共查询到20条相似文献,搜索用时 15 毫秒
1.
Stuart L. Marcus Gregg Lipschik Generosa Trueba Cyrus J. Bacchi 《Biochemical and biophysical research communications》1980,93(4):1027-1035
A single peak of DNA polymerase activity from extracts of , obtained by DEAE-cellulose and phosphocellulose ion-exchange chromatography, was resolved into two peaks differing in KCl concentration necessary to elute them from a DNA-agarose column. Peak I (eluting at 0.2 M KCl) and Peak II (eluting at 0.4 M KCl), differed in response to increasing KCl concentrations, although both functioned optimally with Mg2+ as divalent cation when DNA synthesis was directed either by activated DNA or poly (dC)·(dG)12–18. Due to the potential significance of polyamines in the metabolism of , the effect of exogenous polyamine on rates of DNA synthesis by the peak I and II enzymes was compared with that of murine DNA polymerase alpha. Only the peak I enzyme was significantly stimulated (up to 4-fold) by the biologically active polyamines spermine and spermidine at physiological concentrations. The response of the peak I enzyme resembled that of the alpha polymerase. This result suggests a possible functional difference between peak I and II enzymes, as well as a potential target site for trypanocidal drug development. 相似文献
2.
C O Manahan A A App C C Still 《Biochemical and biophysical research communications》1973,53(2):588-595
Rice callus tissue in suspension culture was labeled with 32PO4?3. Approximately 4% of the total RNA extracted from the tissue could be bound to nitrocellulose filters in the presence of 0.5 M KCl. The base composition of this material was: A, 30.6; U, 32.7; G, 18.0; and C, 18.6%. If the RNA was digested with both T1 and pancreatic ribonucleases prior to binding, the base composition of the bound material was: A, 97.8; U, 0.0; G, 1.6; and C, 0.5%. About 14% of the RNA isolated from purified polyribosomes could be bound to filters in 0.5 M KCl. It is concluded that higher plant RNA contains polyadenylate sequences. 相似文献
3.
Tetsuo Sawai Laura J. Crane J.O. Lampen 《Biochemical and biophysical research communications》1973,53(2):523-530
The plasma membrane-bound penicillinase of has been purified. Amino acid analysis showed no significant differences in composition between the enzyme and exopenicillinase. Enzyme purified from cultures containing H333PO4 or [3H]-glycerol contained 33P or [3H]-glycerol activity and treatment with 8 M urea, 0.2% sodium dodecyl sulfate at 80° C did not remove the 3H-activity from the enzyme protein. Trypsin readily cleaved the glycerol-containing moiety from the enzyme protein, forming enzyme with molecular weight and heat stability like that of the exoenzyme. Phospholipase D and C also produced enzyme resembling the exo-form. 相似文献
4.
A Stevens 《Biochemical and biophysical research communications》1973,54(2):488-493
The content of the sigma subunit (as detected by gel electrophoresis) and activity with T4 DNA were examined with RNA polymerase fractions from both normal and T4 phage-infected . Sigma-containing fractions and core enzymes were obtained by phosphocellulose column chromatography. The sigma-containing fraction of the enzyme from infected cells, although somewhat stimulatory to both core enzymes alone, inhibits the normal sigma-stimulated activity of the core enzyme from infected cells at both low and high KCl concentration. Normal core enzyme activity is inhibited only at high KCl concentration. 相似文献
5.
Frank J. Castora Gary M. Lazarus 《Biochemical and biophysical research communications》1984,121(1):77-86
Mitochondria from human acute lymphoblastic leukemia cells contain an ATP-independent DNA topoisomerase which can relax negative and positive supercoils. This enzyme has been purified 200-fold by carboxymethyl-cellulose or double stranded DNA-cellulose chromatography. In contrast to the molecular weights reported for mitochondrial topoisomerases in other systems, the native leukemia enzyme has a molecular weight of 132,000 daltons as determined by gel permeation chromatography in buffer containing 0.4 M KCl. It also exhibits a sedimentation coefficient of 7.1 S when centrifuged through a 10–30% glycerol gradient in this high salt buffer. The enzyme is presumably a type I topoisomerase analogous to those found in rat liver and mitochondria. 相似文献
6.
H N Lightfoot M Mumby J A Traugh 《Biochemical and biophysical research communications》1975,66(4):1141-1146
Phosphoprotein phosphatase activities which remove phosphoryl groups from ribosomal protein have been partially purified from rabbit reticulocytes by chromatography on DEAE-cellulose. Two major peaks of phosphoprotein phosphatase activity were observed when 40S ribosomal subunits, phosphorylated with cyclic AMP-regulated protein kinases and (γ-32P)ATP, were used as substrate. The phosphatase activity eluting at 0.14 M KCl was characterized further using ribosomal subunits phosphorylated by incubation of intact reticulocytes with radioactive inorganic phosphate. Phosphate covalently bound to 40S ribosomal subunits and 80S ribosomes was removed by the phosphatase activity. The enzyme was not active with phosphorylated proteins associated with 60S ribosomal subunits. 相似文献
7.
Mutational alteration of Bacillus subtilis DNA polymerase 3 to hydroxyphenylazopyrimidine resistance: polymerase 3 is necessary for DNA replication 总被引:18,自引:0,他引:18
A spontaneous mutant of resistant to killing by two hydroxyphenylazopyrimidines has been isolated. The DNA polymerase III of this mutant is resistant to inhibition by these drugs. The Ki for 6-(p-hydroxyphenylazo)-uracil (HPUra) is 20 μM, about 40 times higher than the Ki of the wild-type enzyme. The mutant and wild-type polymerases behave similarly during purification, are sensitive to N-ethylmaleimide and to 0.1 M KCl, and have the same Km for dGTP (0.5 μM). The HPUra inhibition of both enzymes is attenuated competitively by dGTP. We conclude that polymerase III is the target for hydroxyphenylazopyrimidines , and since the drugs specifically inhibit replicative DNA synthesis, polymerase III is necessary for DNA replication. 相似文献
8.
K Werkmeister F Wieland E Schweizer 《Biochemical and biophysical research communications》1980,96(1):483-490
A mixture of two pantetheine-free mutant fatty acid synthetases was dissociated and recombined to form a hybrid apoenzyme complex. the corresponding -mutants exhibit interallelic complementation when crossed with each other and the enzyme synthesized in the resulting diploid contains pantetheine and exhibits overall fatty acid synthetase activity. Accordingly, the hybrid apoenzyme formed could be activated to holo-fatty acid synthetase when incubated with coenzyme A and a partially purified yeast cell extract. The enzyme coenzyme A: fatty acid synthetase apoenzyme 4′-phosphopantetheine transferase has thus been identified in yeast. Further studies on the mechanism of fatty acid synthetase holoenzyme formation will now be possible. 相似文献
9.
Bovine adrenal cortical protein kinase type II catalytic subunit (ATP: Protein Phosphotransferase EC 2.7.1.37) has been purified by a method which relies on differences in net charge for the holoenzyme and the catalytic subunit. The purified subunit migrates as a single band on SDS disc gel electrophoresis (molecular weight, 43,500 daltons). The molecular weight based on gel filtration is 38,600. Isoelectric focusing resolves the subunit into 4 components all of which have the same pH optimum for activity. The apparent Km values for ATP are 24, 25, and 35 for the catalytic subunit, and the holoenzyme assayed in the absence or presence of cyclic AMP respectively; for histone, values of 0.9 and 1.0 mg/ml are obtained for the catalytic subunit and the holoenzyme. The pH-activity profile is broad with optimum activity at pH 6.5. 相似文献
10.
An enriched fraction of plasma membranes was prepared from canine ventricle by a process which involved thorough disruption of membranes by vigorous homogenization in dilute suspension, sedimentation of contractile proteins and mitochondria at followed by sedimentation of a microsomal fraction at . The microsomal suspension was then fractionated on a discontinuous sucrose gradient. Particles migrating in the density range 1.0591–1.1083 were characterized by ( activity and [3H]ouabain binding as being enriched in sarcolemma and were comprised of nonaggregated vesicles of diameter approx. 0.1 μm. These fractions contained which appeared endogenous to the sarcolemma. The enzyme was solubilized using Triton X-100 and 1 M KCl and partially purified. Optimal Ca2+ concentration for enzyme activity was 5–10 μM. Both Na+ and K+ stimulated enzyme activity. It is suggested that the enzyme may be involved in the outward pumping of Ca2+ from the cardiac cell. 相似文献
11.
P P Hipps M R Eveland M H Laird W R Sherman 《Biochemical and biophysical research communications》1976,68(4):1133-1138
-Inositol:NAD(P)+ oxidoreductase (-inositol oxidoreductase) has been identified in bovine brain. This enzyme elutes from DEAE cellulose with 0.3 M KCl in 50 mM Tris buffer, pH 7.5. Using NADH as cofactor -inosose-2 is reduced selectively to -inositol. With NADPH the enzyme forms both -inositol and -inositol, however, at a lower rate. The enzyme was chromatographed on G-100 Sephadex and found to have an apparent molecular weight of 74,000. This enzyme differs in DEAE binding, molecular weight and cofactor specificity from the previously described -inositol oxidoreductase which utilizes NADPH exclusively to produce 3 fold more -inositol than -inositol. 相似文献
12.
The number of water molecules () coupled to the transport of cations across lipid membranes was determined in two different wats: directly from the electro-osmotic volume flux per ion, and, by the use of Onsager's relation, from the open circuit streaming potential produced by an osmotic pressure difference. The results of the two approaches were in general agreement. Monoolein membranes were formed on the ends of polyethylene or Teflon tubing connected to a microliter syringe and the volume change necessary to keep the membrane at a fixed position was measured. It was necesary to make corrections for unstirred layer effects. The results for gramicidin were: for 0.15 M KCl and NaCl, for 3.0 M KCl and NaCl, and for 0.01 M HCl. For nonactin, for both 0.15 and 3.0 M KCl and NaCl. Valinomycin (for 0.15 M KCl) behaved like nonactin. It is shown that for a channel mechanism, in general, is less than or equal to the number of water molecules in a channel that does not contain any cations. Thus, the of 12 for the 0.15 M salts implies that the gramicidin channel can hold at least 12 water molecules. This places an important constraint on models of the channel structure. The of 0 for HCl is consistent with a process in which protons jump along a continuous row of water molecules. The decrease of with the 3.0 M salts may indicate that the channel becomes multiply occupied at high salt concentrations. The of 4 for nonactin and valinomycin means that at least four water molecules are associated with the carrier·cation complex, probably in the interstices between the complex and the disordered lipid. 相似文献
13.
Toluene dioxygenase: purification of an iron-sulfur protein by affinity chromatography 总被引:26,自引:0,他引:26
V Subramanian T N Liu W K Yeh D T Gibson 《Biochemical and biophysical research communications》1979,91(3):1131-1139
Toluene dioxygenase, from , oxidizes toluene to (+)--1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene. The oxygenase-component of this multienzyme system was purified to homogeneity by a two-step procedure that utilized affinity and ion exchange chromatography. The purified enzyme would oxidize toluene only in the presence of NADH, ferrous iron and partially purified preparations of NADH cytochrome c reductase and an iron-sulfur protein (ferredoxinTOL). Spinach NADPH cytochrome c reductase and NADPH could substitute for the reductase and NADH. The molecular weight of the oxygenase-component was determined to be 151,000 and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the enzyme is composed of two subunits with molecular weights of 52,500 and 20,800. The absorption spectrum showed maxima at 550 (Shoulder), 450, 326 and 278 nm and preliminary experiments have indicated the presence of 2 gram atoms of iron and 2 gram atoms of acid-labile sulfur per mole of protein. The results indicate that the oxygenase-component of the toluene dioxygenase enzyme system is an iron-sulfur protein that has been designated ISPTOL. 相似文献
14.
Alfred H. Merrill Kihachiro Horiike Donald B. McCormick 《Biochemical and biophysical research communications》1978,83(3):984-990
Pyridoxamine (pyridoxine) 5′-phosphate oxidase (EC 1.4.3.5) purified from rabbit liver is competitively inhibited by the reaction product, pyridoxal 5′-phosphate. The Ki, 3 μM, is considerably lower than the Km for either natural substrate (18 and 24 μM for pyridoxamine 5′-phosphate and 25 and 16 μM for pyridoxine 5′-phosphate in 0.2 M potassium phosphate at pH 8 and 7, respectively). The Ki determined using a 10% rabbit liver homogenate is the same as that for the pure enzyme; hence, product inhibition is probably not diminished significantly by other cellular components. Similar determinations for a 10% rat liver homogenate also show strong inhibition by pyridoxal 5′-phosphate. Since the reported liver content of free or loosely bound pyridoxal 5′-phosphate is greater than Ki, the oxidase in liver is probably associated with pyridoxal 5′-phosphate. These results also suggest that product inhibition of pyridoxamine-P oxidase may regulate the rate of pyridoxal 5′-phosphate formation. 相似文献
15.
C L Hsin-Hsiung-TaiTai C S Hollander 《Biochemical and biophysical research communications》1974,57(2):457-462
15-Hydroxyprostaglandin dehydrogenase has been purified from swine kidney to a specific activity of near 100 miliunits per mg of protein. The purified enzyme was found to be inhibited by thyroid hormone analogues of which triiodothyroacetic acid was the most potent inhibitor. The concentration required for 50% inhibition was 5 μM for triiodothyroacetic acid. The inhibition by thyroid hormones was uncompetitive and non-competitive with regard to NAD+ and prostaglandin E1, respectively. The sensitivity of this enzyme to thyroid hormones suggests that these hormones may regulate the metabolism of prostaglandins . 相似文献
16.
F.D. Sauer S. Mahadevan J.D. Erfle 《Biochemical and biophysical research communications》1980,95(2):715-721
cells, incubated anaerobically under H2 in 0.1 M KCl or 0.1 M NaCl, above pH 7.5, are interior acid with respect to the incubation medium. The pH gradient thus established can be discharged by either carbonyl cyanide m-chlorophenylhydrazone or valinomycin at high concentration (17μM). In these cells, which actively synthesize CH4 from CO2 and H2, methanogenesis is strongly inhibited when the pH gradient is discharged. 相似文献
17.
William C. Okulicz Robert A. Boomsma Richard G. MacDonald Wendell W. Leavitt 《Biochimica et Biophysica Acta (BBA)/General Subjects》1983,757(1):128-136
This study was undertaken to determine optium conditions for the extraction and measurement of uterine nuclear estrogen receptor at low temperature. We measured the influence of glycero, 0.5 M KCl, 10 mM pyridoxal 5′-phosphate, and 0.5 M NaSCN on the dissocation of estradiol from the receptor at 0°C. The half-time () of estradiol dissociation from the receptor in 0.5 M KCl nuclear extracts containing 30% glycerol was very slow (greater than 250 h). Exclusion of glycerol from the extract (Tris buffer) increased the dissociation rate (). The inhibitory effect of glycerol on estradiol dissociation kinetics predominated over the mild stimulatory effect of KCl; and both effects were independent of the electrical conductivity of the buffer. When pyridoxal phosphate was added to a nuclear KCl extract (barbital fubber) lacking glycerol, dissociation of the estrogen-receptor complex increased such that the ) decreased from 20 to 7.6 h; the receptor extracted from nuclei with 10 mM pyridoxal phosphate exhibited these same rapid dissociation kinetics. The of estradiol dissociation from the receptor at 0°C in the presence of 0.5 M NaSCN was 5.6 h. Following extraction of uterine receptro by KCl, pyridoxal phosphate, or NaSCN, we measured the number of estradiol binding sites at each of two incubation temperatures: 30°C for 1 hr and 0°C for 24 h. We verified that unoccupied receptors was measured reliability in KCl extract during incubation at 0°C in the presence of glycerol. Total receptor can be determined using either pyridoxal phosphate extract or NaSCN extract at low temperature. However, the number of sites recovered in either pyridoxal phosphate or NaSCN extract was twice the number obtained with the KCl procedure at elevated temperature. It is noteworthy that pyridoxal phosphate and NaSCN increased the number of sites when added directly to nuclear KCl extract, and the effect of pyridoxal phosphate and NaSCN was reversed by treatment with L-lysine and dialysis against KCl, respectively. Thus, the lower receptor recovery with the KCl procedure is not due to the inability of KCl to extract these sites from the nucleus but rather is ascribable to the assay procedure itself. Although total receptor can be measured at low temperature with either NaSCN or pyridoxal phosphate, the pyridoxal phosphate method can be used to assay nuclear progesterone receptor in tha same extract. 相似文献
18.
Binding of acetylcholine and related compounds to purified acetylcholine receptor from Torpedo Californica electroplax 总被引:6,自引:0,他引:6
T Moody J Schmidt M A Raftery 《Biochemical and biophysical research communications》1973,53(3):761-772
The binding properties of the purified acetylcholine receptor from were investigated. One type of binding was observed for acetylcholine (KD = 2.3 μM), dimethyl tubocurarine (KD = 6.2 μM), and decamethonium (KD = 55 μM). No cooperativity was observed in ligand binding. By virtue of its ligand binding properties, the purified receptor is nicotinic in nature. 相似文献
19.
The 0.5M KCl wash of rabbit reticulocyte ribosomes (I fraction) catalyzes the deacylation of Met-tRNAfMet. Upon DEAE-cellulose column chromatography, the deacylase activity elutes with the 0.1M KCl wash of the column (f1) and is well-resolved from the peptide chain initiation factors (1–3). The deacylase activity is specific for Met-tRNAfMet (retic., ). Other aminoacyl tRNAs tested including fMet-tRNAfMet (retic., ), Phe-tRNA (), Val-tRNA (retic.), and Arg-tRNA (retic.) are completely resistant to the action of the deacylase. In the presence of the peptide chain initiation factor (IF1) and GTP, retic. Met-tRNAfMet forms the initiation complex Met-tRNAfMet:IF1:GTP (2), and in this ternary complex Met-tRNAfMet is not degraded by the deacylase. Met-tRNAfMet binds to IF1 independent of GTP, and in this complex, this Met-tRNAfMet is degraded by the deacylase.Prior incubation of f1 with Met-tRNAfMet (retic.) strongly inhibited protein synthesis initiation, presumably due to deacylation of the initiator tRNA. This inhibition by f1 was completely prevented when Met-tRNAfMet (retic.) was pre-incubated with peptide chain initiation factors. 相似文献
20.
Massimo Masserini Sandro Sonnino Riccardo Ghidoni Vanna Chigorno Guido Tettamanti 《生物化学与生物物理学报:生物膜》1982,688(2):333-340
GM1 ganglioside was dispersed in different membrane-mimicking systems and the effect of dispersion on GM1 oxidation by galactose oxidase was studied. The following membrane-mimicking systems were used: homogeneous micelles of GM1; mixed micelles (at different proportions of constituents) of GM1 with either GD1a ganglioside (which is resistant to the enzyme), or the non-ionic detergent Triton X-100, or bovine serum albumin; small unilamellar vesicles of egg phosphatidylcholine (PC), containing various proportions of GM1. As a reference substrate not involved in membranous systems and freely interacting with the enzyme, the oligosaccharide portion of GM1 (DesGM1) was employed.The apparent of the enzyme was dramatically dependent on the type of GM1 dispersion. The lowest value was recorded on homogeneous micelles of GM1 and on mixed GM1-GD1a micelles. From this value, the increased 2-fold with GM1-bovine serum albumin lipoprotein micelles, up to 1400-fold with mixed GM1-Triton X-100 (optimal molar ratio, 1:13.8) micelles, and up to 14 000-fold on PC vesicles containing 8 mol% GM1 (this proportion was optimal for enzyme activity on vesicles). The activity developed on these latter vesicles turned out to be still greater (2-fold) than that displayed on DesGM1. The apparent had very similar values in all different membrane systems; in contrast, it was markedly greater on DesGM1. Both Triton X-100 micelles and PC vesicles did not appreciably alter the kinetics of galactose oxidase action on pure galactose, indicating that the above effects are dependent on the intrinsic characteristics of the membrane-like systems containing gangliosides. 相似文献