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1.
E. der Terrossian S.D. Fuller Murray Stewart A.G. Weeds 《Journal of molecular biology》1981,153(1):147-167
Porcine platelet tropomyosin has been isolated by hydroxyapatite chromatography following isoelectric precipitation and ethanol fractionation. A single component (Mr = 30,000 on polyacrylamide gel electrophoresis in sodium dodecyl sulphate) was obtained in a variety of gel electrophoretic systems, including urea/sodium dodecyl sulphate and isoelectric focussing, suggesting the presence of a single polypeptide species. This contrasts with the observations by others using horse platelet tropomyosin or tropomyosins isolated from brain, where two polypeptides were found. The amino acid composition of porcine platelet tropomyosin was virtually identical to that of the horse platelet protein except for the presence of a single cysteine residue in the pig protein, whereas horse tropomyosin contains two. Oxidation of this sulphydryl group produced a molecule of over 60,000 Mr on polyacrylamide gels in sodium dodecyl sulphate, suggesting by analogy with skeletal muscle tropomyosin that the two chains had been linked and therefore existed in register in the coiled-coil structure. Cleavage at the cysteine produced a fragment of Mr = 19,000, indicating that this residue was located about one-third of the distance from a molecular end.Magnesium paracrystals of platelet tropomyosin were examined by electron microscopy following negative staining and found to have a repeat of 345 Å, close to that expected for an extended alpha-helical coiled-coil with an apparent Mr of 30,000. The repeating unit was centrosymmetric and the appearance of broken paracrystals suggested that the molecular ends lie on a dyad axis. Location of the sulphydryl residues in the paracrystals by labelling with N-pyrrolo-isomaleimide showed two bands separated by approximately 80 Å, which was consistent with the location of the molecular ends on a dyad.The amount of tropomyosin present was estimated as 2·2% of the total platelet protein. This implied a molar ratio of G-actin to tropomyosin of about 14:1, based on previous estimates of the actin content in porcine platelets. Assuming that one molecule of tropomyosin will bind six molecules of actin (based on the reduced molecular length of the platelet protein), there was not sufficient tropomyosin to bind to more than half the total actin in the cell. 相似文献
2.
1. Preparations of purified pig kidney aminoacylase (N-Acylamino-acid amidohydrolase, EC 3.5.1.14) were obtained by Sephadex and DEAE-cellulose chromatography in homogeneous form as judged by polyacrylamide gel electrophoresis and immunoelectrophoresis. 2. The apparent molecular weight of the enzyme, determined by gel filtration, was about 86 000. After treatment with mercaptoethanol, performic acid or sodium dodecyl sulphate a band with an apparent molecular weight of approximately 43 000 was observed in polyacrylamide gels containing sodium dodecyl sulphate. Thus pig kidney aminoacylase seems to be composed of two subunits. 3. The amino acid composition of the enzyme was determined. Aminoacylase contains 772 amino acids, which corresponds to a molecular weight of 85 500. 12 tryptophan and 12 half-cystine residues were found. 4. Each subunit of the enzyme contains two -SH groups of different reactivity and two disulfide bonds one of which is easily cleaved by -SH compounds, the second only by performic acid oxidation. 5. Chemical modification of two -SH groups abolishes the catalytic activity of aminoacylase. Cleavage of two disulfide bonds also inactivates the enzyme. It is suggested that the enzyme has two active sites each containing an essential -SH group and disulfide bond. One active site is assumed to be part of each subunit. 相似文献
3.
ISOLATION OF AN ACID-SOLUBLE BASIC PROTEIN FROM MONKEY BRAIN 总被引:2,自引:1,他引:1
—A basic protein, soluble in 0·1 m -perchloric acid, has been purified from brain of Macaca irus. The protein is homogeneous as indicated by ultracentrifugation, gel filtration, gel isoelectric focusing and gel electrophoresis at pH 2·9, 4·3 and 7·5. The molecular weight is estimated to be 16,000 by electrophoresis in sodium dodecyl sulphate–polyacrylamide gels. This result is in agreement with the value of 16,728 obtained from the amino acid analysis. The protein dimerizes under alkaline conditions. The predominant amino acid is glycine (15%) and the protein also contains 4% cysteine. The ratio of acidic to basic amino acids is 1·6, but a high amide content gives the protein a basic character. An isoelectric point of 9·5 is observed in gel isoelectric focusing. 相似文献
4.
The inhibition of platelet cyclo-oxygenase by aspirin is associated with the acetylation of a 72kDa polypeptide in the intracellular membranes. 总被引:3,自引:3,他引:0
Phosphoenolpyruvate carboxykinase was purified from mitochondria of guinea-pig liver by affinity chromatography on GMP-Sepharose. The enzyme was purified 100-fold to a high degree of electrophoretic homogeneity as judged by detection of a single protein band on sodium dodecyl sulphate/polyacrylamide gels. The yield was about 16%. The Mr of the purified enzyme was estimated to be 68500 +/- 680 by analysis on sodium dodecyl sulphate/polyacrylamide gels. Antibodies raised in rabbits against the purified enzyme were highly specific for mitochondrial phosphoenolpyruvate carboxykinase and did not precipitate the cytosolic form of this enzyme from either rat or guinea-pig liver cytosol. The use of this antibody showed that starvation does not increase the amount of the enzyme. However, neonatal-development-dependent increase in its activity is shown to be mediated by accumulation of phosphoenol pyruvate carboxykinase-specific protein. 相似文献
5.
Changes in mammary-gland acetyl-coenzyme A carboxylase associated with lactogenic differentiation 总被引:5,自引:0,他引:5
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The process leading to the rise of acetyl-CoA carboxylase activity in rat mammary tissue after the onset of lactation was investigated. The kinetics of change in enzyme activity and enzyme immunotitratable with antibody against avian liver acetyl-CoA carboxylase were determined during the course of lactogenic differentiation. The antibody inactivates and specifically precipitates acetyl-CoA carboxylase from rat mammary tissue as well as that from chicken liver cytosol. Characterization of the immunoprecipitate of the mammary tissue carboxylase by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis reveals a single biotin-containing polypeptide of about 230000mol.wt. This molecular weight is approximately twice that reported for the avian liver enzyme. However, chicken liver cytosol prepared in the presence of trypsin inhibitor and subjected to immunoprecipitation gives rise to a biotin-containing subunit of 230000mol.wt. as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; omission of proteinase inhibitor leads to a subunit(s) approximately one-half this size. Throughout gestation both carboxylase activity and amounts of immunotitratable enzyme remained low; however, after parturition both parameters rose concomitantly to values 30-40 times the initial values. Therefore the elevated concentration of acetyl-CoA carboxylase appears to result from an increased rate of synthesis of enzyme relative to degradation rather than to activation of a pre-existing form of the enzyme. 相似文献
6.
H R Saibil 《Journal of molecular biology》1982,158(3):435-456
To study the organization of microvilli in the photoreceptor cells of an invertebrate. X-ray diffraction patterns were obtained from aldehyde-fixed squid retinas to a resolution of (40 Å)?1 and correlated with results from electron microscopy and sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Squid photoreceptor microvilli are packed in extensive hexagonal arrays; in addition each microvillus has a hexagonal substructure. Image reconstruction from thin section electron micrographs shows that the microvilli are linked together with specialized membrane junctions at their neighbour contacts, and phosphotungstic acid-stained sections show a central cytoskeleton connected to the membrane by side-arms.The X-ray patterns also reveal two axial periodicities in the microvilli. A weak and diffuse (50 Å)?1 band is tentatively assigned to rhodopsin molecules ordered in the plane of the membrane. In addition, an arc at (85 Å)?1 is attributed to a cytoplasmic or extracellular structure.Sodium dodecyl sulphate/polyacrylamide gel electrophoresis of the isolated microvilli shows that the major component, rhodopsin, comprises about 50% of the total protein. There are two major detergent-insoluble polypeptides with molecular weights of 145,000 and 42,000. The 42,000 component is identified as actin by papain digestion fragment mapping.Cephalopod photoreceptors are highly sensitive to the polarization vector of linearly polarized light. In consequence, the linear rhodopsin chromophores must be aligned relative to the microvillar axes. The membrane junctions and cytoskeleton described here may provide a mechanism for maintaining this rhodopsin alignment. 相似文献
7.
(1) Dopamine, β-hydroxylase (EC 1.14.2.1) was purified from bovine adrenal medullae according to the method of Foldes , Jeffrey , Preston and Austin (1972). (2) The kinetics, pH optimum and the effect of Cu2+ ions on the purified enzyme were found to resemble those of the enzyme isolated by more involved procedures. (3) The sedimentation coefficient (s20) of the homogeneous enzyme in 10 mM-phosphate buffer, pH 7·2, containing 0·1 M-NaCI was found to be 10·24 ± 0·12 (S.E.M. of 10 determinations). (4) The effect of pH on the mol. wt. of the enzyme was investigated and no large deviation was found from the native mol. wt. of 290,000 in the pH range 3·9 to 11·1. (5) The amino acid analysis of dopamine β-hydroxylase is presented, and is contrasted to that of chromogranin A purified from the same chromaffin granule lysate. (6) Treatment with either 8 M-urea or 0·1% (w/v) sodium dodecyl sulphate was found to dissociate the enzyme into three similar, non-active subunits, each of mol. wt. of the order of 100,000. 相似文献
8.
Isolation of collagen glucosyltransferase as a homogeneous protein from chick embryos 总被引:3,自引:0,他引:3
Collagen glucosyltransferase was isolated as a homogeneous protein from chick embryos by a procedure consisting of ammonium sulphate fractionation, two affinity chromatographies and two gel filtrations. The specific activity of the purified enzyme was 32,000 times that of the 15,000 x g supernatant of the embryo homogenate, and the enzyme was pure when examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis using three different gel compositions. The molecular weight of the enzyme was about 72,000-78,000 by sodium dodecyl sulphate polyacrylamide gel electrophoresis, the value being dependent on the gel composition. The apparent molecular weight by gel filtration was dependent on the purity and protein concentration. The sedimentation coefficient S20,w was 4.7. The data suggest that the enzyme molecule consists of one polypeptide chain. 相似文献
9.
Affinity purification and some molecular properties of human liver alkaline phosphatase. 总被引:7,自引:3,他引:4
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Alkaline phosphatase from human liver was purified to homogeneity. The purification procedure included solubilization with butanol, fractionation with acetone, and chromatography on concanavalin A-Sepharose, DEAE-cellulose, Sephadex G-200 and DEAE-Sephadex. Purity was established by standard and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The isoelectric point of the protein was determined to be 4.0. Sephadex-gel filtration gave a mol.wt. of 146000, although a higher value was obtained in the presence of 100mM-NaC1. The subunit mol.wt. 76700, was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Neuraminidase treatment resulted in two enzyme-activity bands on isoelectric-focused gels with isoelectric points of 6.6 and 6.8. The desialylated enzyme gave only one protein band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with a subunit molecular weight indistinguishable from that of the non-neuraminidase-treated protein. The desialylated enzyme was more readily denatured by sodium dodecyl sulphate in the presence of mercaptoethanol than was the native enzyme. 相似文献
10.
F E Verheul J W Draijer A O Muijsers B F Van Gelder 《Biochimica et biophysica acta》1982,681(1):118-129
Bovine heart cytochrome c oxidase consists of 12 stoicheiometric polypeptide chains of at least 11 different types. The enzyme contains 14--16 cysteine residues; the distribution of nearly all cysteine residues over the subunits has been established. In native cytochrome c oxidase two thiol groups reacted rapidly and stoicheiometrically with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). These thiol groups are located in subunits I and III, respectively. This implies that subunit I is not fully buried in the hydrophobic core of the enzyme. After dissociation of the enzyme by sodium dodecyl sulphate more thiol groups became available to DTNB, in addition to those in subunits I and III, at least one in subunit II, two in fraction V/VI and one to two in the smallest subunit fraction. It is shown that separation of the subunits of cytochrome c oxidase by gel permeation chromatography in the presence of sodium dodecyl sulphate depends on the pH of the elution medium. The elution volume of subunits I, III and VII is dependent on pH, that of the others independent. 相似文献
11.
Isolation of lysyl hydroxylase, an enzyme of collagen synthesis, from chick embryos as a homogeneous protein. 总被引:4,自引:3,他引:1
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Two procedures are reported for the purification of lysyl hydroxylase, both procedures involving (NH4)2SO4 fractionation, affinity chromatography on concanavalin A-agarose and elution of the column with ethylene glycol. The additional steps in procedure A consist of gel filtration and chromatography on a hydroxyapatite column, and in procedure B of affinity chromatography on collagen linked to agarose and gel filtration. The best preparations obtained with either of the two procedures were pure when examined by sodium dodecyl sulphate-polyacrylamide-disc-gel or slab-gel electrophoresis, but about half of the preparations obtained by procedure A had minor contaminants. The specific activity of a typical preparation purified by procedure B was 13 4000 times that of the 15 000 g supernatant of the chick-embryo homogenate, with a recovery of about 4%. The molecular weight of the pure enzyme was bout 200 000 by gel filtration, and that of the enzyme subunit about 85 000 by sodium dodecyl sulphate/polyacrylamide-disc-gel or slab-gel electrophoresis. It is suggested that the active enzyme is a dimer consisting of only one type of monomer, and that a previously described enzyme form with an apparent molecular weight of about 550 000 is a polymeric form of this dimer. The catalytic-centre activity of the pure enzyme, as determined with a saturating concentration of a synthetic peptide substrate and under conditions specified, was about 3-4 mol/s per mol. 相似文献
12.
血管紧张素转换酶纯化与性质研究 总被引:5,自引:0,他引:5
为了深入了解猪肺血管紧张素转换酶 (angiotensin converting enzyme,ACE)的性质和功能 ,对猪肺 ACE的分离纯化以及部分酶学性质进行了研究 .猪肺组织匀浆经 1 .6~ 2 .6mol/L硫酸铵分级沉淀等步骤后 ,利用亲和胶进行亲和层析分离 .2 0 0 g猪肺组织中提纯出 0 .79mg ACE,比活力 38.8U/mg,SDS- PAGE电泳鉴定为一条带 ,分子量约 1 80 k D,等电点 (p I)为 p H4.5,糖含量约 2 3.6% ,氨基酸组成分析发现猪肺 ACE分子中含有 1 346个氨基酸 ,其中酸性氨基酸含量较高 ,碘乙酸的修饰结果表明猪肺 ACE中巯基基团未参与酶的催化反应 .酶反应动力学结果显示 ,ACE催化 Fa PGG底物反应时的最适 p H大约为 p H 7.6,反应活化能 Ea=4.37× 1 0 4 J/mol,酶活性部位附近的组氨酸和具有类似 α-氨基性质的氨基酸可能参与了 ACE催化反应 .有关猪肺 ACE的基本生化性质、氨基酸组成以及酶学性质的结果 ,为今后深入研究奠定了基础 . 相似文献
13.
Structural properties of homogeneous protein disulphide-isomerase from bovine liver purified by a rapid high-yielding procedure. 总被引:13,自引:3,他引:10
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Protein disulphide-isomerase from bovine liver was purified to homogeneity as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, two-dimensional electrophoresis and N-terminal amino acid analysis. The preparative procedure, a modification of that of Carmichael, Morin & Dixon [(1977) J. Biol. Chem. 252, 7163-7167], is much faster and higher-yielding than previous procedures, and the final purified material is of higher specific activity. The enzyme has Mr 57 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both in the presence and in the absence of thiol compounds. Gel-filtration studies on Sephadex G-200 indicate an Mr of 107 000, suggesting that the native enzyme is a homodimer with no interchain disulphide bonds. Ultracentrifugation studies give a sedimentation coefficient of 3.5S, implying that the enzyme sediments as the monomer. The isoelectric point, in the presence of 8 M-urea, is 4.2, and some microheterogeneity is detectable. The amino acid composition is comparable with previous analyses of this enzyme from bovine liver and of other preparations of thiol:protein disulphide oxidoreductases whose relation to protein disulphide-isomerase has been controversial. The enzyme contains a very high proportion of Glx + Asx residues (27%). The N-terminal residue is His. The pure enzyme has a very small carbohydrate content, determined as 0.5-1.0% by the phenol/H2SO4 assay. Unless specific steps are taken to remove it, the purified enzyme contains a small amount (5 mol/mol of enzyme) of Triton X-100 carried through the purification. 相似文献
14.
The production of an extracellular chitin deacetylase (CDA) produced by Aspergillus flavus under solid-substrate fermentation (SSF) using wheat bran as substrate was optimized using statistical methods. The CDA production in SSF increased 1.79-fold in comparison to the unoptimized basal level medium. It was purified to a final purity of 3.94-fold by ammonium sulphate precipitation, ion-exchange chromatography, and gel-permeation chromatography (GPC) consecutively and further characterized. The molecular mass of the enzyme was estimated to be about 28?kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and GPC analysis. The optimum pH and temperature of the purified enzyme were pH 8.0 and 50?°C, respectively. Additionally, the effect of some cations and other chemical compounds on the CDA activity was studied. A marginal increase in enzyme activity was observed with metal ions mainly Mn2+ and Zn2+. No inhibition of the enzyme was observed by the end product, that is, acetate up to 70?mM concentration. The Km and kcat values of the enzyme were determined to be 9.45?mg mL?1 and 26.72?s?1 respectively, using colloidal chitin as substrate. Among various substrates tested, glycol chitin and colloidal chitin were deacetylated. 相似文献
15.
Twelve amino acids and amides at 0·1 to 0·75 or 1·0% in 35% sucrose solution were individually tested for their rôle in phagostimulation, growth, and survival in Acyrthosiphon pisum. Leucine and phenylalanine were phagostimulatory at all concentrations tested, tryptophan and valine at 0·1, 0·2, and 0·5%, and threonine at 0·1% only. Methionine was reported earlier by us to be phagostimulatory at 0·05 to 0·5%. Histidine and isoleucine had no effect, whereas arginine and lysine HCl reduced uptake when compared to sucrose alone. The non-essential amino acids, canavanine sulphate and glutamine, reduced uptake at all concentrations, whereas homoserine was phagostimulatory at 0·1 and 0·75%.Arginine, canavanine sulphate, glutamine, histidine, homoserine, isoleucine, leucine, and valine increased weight and prolonged survival, whereas lysine HCl, phenylalanine, threonine, and tryptophan neither promoted growth nor increased survival. Radioactive leucine (14C(U)) was incorporated into the protein fraction of the larval body and exuviae indicating that it took part in protein synthesis. This seems to be the first report in insects where peptide or protein synthesis occurred from single amino acids in sucrose. 相似文献
16.
Concanavalin Å is visibly aggregated by low concentrations of sodium dodecyl sulfate, maximum aggregation being obtained at pH 4.6. Other denaturants, such as urea, guanidine hydrochloride, Triton X-100, cetyltrimethylammonium bromide, Tween 80, and Brij 35 are ineffective in promoting visible aggregation. The sodium dodecyl sulfate-induced aggregation of concanavalin Å requires the presence of an intact, saccharide-ligand binding-site. Rapid and complete reversal of the detergent effect was achieved by use of saccharides which bind to the lectin. Such compounds as tryptophan and o-nitrophenyl β-d-galactopyranoside did not inhibit the aggregation of concanavalin Å by sodium dodecyl sulfate, suggesting that the detergent does not bind the hydrophobic pocket on the surface of the protein. The results suggest that concanavalin Å may have an additional, ligand-binding site which is netal-dependent and which can be modified by the addition of a saccharide ligand. 相似文献
17.
Bovine heart cytochrome c oxidase consists of 12 stoicheiometric polypeptide chains of at least 11 different types. The enzyme contains 14–16 cysteine residues; the distribution of nearly all cysteine residues over the subunits has been established. In native cytochrome c oxidase two thiol groups reacted rapidly and stoicheiometrically with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). These thiol groups are located in subunits I and III, respectively. This implies that subunit I is not fully buried in the hydrophobic core of the enzyme. After dissociation of the enzyme by sodium dodecyl sulphate more thiol groups became available to DTNB, in addition to those in subunits I and III, at least one in subunit II, two in fraction V/VI and one to two in the smallest subunit fraction. It is shown that separation of the subunits of cytochrome c oxidase by gel permeation chromatography in the presence of sodium dodecyl sulphate depends on the pH of the elution medium. The elution volume of subunits I, III and VII is dependent on pH, that of the others independent. 相似文献
18.
D Sha'ag 《Journal of biochemical and biophysical methods》1989,19(1):121-128
In assaying NADPH-oxidase activities using macrophage-derived cell-free systems, sodium dodecyl sulphate dependent NADPH oxidation rates were found to correlate strongly with superoxide-dismutase inhibitable cytochrome-c reduction rates. Optimum sodium dodecyl sulphate concentration was in the range 95-110 microM. A theoretical function for the activation and deactivation of NADPH-oxidase by sodium dodecyl sulphate is suggested. This function may serve to estimate the real Vmax of the enzyme and the number of detergent molecules required for the full activation of the enzymatic complex. 相似文献
19.
Sławomir Orzechowski Joanna Socha-Hanc Andrzej Paszkowski 《Acta Physiologiae Plantarum》1999,21(4):323-330
Alanine aminotransferase (AlaAT, EC 2.6.1.2) from leaves of 14-day-old maize seedlings was purified over 1600-fold to electrophoretical
homogeneity. Specific activity of the purified enzyme measured with L-alanine and 2-oxoglutarate as substrates was 2125 nkat·(mg
protein)−1 at 30 °C. The molecular weights of the native and sodium dodecyl sulfate — denatured AlaAT protein were 95 kDa and 50 kDa
respectively, indicating that the native enzyme is probably a homodimer. AlaAT almost exclusively catalyzed amino group transfer
from L-alanine to 2-oxoglutarate and the reverse reaction. The inhibitory experiments showed that pirydoxal phosphate is directly
involved in the enzymatic catalysis and the enzyme molecule contains essential SH groups. The use of phenylglyoxal demonstrated
the presence of arginine residue as anionic binding site in the active centre of AlaAT.
This work was supported by the State Committee for Scientific Research, a grant No. 5PO6A00510 相似文献
20.
G Webb 《Canadian journal of biochemistry》1978,56(12):1124-1132
The assymmetric 18S and 14S forms of acetylcholinesterase (EC 3.1.1.7) from Electrophorus electricus purified by affinity chromatography on N-methylacridinium Sepharose 2B were subjected to trypsin or collagenase proteolysis and changes in the enzyme composition and structure were monitored by sucrose gradient sedimentation, gel chromatography, and sodium dodecyl sulphate - polyacrylamide gel electrophoresis. A distinction between autolytic and tryptic degradation products is described and the generation of two new forms of acetylcholinesterase from the 18S and 14S enzyme by collagenase proteolysis is reported. The species derived from the 18S form of acetylcholinesterase has a sedimentation coefficient of 21.1S and a Stokes radius of 12.9 nm while the 14S form gives rise to a 17.3S species with a Stokes radius of 11.1 nm. The proteolytically sensitive component ('tail') of the asymmetric forms of acetylcholinesterase is identified with a subunit of 45 000 daltons on sodium dodecyl sulphate - polyacrylamide electrophoresis gels. 相似文献