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1.
  • 1.1. Glutamine synthetase was purified from the diazotroph Azospirillum brasilense.
  • 2.2. The holoenzyme with a Mr of 630,000 is composed of 12 subunits of Mr 52,000.
  • 3.3. A modified subunit of Mr 53,000 was also found by electrophoresis under denaturing conditions.
  • 4.4. It is shown that the Mr 53,000 species is the adenylylated subunit.
  • 5.5. The apparent Km values for glutamate, ATP and ammonia were 2.5 ± 0.3 mM, 200 ± 20 μM and42 ± 2 μM, respectively.
  • 6.6. Levels of glutamine synthetase activity in A. brasilense cells varied by a factor of 8 depending on the nitrogen source and its concentration in the growth medium.
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2.
  • 1.1. Co-isolating proteins (Mr 170,000–220,000) from sodium channel preparations made from the electric organ of the electric eel (Electrophorus electricus) were detected on Western blots using monoclonal a antibodies.
  • 2.2. Similar protein patterns were seen on immunoblots containing immunoprecipitated protein from eel muscle and brain tissues but not heart.
  • 3.3. These co-isolating proteins could be separated from the mature TTX-sensitive channel protein (Mr 280,000) using a lentil lectin-Sepharose column.
  • 4.4. The 180 kDa proteins do not appear to be channel-related and can be detected as contaminants in electroplax sodium channel preparations using the monoclonal antibodies described here.
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3.
  • 1.1. A thermostable orthophosphoric monoester phosphohydrolase (EC 3.1.3.1) from Thermus sp strain Rt41A has been purified 400-fold to give a specific activity of 25 U/mg at 60°C in IM diethanolamine (pH 11.1).
  • 2.2. The enzyme has a Mr of 160,000 and is trimeric.
  • 3.3. The half-life of the enzyme is 5 min at 85°C.
  • 4.4. The enzyme has a wide specificity for a number of phosphate monoesters.
  • 5.5. The Hm of the enzyme is pH dependent, so the pH optimum of the enzyme is affected by the substrate concentration.
  • 6.6. The enzyme is inhibited 50% by 20 mM Ca2+ or Mg2+.
  • 7.7. The Ki for phosphate, EDTA-di sodium salt and arsenate (in 1 M diethanolamine, pH 11.1) is approx 1.2, 1.6 and 4mM respectively.
  • 8.8. Urea (200 mM) is not inhibitory.
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4.
  • 1.1. Malic enzyme purified from the fruit tissue of Mangifera indica was irradiated in dilute solution and the effect of γ-irradiation was investigated.
  • 2.2. The activity of the enzyme decreased exponentially as a function of the applied dose under all conditions investigated. The inactivation yield (Go-value) in neutral solution and in air was 0.069.
  • 3.3. The role of the radicals produced by water radiolysis in the inactivation of the enzyme was investigated by using different gas atmospheres and selective free radical-anions. The hydrogen atom and the hydrated electron (reducing species) were found to be important in the enzyme inactivation; as well as the possible destruction of cysteine and tryptophan residues.
  • 4.4. The irradiated enzyme appears to adopt a more compact conformation as reflected in a slightly lower Mr, Stokes-radius and diffusion coefficient.
  • 5.5. γ-Radiation does not lead to any heterogeneity in the charge and size properties of the enzyme and the pI and the Mr of the subunits were unaffected.
  • 6.6. Some differences in the amino acid composition of the non-irradiated and irradiated enzyme were observed but specific amino acid residues were not preferentially destroyed.
  • 7.7. These changes were also reflected in the ultraviolet spectrum of the enzyme which shifted to lower values.
  • 8.8. The major cause of inactivation seem to be a change in conformation caused by chemical modification of amino acid side chains.
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5.
  • 1.1. The d-lactate dehydrogenase from Leuconostoc lactis has been purified in high yield.
  • 2.2.The enzyme is a dimer of subunits of Mr = 39,000 and each subunit contains a single thiol group. The N-terminal residue is methionine.
  • 3.3. The amino acid composition has been determined and is typical of that of a soluble globular protein.
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6.
  • 1.1. Ferredoxin-glutamate synthase from the green alga Chlamydomonas reinhardii appears to contain as prosthetic groups, 1 FAD, 1 FMN and 1 |3Fe-xS | cluster, per molecule of Mr = 146,000.
  • 2.2. The synthesis of glutamate, catalyzed by this enzyme, proceeds through the formation of an enzyme-bound free radical of flavin semiquinone.
  • 3.3. This enzyme may catalyze the ferredoxin-dependent reduction of 2-oxoglutarate, in the absence of glutamine.
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7.
  • 1.1. Molecular weight estimation and subunit analysis of four yolk phosphoproteins (PP1-PP4) in medaka (Oryzias latipes) eggs were performed.
  • 2.2. PP1 (Mr ≈ 210,000) and PP2 (Mr ≈ 180,000) were found to be heterodimers composed of subunits of 113,000 and 94,000 and subunits of 84,000 and 72,000, respectively.
  • 3.3. PP3 and PP4 [phosvitins of medaka (Murakami et al., 1990, Devl. Growth Differ.32, 619–627)], were monomeric phosphoproteins having mol. wts of about 40,000 and about 20,000, respectively.
  • 4.4. Lipid composition of the mixture of PP1 and PP2, vitellogenin and yolk were found to be almost the same. PP1 and PP2 are probably lipovitellins of medaka.
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8.
  • 1.1. Mammalian major apurinic/apyrimidinic (AP) endonuclease, APEX nuclease (Mr 35.4 kDa) was purified from HeLa cells. A hybrid protein (Mr 36.4 kDa), which was expressed in BW2001 strain cells of E. coli, comprising human APEX nuclease headed by 10 additional amino acids was also purified.
  • 2.2. The purified preparations were frequently associated with 31-, 33- and 35-kDa peptides having AP endonuclease activity.
  • 3.3. The 33- and 35-kDa peptides were suggested to be formed from the hybrid protein or APEX nuclease during their purification processes by proteolytic cleavage with subtilisin-like protease. The 31-kDa peptide was thought to be produced by chemical cleavage of the aspartyl-prolyl bond of APEX nuclease.
  • 4.4. The results support the notion that some of AP endonuclease heterogeneity based on the molecular weight difference are caused by proteolytic (and chemical) cleavage of a species of AP endonucleases during the extraction and purification.
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9.
  • 1.1. A novel glycogen phosphorylase inhibitor was partially purified from crayfish hepatopancreas.
  • 2.2. The inhibitor was found only in two species of crayfish examined, and not in lobster, fresh and salt water clams, mussels or cockroaches.
  • 3.3. The inhibitor is a small protein (Mr = 23,000) which did not show proteolytic activity.
  • 4.4. Preliminary kinetic analysis of the inhibitory mechanism indicated that it bound to both glycogen and the glycogen phosphorylase protein.
  • 5.5. Inhibitor binding to glycogen resulted in a competitive inhibition pattern with respect to glycogen phosphorylase (inhibition constant of ca 10 μg/ml).
  • 6.6. The inhibitor also bound glycogen phosphorylase directly with a binding coefficient of 100 μg/ml resulting in a partially non-competitive inhibition pattern with respect to phosphate.
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10.
  • 1.1. An examination of proteins synthesized by Perinereis cultrifera oocytes incubated in vitro with [3H]leucine clearly shows that these cells are not capable of synthesizing the main yolk protein previously identified in this worm.
  • 2.2. In addition, the detection of radiolabelled vitellin in oocytes after in vitro incubation of an oocyte-coelomocyte cell mixture in presence of [3H]leucine strongly suggests that the coelomocytes, free cells in the coelomic cavity, synthesize and secrete a vitellin precursor, vitellogenin, that is subsequently taken up by the oocytes.
  • 3.3. Two native proteins differing in mol. wt but reacting with anti-vitellin antibodies have been identified in coelomocyte incubation medium. Also found in the coelomic fluid, they have been designated VG1 (Mr = 530,000) and VG2 (Mr = 320,000).
  • 4.4. The two vitellogenins consist of a single type of polypeptide of Mr = 176,000 and are incorporated in the oocytes where they are apparently observed under a single molecular form corresponding to VG1, the highest mol. wt protein similar in size to the initial form of vitellin (VI, 530,000).
  • 5.5. From these data, it seems likely that VG2 is a monomeric molecule that is taken up by the oocytes as a dimer of VG1.
  • 6.6. We conclude that P. cultrifera accumulates vitellin heterosynthetically and that vitellogenin is produced by the coelomocytes. Moreover, a single polypeptide similar in size to the polypeptidic component of secreted vitellogenin has been detected in the coelomocytes.
  • 7.7. Since this polypeptide has been identified previously as the single intraoocytic precursor of the four lower mol. wt products that make up the mature form of vitellin (V5), it appears that P. cultrifera exhibits for vitellogenin a processing pathway in which cleavage of the precursor occurs only after uptake by the oocyte.
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11.
  • 1.1. The degradation of the bone matrix proteins osteocalcin, osteonectin and α2HS-glycoprotein by human cathepsins B and L and human osteoclastoma cathepsins has been investigated.
  • 2.2. Intermediate degradation products (Mr > 12kDa) were not observed during the digestion of α2HS-glycoprotein and osteonectin by cathepsins B and L although they were observed with some of the osteoclastoma cathepsins. Most of the osteoclastoma cathepsins were capable of degrading these two proteins to small peptides at comparable rates.
  • 3.3. Each cathepsin produced a different pattern of osteocalcin degradation products.
  • 4.4. The extensive range of non-collagenous proteins in bone matrix may necessitate the production by osteoclasts of cathepsins with different specificities during bone resorption.
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12.
  • 1.1. Isoenzymes of d-lactate specific dehydrogenase from foot, mantle and hepatopancreas of Patella caerulea have been purified by Chromatographic techniques. d-lactate dehydrogenase (d-Ldh) from P. caerulea tissues was found to be tetrameric with a Mr of ca 140,000 as judged by gel filtration; subunit Mr of ca 37,000 was obtained from SDS-electrophoresis.
  • 2.2. Kinetic studies suggest that P. caerulea foot and mantle d-Ldh is similar to vertebrate muscle-type l-Ldh; furthermore hepatopancreas d-LDH resembles vertebrate heart-type l-LDH since it has a higher affinity for d-lactate and is inhibited by pyruvate.
  • 3.3. The results imply that the P. caerulead-Ldh isoenzymes may have distinct metabolic functions.
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13.
  • 1.1. An inducible Cd-binding compound has been detected in a Cd-treated euryhaline alga, Dunaliella bioculata.
  • 2.2. The apparent molecular mass of this heat-stable Cd-binding compound, as determined by gel filtration, was about 10,000.
  • 3.3. Anion exchange chromatography (on DEAE Sepharose and Mono Q) and autoradiography of non-denaturing PAGE of the Mr 10,000 fractions revealed the presence of a highly acidic Cd-binding protein which differs on its properties from mammal metallothioneins.
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14.
  • 1.1. Exposing adult salamanders, Eurycea bislineata and Desmognathus ochrophaeus, to heat shocks of 1 hr at 2 or 5°C below Critical Thermhal Maximum (CTM) resulted in the induction of two heat shock proteins (hsps) of approx. Mr 70,000 and 30,000.
  • 2.2. Induction patterns in response to similar heat shocks generally differed between the two species.
  • 3.3. The milder heat shocks (5°C below CTM) caused different induction patterns than those from the more severe heat shocks, on a tissue-dependent basis. These results indicate that induction of the two hsps is probably independent.
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15.
  • 1.1. α2-Macroglobulin (α2M) activity is present in the serum of the ostrich, Struthio camelus. The chromogenic synthetic peptide substrates BAPNA and ATNA were hydrolysed by trypsin and chymotrypsin, respectively, in the presence of ostrich serum and the α2M in ostrich serum protected trypsin from being inhibited by soybean trypsin inhibitor. Ostrich α2M proved to be a potent inhibitor of bovine pancreatic trypsin and chymotrypsin.
  • 2.2. α2M was purified to apparent homogeneity by PEG precipitation, DEAE-Toyopearl 650M, Bio-Gel A-5m and Zn2+-affinity chromatography.
  • 3.3. Ostrich α2M migrated as a single band (Mr 779,000) during non-denaturing gradient gel electrophoresis and showed increased mobility after reaction with trypsin. Denaturation dissociated ostrich α2 M into half-molecules. Denaturation with reduction further dissociated the protein into quarter-subunits.
  • 4.4. Isoelectric focusing revealed a pI of 5.3.
  • 5.5. The amino acid composition of ostrich α2M is typical of an α2M, comparing favourably with those of other animal species. The carbohydrate composition of the purified protein, in percentage dry weight of the molecule, was galactose: mannose (1:1), 4.55; N-acetylglucosamine, 2.35; N-acetylneuraminic acid, 0.58; and fucose, 0.77.
  • 6.6. α2M was assessed immunologically by Ouchterlony double-diffusion and Western blot analysis with polyvalent antisera directed against ostrich α2M.
  • 7.7. Ostrich α2M seems to show many physical, chemical and kinetic properties similar to those of other known α2Ms, but is expected to differ from other αMs when considering the primary structure of the bait region, the area differing among α Ms from different species and determining its specificity.
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16.
  • 1.1. Kinetic and physico-chemical studies on human placental microsomal fraction confirmed that the ATPase and ADPase activities detected in this fraction correspond to the enzyme ATP-diphosphohydrolase or apyrase (EC 3.6.1.5). These include substrate specificity, and coincident Mr and pI values of both ATPase-ADPase activities.
  • 2.2. This enzyme hydrolyses both the free unprotonated and cation-nucleotide complex, the catalytic efficiency for the latter being considerably higher.
  • 3.3. Microsomal apyrase is insensitive to ouabain and Ap5A. The highly purified enzyme was only inhibited by o-vanadate, DBS and slightly by DCCD.
  • 4.4. Apyrase seems to be a glycoprotein from its interaction with Concanavalin-A.
  • 5.5. Preliminary studies on the essential amino acid residues suggest the participation of Arg, Lys and His residues, and discard the requirement of −SH, COO, −OH, and probably also Tyr and Trp.
  • 6.6. Two kinetic modulatory proteins of apyrase were detected in placental tissue. An activating protein was found in the soluble fraction and an inhibitory protein was loosely bound to the membranes.
  • 7.7. The proposed in vivo function for apyrase is related to the inhibition of platelet aggregation due to its ADPase activity, which is supported by the direct effect on washed platelets and by its plasma membrane localization.
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17.
  • 1.1. In Musca domestica haemolymph a lipid transfer particle (LTP) is present.
  • 2.2. Musca domestica LTP is able to catalyze the transfer of lipids between different housefly lipophorin forms and also between lipophorins of Diptera and Lepidoptera.
  • 3.3. The lipophorin of larval Dione juno (Lepidoptera) was purified and is composed of two apolipoproteins, apolipophorin I (Mr = 209,000) and apolipophorin II (Mr = 85,000) with a density of 1.124 g/ml.
  • 4.4. The density of housefly lipophorin undergoes variations during the gonotrophic cycle.
  • 5.5. The lipophorin density variation results suggest that when a high rate of lipid utilization occurs, the lipophorin has a higher density value.
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18.
  • 1.1. A 1.7S protein has been purified from mustard seeds (Sinapis alba L.). This protein, soluble in water and dilute salt solutions, is considered as an albumin and constitutes about 10% of the total soluble protein in mustard seeds.
  • 2.2. Its molecular weight is approximately 15,000 and is composed of two polypeptide chains (Mr = 9500 and 5000), linked by two disulfide bridges.
  • 3.3. The amino acid compositions of both subunits as well as of the native protein are reported, showing a strong homology with napins from Brassica napus L.
  • 4.4. The ultraviolet absorption, fluorescence emission and circular dichroism spectra of the purified protein have been obtained. The mustard protein exhibits about 50% α-helix with a very low β-structure content. Based on its structural characteristics, a zein-like packing is proposed for this protein from mustard seeds.
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19.
  • 1.1. In the rat, acetyl-CoA carboxylase (ACC), a rate-limiting enzyme in fatty acid metabolism, exists as at least two different isozymes (Mr 265,000 and 280,000) that display distinct tissue-specific distribution and regulation.
  • 2.2. Based on the study of human tissue and human-derived breast cancer cell lines by enzyme isolation and protein blotting techniques, we have now identified two human isoforms of Mr 265,000 (HACC 265) and 275,000 (HACC 275), each of which is homologous to one of the rat isozymes.
  • 3.3. Human breast carcinoma cell lines show variable expression of these two isoforms, mirrored in the estimation of ACC acetyl-CoA kinetics.
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20.
  • 1.1. Pseudomonas aeruginosa phospholipase C from culture supernatants of bacteria grown in high-Pi basal salt medium with choline, as the sole carbon and nitrogen source, was purified by precipitation with 70% saturation ammonium sulfate in the presence of celite.
  • 2.2. The PLC activity was eluted of this mixture by the use of a reverse gradient of 70-0% ammonium sulfate.
  • 3.3. The peak containing the PLC activity revealed a single protein after SDS-PAGE.
  • 4.4. The method could also be applied to purify PLC produced in a low-Pi complex medium. The resultant preparation was not homogeneous.
  • 5.5. The molecular weight for both PLC preparations was about 70 kDa.
  • 6.6. Both PLC used phosphatydilcholine and sphingomyelin as substrates, displayed hemolytic activity an exhibited an apparent KM of 25 mM for p-nitrophenylphosphorylcholine.
  • 7.7. They were not inhibited by 1% sodium deoxycholate but were 30% inhibited by 1% Triton X-100.
  • 8.8. 2% sodium dodecylsulfate and 1% tetradecyltrimethylammonium bromide inhibited the PLC from the HPl-BSM plus choline but not the enzyme from the LPl-CM.
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