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1.
Size heterogeneity of human cervical mucus glycoproteins. Studies performed with rate-zonal centrifugation and laser light-scattering. 总被引:1,自引:1,他引:0
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Mucus glycoproteins (mucins) from cervical pregnancy mucus were fractionated by using rate-zonal centrifugation in a gradient of guanidinium chloride. The distribution of the macromolecules, as assessed by using sialic acid determination, suggested the presence of three populations of different size. Individual fractions were subjected to laser light-scattering performed as total-intensity measurements as well as photon correlation spectroscopy. The results showed that points of inflexion were present in the distribution of both Mr and DT (translational diffusion coefficient) and that the three populations have Mr values of approx. 24 X 10(6), 16 X 10(6) and 6 X 10(6) respectively. The weight-average Mr for the whole distribution, as calculated from the values obtained for the individual fractions, was 13.6 X 10(6), which is in good agreement with that found for the unfractionated material (11.1 X 10(6]. Plots of log RG (radius of gyration) and log (1/DT) versus log Mr are in keeping with the macromolecules being linear flexible chains. 相似文献
2.
Proteoglycans synthesized by embryonic chicken retina in culture: composition and compartmentalization 总被引:1,自引:0,他引:1
Characteristics of the chondroitin sulfate/dermatan sulfate proteoglycans (CS/DSPGs) and heparan sulfate proteoglycans (HSPGs) from retinas of 14-day chicken embryos were examined following specific lyase digestion of the HSPG and CS/DSPG glycosaminoglycans, respectively. On the basis of gel exclusion chromatography the prevalent CS/DSPGs in the tissue were above Mr 400 X 10(3) with two or three glycosaminoglycan chains of Mr 60-70 X 10(3). The HSPGs existed in two distinct populations in the tissue. Those in the dominant population appeared to be in the range of Mr 250-300 X 10(3) with 9 to 12 glycosaminoglycan chains of Mr 15-25 X 10(3). The other population consisted of free heparan sulfate chains of Mr 15-25 X 10(3). The HSPGs in the medium tended to be intermediate in size. To examine the distribution of proteoglycans, tissues were sequentially homogenized and extracted in saline and reextracted with 4 M guanidine HCl (GdnHCl) and Triton X-100 (TX), or they were washed in heparin solution and dissociated to single cells with trypsin before sequential extraction in saline and GdnHCl with TX. Through comparison of the results of these two extraction methods, CS/DSPGs were found to be almost entirely within the medium or matrix or loosely associated with the cell surface, and most HSPGs were associated with either the basal lamina or the plasma membrane. The single heparan sulfate glycosaminoglycan chains appeared to be intracellular degradation products. These results support reports that CS/DSPGs may be present in the retina interphotoreceptor matrix and that HSPGs may be present in regions of synaptogenesis, associated with cell membranes. 相似文献
3.
Cervical mucus glycoproteins (mucins) were extracted by using slow stirring in 6M-guanidinium chloride supplemented with proteinase inhibitors. Subsequent purification was achieved by isopycnic density-gradient centrifugation in CsCl/guanidinium chloride. The whole mucins (Mr 10 X 10(6) - 15 X 10(6)) were degraded into subunits (Mr 2 X 10(6) - 3 X 10(6)) by reduction. Trypsin digestion of subunits afforded glycopeptides (T-domains) with Mr 0.4 X 10(6). The relationship between the intrinsic viscosity and Mr for the whole mucins and the fragments suggests that cervical mucins are linear flexible macromolecules. This view is supported by hydrodynamic data. 相似文献
4.
Isolation of the heparan sulfate proteoglycans from the extracellular matrix of rat skeletal muscle 总被引:1,自引:0,他引:1
We have previously shown that asymmetric collagen-tailed acetylcholinesterase (AChE) is anchored to the extracellular matrix (ECM) by heparan sulfate proteoglycans (HSPGs). Here we present our studies on the characterization of such PGs from the ECM of rat skeletal muscles. After radiolabeling with 35SO4 for 24h, PGs were extracted from the muscle ECM with 4.0 M guanidine-HCl containing protease inhibitors. PGs were subsequently isolated using sequential DEAE-Sephacel chromatography, digestion with chondroitinase ABC, and Sepharose CL-4B. Two different hydrodynamic size species of HSPGs were found. One type had a Mr of 4-6 X 10(5) (Kav = 0.25) as estimated by gel chromatography in the presence of 1% SDS and accounted for 75% of the total HSPGs. The other HSPG had a Mr 1.5-2.5 X 10(5) (Kav = 0.41). The glycosaminoglycan (GAG) side chains (Mr 20,000 and 12,000) were found composed only of heparan sulfate as determined by nitrous acid oxidation and heparitinase treatment. The large-sized HSPG, which is concentrated in synaptic regions, contains only GAG chains of Mr 20,000, suggesting that each HSPG contains only one kind of heparan sulfate chain in its structure. Our results definitively establish by biochemical criteria that the basement membrane of mammalian skeletal muscle contains HSPGs, the likely matrix receptor for the immobilization of the asymmetric collagen-tailed AChE at the neuromuscular junction. 相似文献
5.
Estradiol-stimulated turnover of heparan sulfate proteoglycan in mouse uterine epithelium 总被引:2,自引:0,他引:2
Heparan sulfate proteoglycans (HSPGs) and dermatan sulfate/chondroitin sulfate proteoglycans may be extracted from the uterine epithelium of immature mice by a 1-min exposure of the luminal surface of excised uteri to 1% Nonidet P-40 detergent. In mice that are treated with estradiol there is a marked increase in free heparan sulfate glycosaminoglycan in the extract. (a) By Sepharose exclusion chromatography the [35S]sulfate-labeled major HSPG had a nominal Mr of 200-250 X 10(3), consisting of a core protein of about 80-90 X 10(3) Mr with about 8-10 heparan sulfate glycosaminoglycan chains (Mr = 13 X 10(3)). The HSPG had a lower bouyant density (less than 1.45 g/ml) than the dermatan sulfate/chondroitin sulfate proteoglycan and was heterogeneous, as was evident in the fact that HSPG attained equilibrium over a wide range of CsCl densities and also showed nonuniform interaction with octyl-Sepharose. (b) Virtually all of the major HSPG was removed when the epithelium was isolated by proteolysis, indicating a cell surface localization. A smaller, less prominent HSPG (nominal Mr = 80 X 10(3)) was synthesized during the first 2 h after isolation. (c) Label and chase experiments with and without chloroquine showed that virtually all of the free heparan sulfate glycosaminoglycan chains derived from endocytosis and lysosomal degradation of the plasma membrane-associated HSPG. We conclude that estradiol stimulates endocytosis of HSPG, predominantly from the basolateral epithelial surface and suggest that this HSPG turnover may reflect changes associated with blastocyst attachment and invasion of the endometrium. 相似文献
6.
Partial characterization of heparan and dermatan sulfate proteoglycans synthesized by normal rat glomeruli 总被引:7,自引:0,他引:7
Rat glomerular heparan sulfate (HS) and dermatan sulfate (DS) proteoglycan synthesis was studied in vitro and in vivo. Incorporation of [35S]sulfate into macromolecules was linear over 16 h in vitro, and DS was the predominant glycosaminoglycan (GAG), while HS dominated in vivo incubations. Proteoglycans were found in the bottom 2/5 (high density) CsCl gradient fractions and eluted as two overlapping peaks from DEAE-Sephacel columns. The proportion of low density 35S-glycoproteins and 35S-proteoglycans increased with time. Two high buoyant density HS proteoglycans were extracted from glomeruli and eluted in DEAE peak I. The first, HS-tIA, had an Mr of 130 X 10(3) with Mr 12.5 X 10(3) GAG chains. This proteoglycan was released from the tissue by trypsin and was partially displaced by heparin treatment. In addition, it was rapidly released into the medium of label-chase experiments after which it migrated slightly more rapidly than HS-tIA in gels, with HS chains similar in length to its tissue counterpart. The second, HS-tIB, had an Mr of 8.6 X 10(3) with little or no attached protein. This proteoglycan was characterized as intracellular as it resisted release by trypsin treatment or heparin extraction in medium and was not detected in the medium of label-chase experiments. Two tissue DS proteoglycans were characterized. The first, DS-tIA, co-purified with HS-tIA and was the predominant proteoglycan synthesized during 4-h in vitro incubations. Like HS-tIA, it was rapidly released into medium and displaced from cell surfaces or tissue "receptors" by heparin or trypsin treatments. A second, Sepharose CL-6B-excluded DS proteoglycan from DEAE peak II, DS-tII, accumulated in tissue over 16 h in vitro. This proteoglycan was self-associating and contained clusters of iduronic acid residues along its Mr 26 X 10(3) DS chains. It resisted extraction from the tissue with heparin, trypsin, and detergent. No DS-tII was detected in the incubation medium. Instead, medium proteoglycans eluted as single Sepharose CL-6B-included peaks. DS chains from medium proteoglycans were shorter (Mr 18 X 10(3)) and had more regularly spaced iduronic acid residues than GAGs from DS-tII. The length and sulfation patterns of DS-mII GAG were similar to GAG from DS-tIA. Thus, glomeruli rapidly synthesized and released Sepharose CL-6B-included heparin-displaceable DS and HS proteoglycans while retaining a Sepharose CL-6B-excluded self-associating DS proteoglycan and an intracellular HS. 相似文献
7.
Hydrodynamic properties of human cervical-mucus glycoproteins in 6M-guanidinium chloride. 总被引:4,自引:3,他引:1
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Cervical mucins and fragments thereof were studied by sedimentation-velocity, rotatory viscometry and laser light-scattering performed as photon-correlation spectroscopy as well as low-angle total-intensity measurements. The Mr of the whole mucins is 10 X 10(6)-15 X 10(6), whereas fragments obtained after reduction of disulphide bonds ('subunits') have Mr 2.1 X 10(6)-2.9 X 10(6), depending on the method used. Subsequent trypsin digestion of subunits afforded glycopeptides with Mr approx. 0.4 X 10(6). The high frictional ratio for the whole mucins is interpreted as a large degree of expansion. The Stokes radius calculated from the diffusion coefficient is approx. 110nm for the whole mucins, which is in agreement with that estimated from the radius of gyration (130nm) by using the concept of the equivalent hydrodynamic sphere. The ratio of the concentration-dependence parameter for the reciprocal sedimentation coefficient (Ks) to the intrinsic viscosity ( [eta] ) for the whole mucins is 1.42, suggesting that the individual macromolecule occupies a spheroidal domain in solution. The relationship between [eta] and Mr for whole mucins, subunits and T-domains suggests that they are linear flexible macromolecules behaving as somewhat 'stiff' random coils. This conclusion is supported by the relationships between the sedimentation coefficients, the diffusion coefficients and the Mr. The hydrodynamic behaviour of the mucins is thus close to that expected for coiling macromolecules entrapping a lot of solvent, which is consistent with the postulated polymeric structure. 相似文献
8.
Biosynthesis of heparan sulfate proteoglycan by human colon carcinoma cells and its localization at the cell surface 总被引:14,自引:6,他引:8
R V Iozzo 《The Journal of cell biology》1984,99(2):403-417
After 24 h of continuous labeling with radioactive precursors, a high molecular weight heparan sulfate proteoglycan (HS-PG) was isolated from both the medium and cell layer of human colon carcinoma cells (WiDr) in culture. The medium HS-PG eluted from a diethylaminoethyl anion exchange column with 0.45-0.50 M NaCl, had an average density of 1.46-1.49 g/ml on dissociative CsCl density-gradient ultracentrifugation, and eluted from Sepharose CL-2B with a Kav = 0.57. This proteoglycan had an estimated Mr of congruent to 8.5 X 10(5), with glycosaminoglycan chains of Mr = 3 X 10(4) which were all susceptible to HNO2 deaminative cleavage. Deglycosylation of the HS-PG with polyhydrogen fluoride resulted in a 3H-core protein with Mr congruent to 2.4 X 10(5). The cell layer contained a population of HS-PG with characteristics almost identical to that released into the medium but with a larger Mr = 9.5 X 10(5). Furthermore, an intracellular pool contained smaller heparan sulfate chains (Mr congruent to 1 X 10(4)) which were mostly devoid of protein core. In pulse chase experiments, only the large cell-associated HS-PG was released (approximately 58%) into the medium as intact proteoglycan and/or internalized and degraded (approximately 42%), with a t1/2 = 6 h. However, the small intracellular component was never released into the medium and was degraded at a much slower rate. When the cells were subjected to mild proteolytic treatment, only the large cell-associated HS-PG, but none of the small component, was displaced. Addition of exogenous heparin did not displace any HS-PG into the medium. Both light and electron microscopic immunocytochemistry revealed that the cell surface reacted with antibody against an HS-PG isolated from a basement membrane-producing tumor. Electron microscopic histochemistry using ruthenium red and/or cuprolinic blue revealed numerous 10-50-nm diam granules and 70-220-nm-long electron-dense filaments, respectively, on the surface of the tumor cells. The results indicate that colon carcinoma cells synthesize HS-PGs with distinct structural and metabolic characteristics: a large secretory pool with high turnover, which appears to be synthesized as an integral membrane component and localized primarily at the cell surface, and a small nonsecretory pool with low turnover localized predominantly within the cell interior. This culture system offers an opportunity to investigate in detail the mechanisms involved in the regulation of proteoglycan metabolism, and in the establishment of the neoplastic phenotype. 相似文献
9.
Physical map of the nrdA-nrdB-ftsB-glpT region of the chromosomal DNA of Escherichia coli 总被引:8,自引:0,他引:8
Seven pLC plasmids (pLC 3-46, 8-12, 8-24, 8-29, 14-12, 19-24 and 42-17) which complemented nrdA, nrdB, ftsB and/or glpT mutations of Escherichia coli were analyzed. A restriction map of each plasmid was constructed and restriction fragments were subcloned into pBR322. A physical map of approx. a 15 X 10(6) Mr segment of the chromosomal DNA was deduced from the overlapping region of the pLC plasmids. The pLC plasmids and newly constructed plasmids were examined for the ability to rescue the mutations. The complementation tests defined the location of the genes in the 15 X 10(6) Mr segment in the following order: nrdA-nrdB-ftsB-glpT. Functional nrdAB and ftsB genes were located in the 3.1 X 10(6) Mr EcoRI-PstI fragment. 相似文献
10.
Spinach chloroplast ribosomal RNAs have been hybridized to restriction endonuclease fragments of spinach chloroplast DNA. All three RNA species (23S, 16S and 5S) hybridized to a single large fragment when the DNA was digested with either Sall or Pstl. Hybridization of 23S RNA to fragments produced by Smal yielded two radioactive bands which corresponded to the bi-molar 2.5 X 10(6) and 1.15 X 10(6) Mr fragments. 16S RNA also hybridized to two, bi-molar Smal fragments (3.4 X 10(6) and 2.5 X 10(6) Mr) and 5S RNA hybridized to the 1.15 X 10(6) Mr bi-molar Smal fragment. The 23S RNA and 16S RNA cistrons were each also shown to contain a single EcoRI site. From the data it was possible to conclude that the ribosomal RNA genes are located on the inverted repeat region of the spinach chloroplast DNA restriction map [1,2], that the sequence of the cistrons is 16S - 23S - 5S and that the size of the spacer between the 16S and 23S RNA cistrons is approximately 0.90 X 10(6) Mr. 相似文献
11.
The synthesis of collagen types IX and X by explants of chick-embryo cartilages was investigated. When sternal cartilage labelled for 24h with [3H]proline was extracted with 4M-guanidinium chloride, up to 20% of the 3H-labelled collagen laid down in the tissue could be accounted for by the low-Mr collagenous polypeptides (H and J chains) of type IX collagen; but no type X collagen could be detected. Explants of tibiotarsal and femoral cartilages were found to synthesize type IX collagen mainly in zones 1 and 2 of chondrocyte proliferation and elongation, whereas type X collagen was shown to be a product of the hypertrophic chondrocytes in zone 3. Pulse-chase experiments with tibiotarsal (zone-3) explants demonstrated a time-dependent conversion of type X procollagen into a smaller species whose polypeptides were of Mr 49 000. The processed chains [alpha 1(X) chains] were shown by peptide mapping techniques to share a common identity with the pro alpha 1(X) chains of Mr 59 000. No evidence for processing of type IX collagen was obtained in analogous pulse-chase experiments with sternal tissue. When chondrocytes from tibiotarsal cartilage (zone 3) were cultured on plastic under standard conditions for 4-10 weeks they released large amounts of type X procollagen into the medium. However, 2M-MgCl2 extracts of the cell layer were found to contain mainly the processed collagen comprising alpha 1(X) chains. The native type X procollagen purified from culture medium was shown by rotary shadowing to occur as a short rod-like molecule 148 nm in length with a terminal globular extension, whereas the processed species comprising alpha 1(X) chains of Mr 49 000 was detected by electron microscopy as the linear 148 nm segment. 相似文献
12.
The porcine ovarian luteinizing hormone/human chorionic gonadotropin receptor II. Is the purified receptor an oligomer of identical subunits? 总被引:1,自引:0,他引:1
J Wimalasena J A Abel J P Wiebe T T Chen 《The Journal of biological chemistry》1986,261(20):9416-9420
Purified porcine luteinizing hormone/human chorionic gonadotropin receptors were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis following reduction and thermal denaturation and stained with Coomassie Brilliant Blue. A major protein of Mr = 77 +/- 4 X 10(3) and a minor protein of Mr = 66 +/- 4 X 10(3) were observed. Iodoreceptor proteins were resolved into a major component of Mr = 77 +/- 3 X 10(3) and a minor component of Mr = 62 +/- 5 X 10(3) after reduction and thermal denaturation. In the absence of reduction, the iodoreceptor had a major component of Mr 63 +/- 3 X 10(3). Purified human chorionic gonadotropin specifically transferred part of the iodoreceptor from the Mr = 63 X 10(3) species to an Mr = 110-120 X 10(3) species. Purified receptors were analyzed by nondenaturing polyacrylamide gel electrophoresis and identified by specific binding of iodo-human chorionic gonadotropin. Three binding species with approximate Mr = 60 X 10(3), 130 X 10(3), and 260 X 10(3) were identified. Iodoreceptors co-migrated with the Mr = 60 X 10(3) species under the same conditions. Similar results were obtained following renaturation of receptors separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis without reduction and thermal denaturation. These results suggest for the first time that the porcine corpus luteum luteinizing hormone/human chorionic gonadotropin receptor may be a hormone binding monomer of Mr = 60-65 X 10(3), and that the monomer may associate to form hormone binding polymeric receptor complexes. 相似文献
13.
Binding of hyaluronate and chondroitin sulphate to liver endothelial cells. 总被引:11,自引:3,他引:8
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Hyaluronate is taken up and metabolized in liver endothelial cells by means of a receptor. To characterize the interaction with the receptor, two preparations of 3H-labelled hyaluronate, of Mr 4 X 10(5) and 6.4 X 10(6), and a series of hyaluronate oligosaccharides were bound to cultured liver endothelial cells at 7 degrees C. The dissociation constant varied between 4.6 X 10(-6) M for an octasaccharide and 9 X 10(-12) M for the largest polymer. The Mr-dependence for the series of oligosaccharides was explained by the increased probability of binding due to the repetitive sequence along the chain. The high affinity of high-Mr hyaluronate for the receptor could also be mainly ascribed to this effect, which rules out any major contribution of co-operative multiple-site attachment to the cell surface. Each liver endothelial cell can bind 10(5) oligosaccharides, about 10(4) molecules with Mr 4 X 10(5) and about 10(3) molecules with Mr 6.4 X 10(6). This is explained by mutual exclusion of large molecules from the cell surface. Chondroitin sulphate is also bound to liver endothelial cells. Inhibition studies showed that it binds to the same receptor as hyaluronate and with an affinity that is about 3-fold higher than that of hyaluronate of the same degree of polymerization. 相似文献
14.
Minor disulfide-bonded collagen (previously termed X1-X7 and now called type IX collagen) was isolated from foetal calf cartilage after pepsin treatment. At least three native fractions, containing, respectively, the X1X2X3, X4, and X5X6X7 chains, were separated; and from further biochemical and physicochemical experiments (differential scanning calorimetry, electrical birefringence, rotary shadowing), we propose a tentative model for their organization within a parent molecule. X1 and X2 are molecules composed of three chains of apparent Mr 62,000 and 50,000 linked by interchain disulfide bonds and containing pepsin-sensitive regions. The cleavage of at least three of these sites, present within X2, gives rise to the X3 and X5X6X7 fractions composed of molecules 80-100 nm and 40-55 nm in length, respectively. The X5X6X7 fraction is not digested by pepsin at 30 degrees C owing to its high thermal stability (certainly explained by its high hydroxyproline + proline content). This organization is in good accordance with that proposed for chicken cartilage type IX collagen; differences could only exist in the number and (or) the location of the pepsin-sensitive sites. 相似文献
15.
M Ohta S Shigeta K Ono T Takao Y Shimonishi S Oka 《Archives of biochemistry and biophysics》1989,275(1):151-165
O-Glycosidically linked oligosaccharide chains were liberated as oligosaccharide alcohols (HPG-beta 2) by beta-elimination treatment from a glycopeptide fraction (HPG, Mr ca. 10(5)) that was isolated from a Pronase digest of a large-size sea squirt antigen termed H-antigen (Mr 10(6)-2 X 10(7)). After HPG-beta 2 was divided into a neutral (HPG-beta 2-N) and an acidic (HPG-beta 2-A) fraction, 11 neutral saccharitols were isolated from HPG-beta 2-N, and their sugar sequences were determined by methylation/GC-MS, FAB-MS, and/or DI/EI-MS. By skin tests specific to patients with sea squirt allergy, it was found that six saccharitols were allergenically active but five were inactive. Furthermore, active saccharitols were all of branched structure containing two nonreducing terminal GalNAc (t-GalNAc) residues, whereas the inactive saccharitols contained one or no t-GalNAc. Since the allergic reaction in skin should depend on the crosslinking of IgE antibodies by certain allergens carrying two or more epitopes, three types of N-acetylgalactosaminyl disaccharide units, GalNAc1----2Fuc1----, GalNAc1----3/4GalNAc1----, and GalNAc1----3/4GlcNAc1----, were nominated as the major components of the allergy-specific epitopes in the active saccharitols. Such multiplicity of the epitope structures might reflect the polyclonality of the specific IgE antibodies. 相似文献
16.
Partial characterization of type X collagen from bovine growth-plate cartilage. Evidence that type X collagen is processed in vivo 总被引:1,自引:0,他引:1
Sequential extraction of bovine growth-plate cartilage with 4 M guanidinium chloride and pepsin was used to identify the intact and pepsinized forms respectively of type X collagen. This collagen occurs predominantly as the processed [alpha 1(X)]3 form in vivo, although the procollagen [pro alpha 1(X)]3 form can also be detected. The bovine pro alpha 1(X) and alpha 1(X) chains have Mr values identical to the corresponding chick species (Mr 59,000 and 49,000). However, the pepsinized alpha 1(X)p chains (Mr 47,000) are larger than those of the chick (Mr 45,000), and the bovine collagen type X is further distinguished by being disulphide-bonded within the triple-helical domain. 相似文献
17.
Molecular movements promoted by metal nucleotides in the heavy-chain regions of myosin heads from skeletal muscle 总被引:1,自引:0,他引:1
D Mornet P Pantel E Audemard J Derancourt R Kassab 《Journal of molecular biology》1985,183(3):479-489
Molecular movements generated in the heavy-chain regions (27-50-20(X 10(3)) Mr) of myosin S1 on interaction with nucleotides ATP, AMPPNP, ADP and PPi were investigated by limited proteolysis of several enzyme-metal nucleotide complexes in the absence and presence of reversibly bound and crosslinked F-actin. The rate and extent of the nucleotide-promoted conversion of the NH2-terminal 27 X 10(3) Mr and 50 X 10(3) Mr segments into products of 22 X 10(3) Mr and 45 X 10(3) Mr, respectively, were estimated to determine the amplitude of the molecular movements. The 22 X 10(3) Mr peptide was identified by amino acid sequence studies as being derived from cleavage of the peptide bond between Arg and Ile (at position 23 to 24). The 45 X 10(3) Mr peptide, previously shown to represent the NH2-terminal part of the 50 X 10(3) Mr region, would be connected to the adjacent C-terminal 20 X 10(3) Mr region by a pre-existing loop segment of about 5 X 10(3) Mr; the proteolytic sensitivity of the latter region is increased particularly by nucleotide binding. The tryptic reaction proved to be a sensitive indicator of the conformational state of the liganded heavy chain as the rate of peptide bond cleavage in the two regions is dependent on the nature of the bound ligand; it decreases in the order: ATP greater than AMPPNP greater than ADP greater than PPi. It depends also on the nature of the metal present, Mg2+ and Ca2+ being much more effective than K+. Binding of F-actin to the S1-MgAMPPNP complex affords significant protection against breakdown of 27 X 10(3) Mr and 50 X 10(3) Mr peptides, but with concomitant hydrolysis of the 50 X 10(3) Mr-20 X 10(3) Mr junction. Additionally, interaction of MgATP with HMM modulates the tryptic fission of the S1-S2 region. The overall data provide a molecular support for the two-state model of the myosin head and emphasize the involvement of the 50 X 10(3) Mr unit in the mechanism of coupling between the actin and nucleotide binding sites. 相似文献
18.
M Ikeda-Saito 《FEBS letters》1986,202(2):245-250
The molecular structure of the spleen green heme protein was reinvestigated by gel-permeation, SDS-polyacrylamide gel electrophoresis, and amino acid analysis. The results showed that the enzyme is a tetramer (Mr 1.5 X 10(5)) with two heavy subunits (Mr 6 X 10(4) with a single prosthetic group per subunit) and two light subunits (Mr 1.5 X 10(4)), and that the tetramer structure is maintained by disulfide bond(s). The amino acid composition of the spleen green heme protein is similar to that of granulocyte myeloperoxidase. The present results contradict the data of Davis and Averill [(1981) J. Biol. Chem. 256, 5992-5996], who reported the enzyme as a monomeric peroxidase with an Mr of 57 000. 相似文献
19.
The substructure of the tubulin molecule was studied by limited proteolysis and high affinity polyclonal antibodies specific for alpha or beta-tubulin. Brief enzymatic cleavage separates the tubulin monomer into two domains of unequal size. Trypsin splits alpha-tubulin into components with Mr values of 36 X 10(3) and 14 X 10(3), chymotrypsin splits beta-tubulin into 31 X 10(3) Mr and 20 X 10(3) Mr fragments. The cleavage occurs at Arg339 (alpha) and Tyr281 (beta), as determined by sequencing several N-terminal residues of the small domains, i.e. the small domains are the C-terminal parts of the molecules, the large ones are the N-terminal parts. There is a second cleavage site of chymotrypsin within Mr 10(3) to 2 X 10(3) of the C terminus of beta-tubulin. The fragments can be separated only under denaturing conditions. They copolymerize into microtubules and incomplete microtubule walls joined by a wall junction, forming S-shapes and hooks in cross-section. The antibodies were raised against electrophoretically purified tubulin monomers. Those produced with alpha-tubulin are directed predominantly against the large domains; they are either specific for alpha-tubulin or cross-react with the large domain of beta-tubulin. Conversely, antibodies raised against beta-tubulin are directed predominantly against the small domains (beta-specific and beta-cross-reacting fractions). Thus the antibodies discriminate not only between the tubulin chains but also between the domains generated by the proteases. The complementary antigenicity correlates well with the stability of the domains. Potential sites of antigenic determinants are located within the polypeptide chains by comparing theoretical predictions with the pattern of immunoblots. Two epitopes of the alpha-cross-reacting antibodies have been located approximately. One is very close to the C terminus (within about 20 residues), the other is close to the N terminus (within about Mr 8 X 10(3) ). The epitope of the beta-cross-reacting antibody is also located within Mr 12 X 10(3) of the C terminus. The antibodies prevent microtubule assembly and cause disassembly of preformed microtubules. A variety of breakdown products are observed by electron microscopy. They include fibres of about 10 nm width, sheets with undefined substructure, thick tapered fibrous bundles and wispy filaments.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献