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31.
S Nagata K Tanizawa N Esaki Y Sakamoto T Ohshima H Tanaka K Soda 《Biochemistry》1988,27(25):9056-9062
The gene for leucine dehydrogenase (EC 1.4.1.9) from Bacillus stearothermophilus was cloned and expressed in Escherichia coli. The selection for the cloned gene was based upon activity staining of the replica printed E. coli cells. A transformant showing high leucine dehydrogenase activity was found to carry an about 9 kilobase pair plasmid, which contained 4.6 kilobase pairs of B. stearothermophilus DNA. The nucleotide sequence including the 1287 base pair coding region of the leucine dehydrogenase gene was determined by the dideoxy chain termination method. The translated amino acid sequence was confirmed by automated Edman degradation of several peptide fragments produced from the purified enzyme by trypsin digestion. The polypeptide contained 429 amino acid residues corresponding to the subunit (Mr 49,000) of the hexameric enzyme. Comparison of the amino acid sequence of leucine dehydrogenase with those of other pyridine nucleotide dependent oxidoreductases registered in a protein data bank revealed significant sequence similarity, particularly between leucine and glutamate dehydrogenases, in the regions containing the coenzyme binding domain and certain specific residues with catalytic importance. 相似文献
32.
Protoplasts and vacuoles were isolated from immature apple fruit(Malus pumila Mill. cv. Golden Delicious). ATP-stimulated Ca2+uptake was identified in both protoplast vesicles and tonoplastvesicles. The apparent Km for Ca2+ of the tonoplast transportsystem was 43.4 µM. The pH optima were 7.2 and 6.7 forCa2+ transport by protoplast and tonoplast vesicles, respectively.Ca2+ transport in tonoplast vesicles was strongly inhibitedby the calmodulin antagonists fluphenazine and N-(6-aminohexyl)-5-chloro-l-naphthalensulfonamidehydrochloride (W-7), while N-aminohexyl)-l-naphthalensulfonamidehydrochloride (W-5) was relatively ineffective. Addition ofexogenous calmodulin stimulated transport by 35%. Ca2+ uptakewas inhibited by vanadate, but not by the ionophores carbonylcyanidem-chlorophenyl hydrazone (CCCP) or valinomycin. The resultsindicate that apple tonoplasts have a Ca2+ transport systemthat is driven by the direct hydrolysis of ATP, and may be calmodulindependent.
1Present address: Morioka Branch, Fruit Tree Research Station,Ministry of Agriculture, Forestry and Fisheries, Shimokuriyagawa,Morioka 020-01, Japan. To whom reprint requests should be addressed. (Received October 18, 1985; Accepted January 29, 1986) 相似文献
33.
Improved method for the isolation and preliminary characterization of human DAF (decay-accelerating factor) 总被引:5,自引:0,他引:5
DAF (decay-accelerating factor) is one of the integral membrane proteins of erythrocytes, and is considered to play an important role in the regulation of complement activation. The purification of DAF has been impeded by the difficulty in removing glycophorin. We devised an effective method for removing glycophorin. Through the limited trypsinization of stromata prior to the extraction of DAF, glycophorin was readily digested so that the DAF could be purified free of glycophorin by DEAE-Sephacel and Bio-Gel A 0.5 m chromatographies. On SDS-PAGE, DAF from trypsinized stromata showed the same mobility as that from native stromata: its molecular weight was estimated to be about 70 kDa. Amino acid analysis of DAF showed high contents of serine and glutamic acid. The amino-terminal sequence of DAF prepared by the present method, determined for the 29 residues, did not show significant homology with that of glycophorin. 相似文献
34.
Structures of deepoxytrichothecene metabolites from 3'-hydroxy HT-2 toxin and T-2 tetraol in rats. 总被引:3,自引:3,他引:0 下载免费PDF全文
3'-Hydroxy HT-2 toxin and T-2 tetraol, in vivo metabolites of T-2 toxin, were orally administered to Wistar rats, and four metabolites having a trichothec-9,12-diene nucleus, which were termed deepoxytrichothecenes, were newly found in the excreta. Their structures were confirmed as 3'-hydroxy-deepoxy HT-2, 3'-hydroxy-deepoxy T-2 triol, 15-acetyl-deepoxy T-2 tetraol, and deepoxy T-2 tetraol on the basis of mass and nuclear magnetic resonance spectroscopy. Resolution of T-2 metabolites and corresponding deepoxytrichothecenes by gas-liquid and thin-layer chromatography was also described. 相似文献
35.
K Yamashita A Hitoi N Tateishi T Higashi Y Sakamoto A Kobata 《Archives of biochemistry and biophysics》1985,240(2):573-582
Paper electrophoresis and Bio-Gel P-4 column chromatography of the oligosaccharides released from mouse kidney gamma-glutamyltranspeptidase by hydrazinolysis gave fractionation patterns quite distinct from those of the bovine and rat kidney enzymes. Structural studies of the fractionated oligosaccharides by sequential exoglycosidase digestion in combination with methylation analysis showed that mouse kidney gamma-glutamyltranspeptidase contains a series of bisected complex-type asparagine-linked sugar chains with the following oligosaccharides as their outer chain moieties: GlcNAc beta 1----, Sia alpha 2----Gal beta 1----4GlcNAc beta 1----, Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----, and sialylated N-acetyllactosamine repeating sugar chains. Some of these sugar chains were found for the first time in glycoproteins. 相似文献
36.
Internodal cells of Chara australis were subjected to two consecutiveintracellular perfusions with a Ca2+-free EGTA medium whichdisintegrated the tonoplast within about 10 minutes and thenwith a Ca2+-buffered medium. All perfusion media usually contained1 mM ATP. To stop the electrogenic pump, the internode was depletedof intracellular ATP. The excitability of the plasmalemma wasnot significantly influenced by intracellular free Ca2+ concentrationsup to 104 M. To trigger action potentials, minimum currentdensities of 1 to 2 µA cm2 had to be applied atall tested Ca2+ concentrations. In the absence of cytoplasmicATP, excitability was completely lost at all Ca2+ concentrations.
1 Present address: Botanisches Institut der Universit?t Bonn,Venusbergweg 22, D-5300 Bonn, FRG. (Received September 22, 1984; Accepted March 6, 1985) 相似文献
37.
Ionic Activity Gradients across the Surface Membrane of Cytoplasmic Droplets Prepared from Chara australis 总被引:1,自引:0,他引:1
The membrane potential and the ionic activity gradients of K+and Cl across the surface membrane of cytoplasmic dropletsprepared from Chara australis internodal cells, were measuredin high and low ionic strength bathing solutions using liquidion exchange microelectrodes selective for K+ and Cl.Our results indicate that K+ is close to electrochemical equilibriumwhereas Cl is not.
1 Present address: ICI Japan, Palace Hotel Annex Building, Marunouchi,Tokyo, Japan. 相似文献
38.
The absence of any effect of alpha-human atrial natriuretic peptide on sodium transport and osmotic water flow in the toad bladder 总被引:1,自引:0,他引:1
Synthesized alpha-human atrial natriuretic peptide (alpha-hANP), at 10(-6) M, failed to inhibit short-circuit current and basal and 10 mU/ml vasopressin-stimulated osmotic water flow in the bladder either pretreated with cyclooxygenase inhibitor, or preincubated with arachidonic acid, a precursor of PGE2. These results indicate alpha-hANP to have no direct effect on sodium transport and water permeability in the bladder, and no evidence was obtained indicating that alpha-hANP suppresses vasopressin-stimulated water flow by increasing PGE2 production. 相似文献
39.
40.
S. Fujimori I. Akaoka K. Sakamoto H. Yamanaka K. Nishioka N. Kamatani 《Human genetics》1985,71(2):171-176
Summary 2,8-Dihydroxyadenine urolithiasis associated with partial deficiencies of adenine phosphoribosyltransferase (APRT) has been found only among Japanese families. All Caucasian patients with the same lithiasis are completely deficient in this enzyme. Partially purified APRT from one of the Japanese families with the lithiasis associated with a partial deficiency of APRT had a reduced affinity for 5-phosphoribosyl-1-pyrophosphate (PRPP). In the present investigations, we have shown that this characteristic is common in mutant enzymes from all the four separate Japanese urolithiasis families associated with partial APRT deficiencies so far tested. The mutant enzymes also had several other characteristics in common including increased resistance to heat in the absence of PRPP and reduced sensitivity to the stabilizing effect of PRPP. These data suggest that these families have a common mutant allele (APRT
*
J) at the APRT gene locus. 相似文献