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1.
Three types of invertase (invertase I, II and III) are separatedfrom the soluble and insoluble fractions (4,500xg, 10 min supernatantand pellets of the homogenate, respectively) of baker's yeastby a DEAE cellulose column chromatography. The invertases Iand II are eluted with 0.1 M sodium acetate buffer (pH 3.9)and with 0.1 M sodium acetate buffer (pH 6.2) containing 0.1M NaCl from DEAE cellulose respectively, whereas the invertase-IIIremains adsorbed on the cellulose under these conditions. Theyare present in proportions of 2.5: 1 : 0.06 in the soluble fractionand 1.4: 1 : 0.12 in the insoluble fraction of the fresh baker'syeast cells. While in-vertase-II remains at a constant level,invertases I and III in the soluble fraction increase upon incubationof cells for the formation of invertase under the continuoussupply of sucrose. Invertases I and II differ from each other considerably in theoptimum pH and slightly in the response to (activation and inactivationby) crude papain and are identical with respect to the heatstability and probably to the affinity for sucrose.
1Present address: Chemical Laboratory, Nippon Medical School,Konodai, Ichikawa-shi, Chiba-ken. 相似文献
2.
SYNOPSIS. DNA-dependent RNA polymerases have been solubilized from homogenates of Crithidia fasciculata using gentle extraction procedures. RNA polymerase I and II are separated on DEAE cellulose at 0.07M (NH4)2SO4 and 0.13M (NH4)2SO4 respectively. RNA polymerase II is inhibited 80% by α-amanitin (25 μg/ml). Both RNA polymerases require DNA as a template, ribonucleoside triphosphates and Mn2+. The synthesis of RNA as a product is inhibited by DNase. RNase, pronase and actinomycin D. Purified kinetoplast and nuclear DNA can serve as templates for the RNA polymerases. Denatured DNA templates are preferred. The synthesis of RNA continues for at least an hour and is inhibited by trypanocidal drugs including suramin. antrycide, acriflavine, ethidium bromide and berenil. Complementary RNA synthesized in vitro from C. fasciculata kinetoplast DNA hybridizes with C. fasciculata kinetoplast DNA but not with C. fasciculata nuclear DNA or Blastocrithidia culicis kinetoplast DNA, Escherichia coli, T4 or calf thymus DNAs. The complementary RNA synthesized in vitro from C.fasciculata kinetoplast DNA sediments at 4–5S. 相似文献
3.
Structure-activity study of cytotoxicity and microtubule assembly in vitro by taxol and related taxanes 总被引:1,自引:0,他引:1
J Parness D G Kingston R G Powell C Harracksingh S B Horwitz 《Biochemical and biophysical research communications》1982,105(3):1082-1089
Fractionation of bovine brain cytosol by DEAE cellulose chromatography revealed the presence of a calcium-dependent protein kinase. This soluble neuronal protein kinase selectively phosphorylated several endogenous substrates. The most prominent substrate was a polypeptide with an apparent Mr of 45,000 which was stimulated 20-fold by addition of both calcium and calmodulin. Activation was dose-dependent, with half-maximal phosphorylation occurring at 0.9 μM free Ca2+ and 60nM calmodulin. The effect of calmodulin was competitively inhibited by a variety of calmodulin inhibitors, in a manner characteristic of most calmodulin-dependent enzymes. This calcium- and calmodulin-dependent protein kinase is distinct from any previously described protein kinase. 相似文献
4.
Poly(A) polymerase activity was first detected in yeast extracts, primarily in association with the ribosomal fraction, by Twu and Bretthauer in 1971 (Twu, J. S., and Bretthauer, RK. (1971) Biochemistry 10, 1576-1582). This activity has now been separated into three distinct enzymes by chromatography on DEAE-cellulose. Each of the three enzymes can catalyze the incorporation of adenylate residues from ATP into a polyadenylate (poly(A)) tract at the 3' terminus of a primer RNA. Enzyme I elutes at 0.07 M ammonium sulfate from the DEAE-cellulose column, utilizes the mixed polynucleotide poly(A,G,C,U) or ribosomal RNA most efficiently in vitro, and may be responsible in vivo for the initiation of the poly(A) tracts found on yeast messenger RNA. Enzyme II elutes from the column at 0.20 M ammonium sulfate, requires poly(A) itself or an RNA primer containing a 3'-oligo(A) tract, and may be responsible in the nucleus for the elongation of tracts initiated by enzyme I. Enzyme III elutes from the column at 0.56 M ammonium sulfate and is present in low amounts in nuclear extracts. It may be involved in adding poly(A) tracts to messenger RNA in mitochondria. These enzymes also have the intrinsic capacity for the incorporation of cytidylate residues from CTP, which correlates with the finding of cytidylate residues in the poly(A) tracts present in the yeast RNA which is rapidly labeled in vivo. About 75% of the total poly(A) polymerase activity of yeast is enzyme I, most of which is present in the soluble protein fraction of the whole yeast extract. About 20% of the total poly(A) polymerase is enzyme II, and 1 to 5% is enzyme III. All three of the yeast poly(A) polymerases require an RNA primer with a free 3'-hydroxyl group, show no requirement for a DNA template, require Mn-2+ for optimal activity, have pH optima of 8.5, and are inhibited by GTP, CTP, UTP, and native yeast DNA. Polymerases I and II have similar molecular weights by gel filtration. 相似文献
5.
The study addressed the question of whether35SO4 labeled molecules that the have been delivered to the goldfish optic nerve terminals by rapid axonal transport include soluble proteoglycans. For analysis, tectal homogenates were subfractionated into a souluble fraction (soluble after centrifugation at 105,000g), a lysis fraction (soluble after treatment with hypotonic buffer followed by centrifugation at 105,000g) and a final 105,000g pellet fraction. The soluble fraction contained 25.7% of incorporated radioactivity and upon DEAE chromatographys was resolved into a fraction of sulfated glycoproteins eluting at 0–0.32 M NaCl and containing 39.5% of total soluble label and a fraction eluting at 0.32–0.60 M NaCl containing 53.9% of soluble label. This latter fraction was included on columns of Sepharose CL-6B with or without 4 M guanidine and after pronase digestion was found to have 51% of its radioactivity contained in the glycosaminoglycans (GAGs) heparan sulfate and chondroitin (4 or 6) sulfate in the ratio of 70% to 30%. Mobility of both intact proteoglycans and constituent GAGs on Sepharose CL-6B indicated a size distribution that is smaller than has been observed for proteoglycans and GAGs from cultured neuronal cell lines. Similar analysis of lysis fraction, containing 11.5% of incorporated35SO4, showed a mixture of heparan sulfate and chondroitin sulfate containing proteoglycans, apparent free heparan sulfate and few, if any, sulfated glycoproteins. Overall, the result support the hypothesis that soluble proteoglycans are among the molecules axonally transported in the visual system. 相似文献
6.
DNA polymerase γ, cytochrome c oxidase and mitochondrial integrity in rabbit spleen lymphocytes stimulated with concanavalin A 总被引:1,自引:0,他引:1
N. Hardt D. De Kegel L. Vanheule G. Villani S. Spadari 《Experimental cell research》1980,127(2):269-276
The activities of two mitochondrial enzymes: DNA polymerase γ and cytochrome c oxidase, have been compared with the kinetics of nuclear DNA synthesis and levels of the nuclear DNA polymerases α and β in rabbit spleen lymphocytes untreated or stimulated with concanavalin A (ConA). At early initiation of the culture, before the maximum increase of both the replicative α-polymerase and nuclear DNA synthesis, a simultaneous enhancement in the activities of the two mitochondrial enzymes was observed, just preceding and paralleling the expansion of the ATP pool. It is suggested that this is a reflection of increased mitochondrial activity providing the energy which results in an elevation of the concentration of dNTPs to values which are optimal for the replicative α-polymerase. During the later stages of incubation of lymphocytes, the activities of both DNA polymerase γ and cytochrome c oxidase decrease, paralleling a progressive loss of integrity of the mitochondrial structures within an increasing proportion of the lymphocyte population. 相似文献
7.
L C Yeoman S Seeber C W Taylor D J Fernbach J M Falletta J J Jordan H Busch 《Experimental cell research》1976,100(1):47-55
Two-dimensional polyacrylamide gel electrophoresis comparisons were made for the non-histone “Chromatin fraction II” proteins of normal, phytohemagglutinin-stimulated and acute leukemic lymphocytes. The “Chromatin fraction II” proteins were extracted from the nuclear residue fraction after initial treatment with (a) 0.075 M NaCl containing 0.025 M EDTA, pH 8; (b) 0.01 M Tris-HCl, pH 8; and (c) 0.4 N H2SO4. Most of the proteins found earlier in the “Chromatin fraction II” of rodent liver and hepatomas were also found in the human cells. Some changes such as the decrease in amount of protein BA of normal rodent cells were found in the comparison of normal and stimulated human cells. By comparison with normal lymphocytes, the phytohemagglutinin-treated cells had decreased spot densities and sizes for proteins BA and Bv and an increase in densities and sizes of proteins CB, C25, CS and CT. In the acute lymphocytic leukemic cells there was a decrease in spots A24, BA, Bv, CD and CD′ by comparison with the normal lymphocytes. Protein CG′ which was found earlier in the hepatomas was found in acute lymphocytic leukemic cells but not in the control or phytohemagglutinin-treated cells. These studies show that there is a loss in specific Chromatin proteins BA and Bv from the Chromatin of rapidly turning over cells. Concomitantly, increases are observed for the amounts of protein spots CB, C25, CS and CT in the actively growing cell samples. 相似文献
8.
Transformation of normal human peripheral lymphocytes by phytohemagglutinin (PHA) to blast-like cells is accompanied by an enhancement of the protein phosphokinase activity. This activity becomes maximal approx. 70 h after exposure to the mitogen and amounts to a 3- to 4-fold stimulation in the 10 000 g supernatant and 8- to 10-fold in the nuclear fraction. This augmented activity is due to the increased level of some of the multiple forms of lymphocyte protein kinase, specially the one which is active on exogenous casein and elutes from DEAE-cellulose at 125 mM phosphate (casein-kinase S-C3 and N-C2). Acute lymphoblastic leukemic cells have a protein kinase pattern upon chromatography on DEAE-cellulose which is similar, but not identical, to that of PHA-stimulated lymphocytes. The most remarkable difference is the presence in the 10 000 g supernatant of leukemic cells of a protein kinase form which was either absent or barely detectable in resting or PHA-treated normal lymphocytes. This protein kinase is active on casein and is not stimulated by cAMP. The results obtained are discussed in connection both with the known enhanced protein phosphorylation in PHA-stimulated cells and with the protein kinase changes observed in other cellular systems. 相似文献
9.
Nuclear and cytoplasmic protein kinases were measured during the traverse of synchronous CHO cultures through G1 into S phase. Cells were synchronized by selective detachment of cells blocked in metaphase using colcemid. Nuclei were isolated and the protein kinases extracted from the nuclear preparation with 0.6 M NaCl. This procedure solubilized greater than 90% of the total protein kinase activity present in the nuclear preparation. DEAE chromatography of this extract showed 5 apparently different ionic forms of nuclear protein kinases. The nuclear protein kinases preferred casein and phosvitin to histone as substrates and were cyclic AMP-independent. Nuclear protein kinase activities increased greater than two-fold, when expressed as units of activity per cell nucleus, during G1 phase traverse, concomitant with a 70% increase in nuclear non-histone proteins (those soluble in 0.6 M NaCl). This resulted in only a 40% increase in the specific activities (units/microgram protein in 0.6 M NaCl extractable nuclear fraction) of these enzymes as cells progressed through G1 into S phase. This was in contrast to cytoplasmic cyclic AMP-dependent protein kinase activities which also increased two-fold during progression through G1 phase while total cellular protein increased less than 20%. Activation of, as well as synthesis of, cyclic AMP-dependent cytoplasmic protein kinases during G1 phase suggests a regulatory mechanism for precise temporal phosphorylation, whereas the constant specific activity in nuclear kinases during cell cycle is more compatible with the maintenance of bulk phosphorylation processes in the nucleus. 相似文献
10.
Schizophyllum commune produced an esterase which released ferulic acid from starch-free wheat bran and from a soluble ferulic acid-sugar ester that was isolated from wheat bran. The preferred growth substrate for the production of ferulic acid esterase was cellulose. Growth on xylan-containing substrates (oat spelt xylan and starch-free wheat bran) resulted in activity levels that were significantly lower than those observed in cultures grown on cellulose. Similar observations were made for endoglucanase, p-nitrophenyllactopyranosidase, xylanase, and acetyl xylan esterase. Of the enzymes studied, only arabinofuranosidase was produced at maximum levels during growth on xylan-containing materials. Ferulic acid esterase that had been partially purified by DEAE chromatography released significant amounts of ferulic acid from wheat bran only in the presence of a xylanase-rich fraction, indicating that the esterase may not be able to readily attack high-molecular-weight substrates. The esterase acted efficiently, without xylanase addition, on a soluble sugar-ferulic acid substrate. 相似文献
11.
The soluble hydroperoxide isomerase and 15-lipoxygenase activities were partially purified from the oomycete Saprolegnia parasitica and some of their properties characterized. Both enzymes co-eluted with a molecular weight of 145,000–150,000 on Sephacryl S-300 chromatography. The enzyme activities also co-eluted on DEAE Sephadex ion exchange chromatography and hydroxylapatite chromatography. Both activities showed similar responses to pH and temperature. Both enzymes showed parallel inhibition by p-hydroxymercuribenzoate and eicosatetraynoic acid. The partially purified hydroperoxide isomerase showed an apparent km of 166 μM and a Vmax of 5.3 μmol/min/mg protein for exogenous 15-HPETE. It was not stimulated by calcium. These results suggest that the soluble hydroperoxide isomerase and 15-lipoxygenase activities from S. parasitica are both contained on the same protein or protein complex. 相似文献
12.
Rat lymph chylomicrons were separated into two fractions using heparin-Sepharose chromatography: a major fraction which elutes from the column with the void volume at 0.05 M NaCl, and a smaller fraction which binds to the column at 0.05 M NaCl and elutes at 0.3 M NaCl. These two fractions differ in mean particle size, and lipid and protein compositions. Both fractions share apolipoproteins B, A-IV, E, A-I, and C, but the fraction which binds to heparin-Sepharose contains two additional proteins: protein I (Mr = 6.0 X 10(4)), and protein II (Mr = 8.0 X 10(4)). Both proteins are also present in the lipoprotein-free fraction of rat serum. Proteins I and II bind to heparin-Sepharose, and are highly amphiphilic: they bind with high affinity to phospholipid surfaces and form stable monolayers at the air-water interface. The molecular weight, amino acid composition, heparin binding, and amphiphilicity of protein I resemble that of beta 2-glycoprotein I; in addition, protein I from rat lymph chylomicrons cross-reacts with rabbit antiserum to human beta 2-glycoprotein I, suggesting that these two proteins are homologous. Protein II appears to be a previously undescribed protein. The possible functions of these two proteins are discussed. 相似文献
13.
Alkaliphilic and halophilic Bacillus sp. BG-CS10 was isolated from Zabuye Salt Lake, Tibet. The gene celB, encoding a halophilic cellulase was identified from the genomic library of BG-CS10. CelB belongs to the cellulase superfamily
and DUF291 superfamily, with an unknown function domain and less than 58% identity to other cellulases in GenBank. The purified
recombinant protein (molecular weight: 62 kDa) can hydrolyze soluble cellulose substrates containing beta-1,4-linkages, such
as carboxylmethyl cellulose and konjac glucomannan, but has no exoglucanase and β-glucosidase activities. Thus, CelB is a
cellulase with an endo mode of action and glucomannanase activity. Interestingly, the enzyme activity was increased approximately
tenfold with 2.5 M NaCl or 3 M KCl. Furthermore, the optimal temperatures were 55°C with 2.5 M NaCl and 35°C without NaCl,
respectively. This indicates that NaCl can improve enzyme thermostability. The K
m
and k
cat values of CelB for CMC with 2.5 M NaCl were 3.18 mg mL−1 and 26 s−1, while the K
m
and k
cat values of CelB without NaCl were 6.6 mg mL−1 and 2.1 s−1. Thus, this thermo-stable, salt and pH-tolerant cellulase is a promising candidate for industrial applications, and provides
a new model to study salt effects on the structure of protein. 相似文献
14.
Masayoshi Takakuwa Youichi Tamai Masahisa Kushibe 《Bioscience, biotechnology, and biochemistry》2013,77(10):2071-2073
Intracellular arylsulfatases from Klebsiella aerogenes W70 cells grown in methionine medium (M enzyme) and inorganic sulfate medium containing tyramine (T enzyme) were purified respectively by fractionation with (NH4)2SO4, followed by successive chromatographies on DEAE cellulose, hydroxylapatite, Sephadex G-100 and DEAE Sephadex A-25. On polyacrylamide gel electrophoresis, the two enzymes gave single bands with the same mobilities. Molecular weights of both, determined by SDS gel electrophoresis and by Sephadex G-100 chromatography, were 47,000 and 45,000, respectively. Their activities were maximal at pH 7.5. The affinities of the enzymes (M and T enzymes) for their substrate (Km) and the maximum velocity of hydrolysis (Vmax) were enhanced by addition of electron withdrawing substituents. The enzymes were inhibited by inorganic phosphate, cyanide, hydroxylamine and tyramine. The inhibition by tyramine was competitive (Ki = 1.0 × 10?4 m). These results show that the two enzymes were identical. This was confirmed by the fact that mutant strains, which were unable to synthesize arylsulfatase when grown with methionine, could also not synthesize the enzyme when grown with tyramine. 相似文献
15.
The caseinolytic enzymes of the midgut lumina and epithelia of Leucophaea were purified through precipitation by 60% saturated (NH4)2SO4, followed by gel permeation on Sephadex G-200 and subsequent DEAE anionexchange chromatography. At least four peaks with enzyme activity were eluted from anionexchange chromatography columns. Gregarines of the midgut lumen apparently do not contribute to the caseinolytic activity within the midgut. Elution profiles of lumen and epithelial enzymes were nearly identical. The same enzymes were identified in the lumina of epithelial microsomal vesicles. This allows the conclusion that these enzymes are produced by the midgut epithelia.Practically all protease activity of the midgut was found in the posterior half, both in the lumen and epithelium. Feeding stimulated protease production primarily in the posterior midgut. The pH optimum of the proteases lay between 9.0 and 9.5 which was closely matched by the observed pH of the posterior midgut where most of the activity is seen. The anterior midgut pH was determined to be around 8.0.The anterior midgut of Leucophaea contained a heatstable protease inhibitor with characteristics of a competitive inhibitor. This inhibitor was precipitable by 60% saturated (NH4)2SO4 and eluted from a Sephadex G-200 column more or less together with the proteases. From a DEAE anionexchange column it was eluted by 0.8 M NaCl, i.e. after the main portion of the proteases. The biological significance of the protease inhibitor in the anterior portion of the midgut is obscure. 相似文献
16.
Villani G Canosi U Fortunato A Mazza G Polsinelli M Falaschi A 《Nucleic acids research》1974,1(3):461-477
We have isolated a mutant of Bacillussubtilis deficient in DNA polymerase I, denominated polA42, which shows a reduced ability to repair the damage to DNA by UV radiation, MMS and mitomycin C;the ability to perform recombination is not appreciably impaired.DEAE cellulose chromatography allows the separation of polymerases I and II from the parental strain;a simple procedure is also described which allows to separate rapidly the polymerases II and III of the mutant strain. The three separated polymerases have similar catalytic properties but they can be distinguished for their sensitivity to inhibitors: PCMB inhibits polymerases II and III but not polymerase I; HPUra inhibits only polymerase III. All three enzymes are unaffected by nalidixate. The DNA synthesis occurring in cells of the polA42 strain permeabilized with toluene is inhibited by nalidixate, whereas the synthesis occurring in polA+ toluenized cells is unaffected by the drug. The polA gene has been mapped by transduction and localized between the phe12 and argA3 genes. 相似文献
17.
Globulins were prepared by repeated precipitation with 1.3 M (NH4)2SO4 from a 0.7 M NaCl extract of milled rice. Isoelectric precipitation at pH 4.5 did not effectively remove the α-globulin from the others. A major fraction that remained in solution during dialysis of the globulin precipitate against water was similar in properties to the globulin soluble at pH 4.5 during the isoelectric precipitation process. Some properties of this water-soluble globulin fraction are reported. Proteins extracted from milled rice at 50° with 0.5 M NaCl and precipitated as 1- to 3-μm particles on cooling were verified to be globulins. 相似文献
18.
DNA polymerases were purified several hundred-fold from the10 000 x g soluble (polymerase I) and particulate (polymeraseIII) fractions prepared from virus PBCV-1 infected ChlorellaNC64A extracts. Both DNA polymerases exhibited optimal activitywith activated calf thymus DNA at pH 8.5. DNA polymerase I required3.0 mol m3 MgSO4 and 150 to 250 mol m3 KCl foroptimum activity whereas, DNA polymerase III required 2.0 molm3 MgSO4 and 150 mol m3 KCl. Both enzymes wereinhibited by pyrophosphate, actinomycin D, ethidium bromide,dideoxythymidine triphosphate, and N-ethylmaleimide but wererelatively insensitive to aphidicolin. DNA polymerase I differedfrom DNA polymerase III in its response to cations (particularlyNH4Cl), elution from a DEAE cellulose column, and molecularweight. Key words: Algal virus, DNA polymerase, Chlorella 相似文献
19.
《Journal of Fermentation Technology》1986,64(3):219-225
d-Xylulose reductase (EC 1.1.1.9) from Pachysolen tannophilus IFO 1007 was purified by Sephadex G-100 gel chromatography with three columns and DEAE cellulose chromatography. The purified enzyme was entirely homogeneous on disc gel electrophoresis. It was most active at pH 9.1–10.0 and 55°C, and stable at pH 7–9 and below 25 °C. Its activity was stimulated by NH4Cl,NaCl,MgCl2,KCl, glutathione, cysteine and glycine, and inhibited remarkably by SH inhibitor such as lead acetate, HgCl2 and AgNO3. It oxidized xylitol, sorbitol, ribitol and glycerine but not mannitol, inositol, arabitol and erythritol. Its Km values of enzyme against xylitol, sorbitol and ribitol were 1.1 × 10−2 M, 3.0 × 10−2 M and 5.0 × 10−2 M, respectively. Its molecular weight was determined to be 120,000 by Sephadex G-200 column chromatography, and that of its subunit was 40,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. 相似文献
20.
Media of pig aorta was extracted with 1 M NaCl and 2 M MgCl2 to remove most of the soluble collagen, proteoglycans and glycoproteins. The glycoproteins remaining in the residue were extracted with 6 M urea-0.1 M mercaptoethanol. The urea soluble proteins were precipitated by dialysis, redissolved in 4 M guanidine-0.05 M DTT and were S-carboxamidomethylated (CM-guanidine extract). This extract was further fractionated by a variety of methods in order to separate a glycoprotein from collagen and proteoglycans. Caesium chloride density-gradient ultracentrifugation of the CM-guanidine extract separated a minor proteoglycan peak from a major glycoprotein fraction still containing some hydroxyproline. This major glycoprotein fraction was excluded as a single peak from Sephadex G 100 and G 200 in 4 M guanidinium chloride or in 6 M urea-0.2 per cent SDS. Sodium dodecylsulphate gel electrophoresis separated this high molecular weight Sephadex fraction into a major low molecular weight (approximately 35000 daltons) component and a minor high molecular weight component. This glycoprotein fraction could also be separated from a collagenous fraction and from proteoglycans by ion exchange chromatography on DEAE cellulose or by gelfiltration on Sepharose 4 B in 6 M urea-0.02 M EDTA-0.2 per cent SDS at pH 7.0. The isolated glycoprotein fraction is rich in dicarboxylic amino acids, contains galactose, mannose, (glucose), N-acetylglucosamine and sialic acid. The S-carboxamidomethyl glycoprotein preparation interacts with acid soluble calf skin collagen on isoelectric focusing in sucrose gradient in urea. This interaction is in favour of the biological role claimed for structural glycoproteins during fibrogenesis and differentiation. 相似文献