecular weight two-chain u-PA (tcu-PA), catalytic site-inactivated tcu-PA, an amino-terminal fragment of u-PA (amino acids 1-143), and a smaller peptide (amino acids 4-42) corresponding primarily to the epidermal growth factor-like domain. Binding was not inhibited by low molecular weight urokinase or by a recombinant scu-PA missing amino acids 9-45. Cell-bound scu-PA migrated at its native molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of plasminogen, scu-PA bound to endothelial cells generated greater plasmin activity than did scu-PA in the absence of cells. In contrast, when tcu-PA was added directly to HUVEC, sodium dodecyl sulfate-stable complexes formed with cell or matrix-associated plasminogen activator inhibitors with a loss of plasminogen activator activity. These studies suggest that endothelial cells in culture express high affinity binding sites for the epidermal growth factor domain of scu-PA. Interaction of scu-PA with these receptors may permit plasminogen activator activity to be expressed at discrete sites on the endothelial cell membrane. 相似文献
16.
Skeletal muscle glycogen content and structure, and the activities of several enzymes of glycogen metabolism are reported for the hepatic glycogen phosphorylase b kinase deficient (gsd/gsd) rat. The skeletal muscle glycogen content of the fed gsd/gsd rat is 0.50 +/- 0.11% tissue wet weight, and after 40 hours of starvation this value is lowered 40% to 0.30 +/- 0.05% tissue wet weight. In contrast the gsd/gsd rat liver has an elevated glycogen content which remains high after starvation. The skeletal muscle phosphorylase b kinase, glycogen phosphorylase, glycogen synthase and acid alpha-glucosidase activities are 17.2 +/- 2.9 units/g tissue, 119.9 +/- 6.4 units/g tissue, 12.2 +/- 0.4 units/g tissue and 1.4 +/- 0.4 milliunits/g tissue, respectively, with approx. 20% of phosphorylase and approx. 24% of synthase in the active form (at rest). These enzyme activities resemble those of Wistar skeletal muscle, and again this contrasts with the situation in the liver where there are marked differences between the Wistar and the gsd/gsd rat. Fine structural analysis of the purified glycogen showed resemblance to other glycogens in branching pattern. Analysis of the molecular weight distribution of the purified glycogen indicated polydispersity with approx. 66% of the glycogen having a molecular weight of less than 250 X 10(6) daltons and approx. 25% greater than 500 X 10(6) daltons. This molecular weight distribution resembles those of purified Wistar liver and skeletal muscle glycogens and differs from that of the gsd/gsd liver glycogen which has an increased proportion of the low molecular weight material.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
17.
Particle masses of the Escherichia coli pyruvate dehydrogenase (PDH) complex and its component enzymes have been measured by scanning transmission electron microscopy (STEM). The particle mass of PDH complex measured by STEM is 5.28 X 10(6) with a standard deviation of 0.40 X 10(6). The masses of the component enzymes together with their standard deviations are (2.06 +/- 0.26) X 10(5) for the dimeric pyruvate dehydrogenase (E1), (1.15 +/- 0.17) X 10(5) for dimeric dihydrolipoyl dehydrogenase (E3), and (2.20 +/- 0.17) X 10(6) for dihydrolipoyl transacetylase (E2), the 24-subunit core enzyme. The latter value corresponds to a subunit molecular weight of (9.17 +/- 0.71) X 10(4) for E2. The subunit molecular weight measured by polyacrylamide gel electrophoresis in sodium dodecyl sulfate is 8.6 X 10(4). STEM measurements on PDH complex incubated with excess E3 or E1 failed to detect any additional binding of E3 but showed that the complex would bind additional E1 under forcing conditions (high concentrations with glutaraldehyde). The additional E1 subunits were bound too weakly to represent binding sites in an isolated or isolable complex. The mass measurements by STEM are consistent with the subunit composition 24:24:12 when interpreted in the light of the flavin content of the complex and assuming 24 subunits in the core enzyme (E2). 相似文献
18.
The kinetic parameters of three different molecular forms of urokinase (UK) for the activation of native Glu-plasminogen were compared. The apparent Michaelis constant (Km. app.) of each UK was almost of the same order of magnitude (31-38 microM), but the catalytic constants (kc) were observed to be different: UKh (high molecular weight form, molecular weight 53,000), 2.4 +/- 0.2 s-1; UK+ (low molecular weight form, molecular weight 33,000), 0.83 +/- o.10 s-1, and UKl (trypsin-digested form, molecular weight 36,000), 0.91 +/- 0.18 s-1. The overall second order rate constant, kc/Km calculated for UKh was 7.7 X 10(4) M-1 s-1, higher than for UKl (2.2 X 10(4) M-1 s-1) or UKt (2.4 X 10(4) M-1 s-1), indicating the possibility of a much higher degree of enzymatic specificity and efficiency. 相似文献
19.
Malate synthase from baker's yeast has been investigated in solution by the small-angle X-ray scattering technique. Size, shape and structure of the native substrate-free enzyme and of various enzyme-substrate complexes have been determined. As the enzyme was found to be rather unstable against X-rays, several precautions as well as sophisticated evaluation procedures had to be adopted to make sure that the results were not influenced by radiation damage. These included use of low primary intensity, short time of measurement, the presence of high concentrations of dithiothreitol, combined use of the conventional slit-collimation system and the new cone-collimation system. 1. For the native substrate-free enzyme the following molecular parameters could be established: radius of gyration R = 3.96 +/- 0.02 nm, maximum particle diameter D = 11.2 +/- 0.6 nm, radius of gyration of the thickness Rt = 1.04 +/- 0.04 nm, molecular weight Mr = 187000 +/- 3000, correlation volume Vc = 338 +/- 5 nm3, hydration x = 0.35 +/- 0.02 g/g, mean intersection length - l = 5.0 +/- 0.2 nm. Comparison of the experimental scattering curve with theoretical curves for various models showed that the enzyme is equivalent in scattering to an oblate ellipsoid of revolution rather than to a circular cylinder. The semiaxes of this ellipsoid are a = b = 6.06 nm and c = 2.21 nm. Thus with an axial ratio of about 1:0.36 the enzyme is of very anisometric shape. 2. Binding of the substrates (acetyl-CoA, glyoxylate) or the substrate analogue pyruvate causes slight structural changes of the enzyme. These changes are reflected mainly by a slight decrease of the radius of gyration (0.3--1.3%, as established both with the slit-smeared and the desmeared curves). Concomitantly there occurs a decrease of the maximum particle diameter and an increase of the radius of gyration of the thickness. These changes imply an increase of the axial ratio by 2.2--6.9%, i.e. substrate binding induces a decrease of anisometry. While the particle volume appears to be unchanged on binding glyoxylate or its analogue pyruvate, binding of acetyl-CoA causes slight changes of this parameter. In a similar manner the binding of acetyl-CoA leads to a slight enhancement of the molecular weight; this increase corresponds to the binding of 2.7 +/- 1 molecules of acetyl-CoA. 相似文献
20.
A high affinity thyroid hormone binding protein in the cytosol of embryonic rat brain cells in primary cultures 总被引:2,自引:0,他引:2
A M Lennon F Chantoux J Osty J Francon 《Biochemical and biophysical research communications》1983,116(3):901-908
A thyroid hormone binding protein(s) has been characterized in the cytosol of fetal rat brain cells in primary cultures. This protein is closely related to the one described in brain supernatants with respect to its electrophoretic mobility, binding kinetic parameters and estimated molecular weight (65 000 daltons). However, in contrast to the brain cytosolic binding protein, two classes of affinity sites for triiodothyronine (T3) and thyroxine (T4) have been demonstrated: a high affinity site (KA = 1.2-3.7(3) X 10(9) M-1 for T3 and KA = 3.7-5 X 10(8) M-1 for T4) and a low affinity site (KA = 0.8-1.4 X 10(8) M-1 for T3 and 1.6-2.9 X 10(7) M-1 for T4). The results are discussed with respect to their cellular significance. 相似文献