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131.
We have identified a new thymosin beta 4-like peptide in pork spleen. The new peptide (12 mg) and thymosin beta 4 (33 mg) were isolated from 230 g of spleen by solid phase extraction, preparative isoelectric focusing, and HPLC. The new peptide was termed thymosin beta 9 Met to indicate its close relationship to thymosin beta 9 from calf. The only difference from thymosin beta 9 is the substitution of leucine by methionine at position 6. This peptide replaces thymosin beta 10 which is the minor thymosin beta 4-like peptide in most mammals, e.g., in man, rat, mouse, cat, and rabbit. The structure was determined by amino acid analysis, tryptic digestion, and carboxypeptidase digestion. Pork spleen contains 192 micrograms of thymosin beta 4 and 117 micrograms of thymosin beta 9 Met per gram of tissue. 相似文献
132.
R N Puri F X Zhou R F Colman R W Colman 《Biochemical and biophysical research communications》1989,162(3):1017-1024
Thrombin-induced platelet aggregation is accompanied by cleavage of aggregin, a surface membrane protein (Mr = 100 kDa), and is mediated by the intracellular activation of calpain. We now find that agents that increase intracellular levels of platelet cAMP by stimulating adenylate cyclase, also inhibit thrombin binding and platelet activation by destabilizing thrombin receptors on the platelet surface. Iloprost (a stable analog of PGI2) and forskolin each completely inhibited platelet aggregation by 2 nM thrombin and markedly decreased cleavage of aggregin. Thrombin inactivated by D-phenylalanine-L-prolyl-L-arginine chloromethyl ketone (PPACK-thrombin) binds to the highest affinity site for thrombin on the platelet surface, but thrombin modified by N alpha-tosyl-L-lysine chloromethylketone (TLCK-thrombin) does not. We now demonstrate that preincubation of platelets with PPACK-thrombin blocked platelet aggregation and cleavage of aggregin induced by 2 nM thrombin. In contrast, TLCK-thrombin neither blocked platelet aggregation nor the cleavage of aggregin. These results show that a) platelet aggregation and cleavage of aggregin by thrombin (2nm) involves the occupancy of high affinity alpha-thrombin receptors on the platelet surface, and b) stimulators of adenylate cyclase which increase cAMP, inhibit thrombin-induced platelet aggregation and cleavage of aggregin by mechanisms which include inhibiting the binding of thrombin to its receptors. 相似文献
133.
Chromium (V) and hydroxyl radical formation during the glutathione reductase-catalyzed reduction of chromium (VI) 总被引:3,自引:0,他引:3
Electron spin resonance measurements provide evidence for the formation of long-lived Cr(V) intermediates in the reduction of Cr(VI) by glutathione reductase in the presence of NADPH and for the hydroxyl radical formation during the glutathione reductase catalyzed reduction of Cr(VI). Hydrogen peroxide suppresses Cr(V) and enhances the formation of hydroxyl radicals. Thus Cr(V) intermediates catalyze generation of hydroxyl radicals from hydrogen peroxide through a Fenton-like reaction. Thus the mechanism of Cr(VI) toxicity might involve the interaction between macromolecules and the hydroxyl radicals. 相似文献
134.
Solution structure of the chromomycin-DNA complex 总被引:8,自引:0,他引:8
The structure of the chromomycin-DNA complex at the deoxyoctanucleotide duplex level has been determined from one- and two-dimensional proton NMR studies in Mg-containing aqueous solution. The NMR results demonstrate that the antitumor agent binds as a symmetrical dimer to the self-complementary d[T-T-G-G-C-C-A-A] duplex with retention of the 2-fold symmetry in the complex. A set of intermolecular nuclear Overhauser enhancements (NOEs) establishes that two chromomycin molecules in the dimer share the minor groove at the G-G-C-C.G-G-C-C segment in such a way that each hydrophilic edge of the chromophore is located next to the G-G.C-C half-site and each C-D-E trisaccharide chain extends toward the 3'-direction of the octanucleotide duplex. In addition, the A-B disaccharide segment and the hydrophilic side chain of the antitumor agent are directed toward the phosphate backbone. The observed changes in nucleic acid NOEs and coupling patterns on complex formation establish a transition to a wider and shallower minor groove at the central G-G-C-C.G-G-C-C segment required for accommodating the chromomycin dimer. The present demonstration that chromomycin binds as a dimer and switches the conformation of the DNA at its G.C-rich minor groove binding site provides new insights into antitumor agent design and the sequence specificity of antitumor agent-DNA recognition. 相似文献
135.
G Carrasquer X Y Wu D Kissel W S Rehm M Schwartz M A Dinno 《Biochimica et biophysica acta》1989,982(1):73-78
Addition of 10(-5) M amphotericin B to the tear solution of an in vitro preparation of the frog cornea increased the transepithelial conductance, gt, and decreased the apical membrane fractional resistance, f(R0), in the presence or absence of tear Na+ and Cl-. In the presence of tear Na+ and Cl-, amphotericin B increased the short-circuit current, Isc, from 3.9 to 8.8 microA.cm-2 and changed the intracellular potential, V0, from -48.5 to -17.9 mV probably due to a higher increase in the Na+ than in the K+ conductance. In the absence of tear Na+ and Cl-, amphotericin B decreased Isc from 5.5 to about 0 microA.cm-2 due to K+ (and possibly Na+) flux from cell to tear and changed V0 from -35.4 to -63.6 mV due to the increase in conductance of both ions. Increase in the tear K+ from 4 to 79 mM (in exchange for choline), in the presence of amphotericin B and absence of tear Na+ and Cl-, decreased f(R0) from 0.09 to 0.06, increased gt from 0.23 to 0.31 mS, increased Isc from 0.63 to 7.3 microA.cm-2, and changed V0 from -65.5 to -17.3 mV due to the change in EK in the presence of a high conductance in the tear membrane. Similar effects were observed with an increase of tear Na+. Results support the concept that the Na+ conductance opened by amphotericin B in the apical membrane is greater than the K+ conductance. Previously observed transepithelial effects of the ionophore may be explained mostly on the basis of its effect on the apical membrane. 相似文献
136.
We have examined the kinetics and mechanism by which iron can displace copper at the specific metal-binding sites of ovotransferrin. Fe2+ was added to Cu2+-ovotransferrin-CO3(2-) in the presence of NaHCO3 and ambient O2. The reaction has been followed by standard and stopped-flow spectrophotometry, EPR spectroscopy and analysis of chromogen-reactive Fe2+. The reaction is best described as triphasic. An initial jump in absorbance takes place in the first 2 s. In the next minute there is a further increase in absorbance and shift in the spectral maximum from 440 to 446 nm. The third phase is complex. The bulk of the spectrophotometric change, a decrease in absorbance with a shift to a maximum of 453 nm, lasts approx. 3 min. Minor spectral and EPR changes, however, take place over the next several hours. Chromogenic analysis of Fe2+ indicates that approx. 1 min is required to oxidize the Fe2+. EPR spectra reveal the formation of an Fe3+-ovotransferrin complex within the first 20 s; however, this lacks the characteristic doublet of specific Fe3+-ovotransferrin-CO3(2-). The simultaneous presence of specific Cu2+-ovotransferrin-CO3(2-) and Fe3+-ovotransferrin-CO3(2-) signals suggests a period in which the protein specifically binds both metal ions perhaps resulting from a differential reactivity of the two metal-binding sites. The addition of Cu(NO3)2 to Fe3+-ovotransferrin-CO3(2-) resulted in a complex with specific Fe3+ and non-specific Cu2+. The EPR spectrum of this complex and the final product of our displacement reaction were virtually identical. Distinct parallels in reaction of Cu2+-ovotransferrin-CO3(2-) with Fe(NH4)2(SO4)2, Fe(NO3)3 and Fe3+-nitrilotriacetic acid were observed. A reaction sequence involving the binding and oxidation of non-specific Fe2+ followed by Cu2+ displacement by Fe3+ at the specific sites and binding of non-specific Cu2+ is suggested. 相似文献
137.
Classical Raman spectroscopic studies of NADH and NAD+ bound to lactate dehydrogenase by difference techniques 总被引:2,自引:0,他引:2
The binding of the coenzymes NAD+ and NADH to lactate dehydrogenase causes significant changes in the Raman spectra of both of these molecules relative to spectra obtained in the absence of enzyme. The molecular motions of the bound adenine moiety of both NAD+ and NADH as well as adenine containing analogues of these coenzymes produce Raman bands that are essentially identical, suggesting that the binding of adenine to the enzyme is the same regardless of the nicotinamide head-group nature. We also have observed that the molecular motions of the bound adenine moiety are different from both those obtained when it is in either water, various hydrophobic solvents, or various other solvent compositions. Protonation of the bound adenine ring at the 3-position is offered as a possible explanation. Significant shifts are observed in both the stretching frequency of the carboxamide carbonyl of NAD+ and the rocking motion of the carboxamide NH2 group of NADH. These shifts are probably caused by hydrogen bonding with the enzyme. The interaction energies of these hydrogen-bonding patterns are discussed. The aromatic nature of the nicotinamide moiety of NAD+ appears to be unchanged upon binding. Pronounced changes in the Raman spectrum of the nicotinamide moiety of NADH are observed upon binding; some of these changes are understood and discussed. Finally, these results are compared to analogous results that were recently reported for liver alcohol dehydrogenase [Chen et al. (1987) Biochemistry 26, 4776-4784]. In general, the coenzyme binding properties are found to be quite similar, but not identical, for the two enzymes. 相似文献
138.
3 beta,20 alpha-Hydroxysteroid oxidoreductase was purified to homogeneity from fetal lamb erythrocytes. The Mr 35,000 enzyme utilizes NADPH and reduces progesterone to 4-pregnen-20 alpha-ol-3-one [Km = 30.8 microM and Vmax = 0.7 nmol min-1 (nmol of enzyme)-1] and 5 alpha-dihydrotestosterone to 5 alpha-androstane-3 beta, 17 beta-diol [Km = 74 microM and Vmax = 1.3 nmol min-1 (nmol of enzyme)-1]. 5 alpha-Dihydrotestosterone competitively inhibits (Ki = 102 microM) 20 alpha-reductase activity, suggesting that both substrates may be reduced at the same active site. 16 alpha-(Bromoacetoxy)progesterone competitively inhibits 3 beta- and 20 alpha-reductase activities and also causes time-dependent and irreversible losses of both 3 beta-reductase and 20 alpha-reductase activities with the same pseudo-first order kinetic t1/2 value of 75 min. Progesterone and 5 alpha-dihydrotestosterone protect the enzyme against loss of the two reductase activities presumably by competing with the affinity alkylating steroid for the active site of 3 beta,20 alpha-hydroxysteroid oxidoreductase. 16 alpha-(Bromo[2'-14C]acetoxy) progesterone radiolabels the active site of 3 beta,20 alpha-hydroxysteroid oxidoreductase wherein 1 mol of steroid completely inactivates 1 mol of enzyme with complete loss of both reductase activities. Hydrolysis of the 14C-labeled enzyme with 6 N HCl at 110 degrees C and analysis of the amino acid hydrolysate identified predominantly N pi-(carboxy[2'-14C]methyl)histidine [His(pi-CM)].(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
139.
The primary structure of human apolipoprotein A-IV 总被引:2,自引:0,他引:2
C Y Yang Z W Gu I S Chong W J Xiong M Rosseneu H X Yang B R Lee A M Gotto L Chan 《Biochimica et biophysica acta》1989,1002(2):231-237
Human apolipoprotein (apo) A-IV was purified from chylous ascites fluid. Proteolytic peptides produced by trypsin and Staphylococcus aureus V8 proteinase digestions were purified by high-performance liquid chromatography and sequenced. Human apoA-IV contains 376 amino acid residues. The peptide-derived sequence generally matches two previously reported DNA-derived amino acid sequences except for discrepancies in five positions. In order to examine these discrepancies further, one complete apoA-IV cDNA clone and another partial clone were sequenced. Comparison of all the available information indicates that the peptide-derived sequence reported here is accurate. Sequencing errors probably account for some of the discrepancies between the two primary sequences predicted by earlier nucleotide analyses. In certain positions, however, bona fide sequence heterogeneity or cloning artifact cannot be excluded. 相似文献
140.