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21.
In vitro translation of human pheochromocytoma messenger RNAs: characterization of tyrosine-hydroxylase and dopamine-beta-hydroxylase 总被引:1,自引:0,他引:1
mRNAs extracted from human pheochromocytoma were translated in vitro in a lysate of a rabbit reticulocytes. Two enzymes of the biosynthetic pathway of the catecholamines, tyrosine-hydroxylase (TH) and dopamine-beta-hydroxylase (DBH), were characterized as translation products after immunoprecipitation by specific antisera and electrophoretic analysis. The precursor of TH is a polypeptide having a molecular mass of 62,000 identical to that found for the mature protein. The molecular mass of the precursor of DBH 73,000 while that of the mature form is 79,000. TH and DBH have been translated from mRNAs having sedimentation coefficients of 22S and 25S, respectively. 相似文献
22.
A technique involving culture in soft agar was used for the assay of forward mutation of V79 cells to 6-thioguanine (6TG) resistance. The main reason for the use of soft agar was to prevent reduction in recovery of mutants depending on the cell density plated for mutation selection, which is the chief problem in the liquid method, and which results mainly from metabolic co-operation due to cell-to-cell contact.V79 cells grew well in fortified soft agar medium (DMEM + 20% FBS) showing cloning efficiencies (>80%) as high as in liquid culture. Therefore, V79/HGPRT mutagenesis could be assayed quantitatively in soft agar culture.The frequency of 6TG-resistant colonies in agar selective medium increased linearly with increase in concentration of EMS. Toxicity and mutagenic responses were greater in soft agar than in liquid culture.In cultures of untreated and EMS-treated cells, more than 95% of the 6TG-resistant colonies isolated were aminopterin-sensitive.Use of soft agar for selection prevented the reduction in the number of mutants with increase in the size of incula on plating up to 1?2 × 106 cells per 9-cm dish: in liquid culture, even with a lower plating number (2 × 105 cells per 9-cm dish), a notable reduction in numbers of mutants was observed. This character was re-examined in a reconstruction experiment. The results show that, when up to 2 × 106 cells were plated per 9-cm dish, 6TG-resistant cells were almost completely recovered from the soft agar medium, whereas only 10% were recovered from liquid culture. 相似文献
23.
Ralf Morgenstern Claes Guthenberg Bengt Mannervik Joseph W. DePierre 《FEBS letters》1983,160(1-2):264-268
The amount and nature of glutathione transferases in rat liver microsomes were determined using immunological techniques. It was shown that cytosolic glutathione transferase subunits A plus C, and B plus L were present at levels of 2.4 ± 0.6 and 1.5 ± 0.1 μg/mg microsomal protein, respectively. These levels are 10-times higher than those for non-specific binding of cytosolic components judging from the distribution of lactate dehydrogenase, a cytosolic marker. The possibility that a portion of these glutathione transferases is functionally localized on the endoplasmic reticulum is discussed. A previously described microsomal glutathione transferase which is distinct from the cytosolic enzymes is present in an amount of 31 ± 6 μg/mg microsomal protein. 相似文献
24.
Michael N. Horst 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1990,159(6):777-788
Summary Then-acetyl-d-glucosamine-1-phosphate: dolichol phosphate transferase fromArtemia has been partially purified and characterized. The enzyme is solubilized from crude microsomes using Triton X-100, and after detergent removal appears to be associated with phospholipids. Using dolichol phosphate and UDP-n-acetyl-d-glucosamine as substrates, the enzyme catalyzes the formation of dolichol-pyrophosphate-n-acetyl-d-glucosamine. the product identity has been verified by TLC and paper chromatography following mild acid hydrolysis. Under the incubation conditions used only one product is made, i.e., Dol-p-p-GlcNAc. The formation of product is linear with increasing amounts of added protein and with time of incubation. The enzyme requires magnesium ions for activity. Activity of the enzyme is stimulated 6-fold by exogenous dolichol phosphate and is also stimulated by added phospholipids, with optimal activity being obtained in the presence of mixtures of phosphatidylcholine and phosphatidylglycerol. Enzymatic activity is not increased upon addition of GDP-mannose or dolichol phosphate mannose. The enzyme is rapidly inactivated by exposure to several detergents, including Triton X-100 and deoxycholate. The activity is inhibited by tunicamycin and by the purified B2 homologue of this antibiotic. Other antibiotic inhibitors such as diumycin and polyoxin D have little effect on the enzyme. Both the microsomal and solubilized enzyme preparations are inactivated by 70% upon treatment with phospholipase A2; activity may be restored by addition of phospholipids. Following hydrophobic interaction chromatography on Phenyl Sepharose, gel filtration chromatography on Sepharose CL-4B indicated that the enzyme, purified 81-fold, contained phophatidylcholine and phosphatidyl-ethanolamine.Abbreviations SDS
sodium dodecyl sulfate
- PMSF
phenyl methanesulfonylfluoride
- HEPES
4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid
- GlcNAc
N-acetyl-d-glucosamine
- Dol-PP-GlcNAc
dolichol pyrophosphate N-acetyl-d-glucosamine
- Dol-P-man
dolichol-phosphate-mannose
- Dol-PP- (GlcNAc)2
dolichol-pyrophosphate-di-N- acetylchitobiose
- DMSO
dimethylsulfoxide
- C:M (2:1)
chloroform:methanol (2:1)
- C:M:W (10:10:3)
chloroform:methanol:water (10:10:3)
- GlcNAc-1-P
N-acetyl-d-glucosamine-1-phosphate
- Dol-P
dolichol phosphate
- EGTA
ethylene glycol bis (b-aminoethyl ether)-NNNN tetraacetic acid 相似文献
25.
Heterogeneity of the glutathione transferase genes encoding enzymes responsible for insecticide degradation in the housefly 总被引:6,自引:0,他引:6
Michael Syvanen Zonghan Zhou Jonathan Wharton Claire Goldsbury Alan Clark 《Journal of molecular evolution》1996,43(3):236-240
One of the four glutathione-S-transferases (GST) that is overproduced in the insecticide-resistant Cornell-R strain of the housefly (Musca domestica) produces an activity that degrades the insecticide dimethyl parathion and conjugates glutathione to lindane. In earlier
work, it was shown that the resistant Cornell-R carries an amplification, probably a duplication, of one or more of its GST
loci and that this amplification is directly related to resistance. Using polymerase chain reaction (PCR) amplification with
genomic DNA, multiple copies of the gene encoding the parathion-degrading activity (called MdGst-3) were subcloned from both
the ancestral, insecticide-susceptible strain BPM and from the insecticide-resistant Cornell-R. In BPM, three different MdGst-3
genes were identified while in Cornell-R, 12 different MdGst-3 sequences were found that, though closely related to ancestral
genes, had diverged by a few nucleotides. This diversity in MdGst-3 genomic sequences in Cornell-R is reflected in the expressed
sequences, as sampled through a cDNA bank. Population heterozygosity cannot account for these multiple GST genes. We suggest
that selection for resistance to insecticides has resulted in not only amplification of the MdGst-3 genes but also in the
divergence of sequence between the amplified copies.
Received: 22 November 1995 / Accepted: 23 February 1996 相似文献
26.
Characterisation of glutathione transferases and glutathione peroxidases in pea (Pisum sativum) 总被引:2,自引:0,他引:2
Robert Edwards 《Physiologia plantarum》1996,98(2):594-604
27.
Wim Van den Ende Dominik Van Wonterghem Peter Verhaert Erna Dewil André Van Laere 《Planta》1996,199(4):493-502
Fructan: fructan fructosyl transferase (FFT, EC 2.4.1.100) was purified from chicory (Cichorium intybus L. var. foliosum cv. Flash) roots by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, and anion- and cation-exchange chromatography. This protocol produced a 60-fold purification and a specific activity of 14.5 mol·(mg protein) –1·min–1. The mass of the enzyme was 69 kDa as estimated by gel filtration. On sodium dodecyl sulfatepolyacrylamide gel electrophoresis and mass spectrometry, 52-kDa and 17-kDa fragments were found, suggesting that the enzyme was a heterodimer. Optimal activity was found between pH 5.5 and 6.5. The enzyme used 1-kestose, 1,1-nystose, oligofructan and commercial chicory root inulin (degree of polymerization 10) as donors and acceptors. Sucrose was the best acceptor but could not be used as a donor. However, at higher concentrations sucrose acted as a competitive inhibitor for donors of FFT. 1-Kestose was the most efficient and 1,1-nystose the least efficient donor. The purified enzyme exhibited -fructosidase activity, specially at higher temperatures and lower substrate concentrations. The synthesis of fructans from 1-kestose decreased at higher temperatures (5–50°C). Therefore enzyme assays were performed at 0°C. The same fructan oligosaccharides, with a distribution similar to that observed in vivo, were obtained upon incubation of the enzyme with sucrose and commercial chicory root inulin.Abbreviations Con A
concanavalin A
- DP
degree of polymerization
- FFT
fructan: fructan fructosyl transferase
- Fru
fructose
- Glc
glucose
- Kes
1-kestose
- MALDI-TOF MS
matrix-assisted laser desorption ionisation time of flight mass spectrometry
- Nys
1,1-nystose
- pI
isoelectric point
- SST
sucrose: sucrose fructosyl transferase
- Suc
sucrose
The authors would like to thank E. Nackaerts for valuable assistance. W. Van den Ende is also grateful to the National Fund for Scientific Research (NFSR Belgium) for giving a grant for research assistants. P. Verhaert is a research associate of the NFSR. This work was also supported by grant OT/91/18 from the Research Fund K.U. Leuven. 相似文献
28.
ADP-ribosylation reaction, that is the transfer of the ADP-ribose moiety of NAD+ to acceptor protein, is catalyzed by two classes of ADP-ribosyltransferases,i.e., poly(ADP-ribose) synthetase and mono (ADP-ribosyl)transferases. These two types differ not only in the number of transferring ADP-ribose units but also in the acceptor amino acid(s) and protein. Their in hibitors, particularly those of poly(ADP-ribose) synthetase, have been successfully employed in studies on biological functions of the enzymes and other related fields of research. Recently, we found many potent and specific inhibitors of poly-(ADP-ribose) synthetase, and broadened their chemical as well as biochemical variety. More recently, we found several potent inhibitors of arginine-specific mono(ADP-ribosyl)transferases and activators of poly(ADP-ribose) synthetase. 相似文献
29.
30.
Ke Zeng John P. Rose Hong-Chi Chen Corey L. Strickland Chen-Pei D. Tu Bi-Cheng Wang 《Proteins》1994,20(3):259-263
A chimeric enzyme (GST121) of the human α-glutathione S-transferases GST1-1 and GST2-2, which has improved catalytic efficiency and thermostability from its wild-type parent proteins, has been crystallized in a space group that is isomorphous with that reported for crystals of GST1-1. However, a single-site (G82R) mutant of GST121, which exhibits a significant reduction both in vitro and in vivo in protein thermostability, forms crystals that are not isomorphous with GST1-1. The mutant protein crystallizes in space group P212121, with cell dimensions a = 49.5, b = 92.9, c = 115.9 Å, and one dimer per asymmetric unit. Preliminary crystallographic results show that a mutation of the surface residue Gly 82 from a neutral to a charged residue causes new salt bridges to be formed among the GST dimers, suggesting that the G82R mutant might aggregate more readily than does GST121 in solution resulting in a change of its solution properties. © 1994 Wiley-Liss, Inc. 相似文献