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11.
Aeromonas aminopeptidase contains two nonidentical metal binding sites that have been shown by both spectroscopy and kinetics to be capable of interacting with one another [Prescott, J.M., Wagner, F.W., Holmquist, B., & Vallee, B.L. (1985) Biochemistry 24, 5350-5356]. The effects of metal ion substitutions on the susceptibility of the p-nitroanilides of L-alanine, L-valine, and L-leucine--substrates that are hydrolyzed at widely differing rates by native Aeromonas aminopeptidase--were studied by determining values of kcat and Km for the 16 metalloenzymes that result from all possible combinations of Zn2+, Co2+, Ni2+, and Cu2+ in each of the two sites. The different combinations of metal ions and substrates yield a broad range in kinetic values; kcat varies by more than 1800-fold, Km by 3000-fold, and kcat/Km ratios by more than 10,000. L-Leucine-p-nitroanilide is by far the most susceptible of the three substrates, and the hyperactivation previously observed with aminopeptidase containing either Ni2+ or Cu2+ in the first binding site and Zn2+ in the second site occurs only with the two poorer substrates, L-alanine-p-nitroanilide and L-valine-p-nitroanilide. Although the enzyme with Zn2+ in both sites hydrolyzes the substrates with N-terminal alanine and valine poorly, it is extremely effective toward L-leucine-p-nitroanilide. Neither metal binding site can be identified as controlling either Km or kcat; both parameters are influenced by the identity of the metal ions, by the site each occupies, and, most strongly, by the substrate.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
12.
Bestatin reversibly inhibits Aeromonas aminopeptidase (EC 3.4.11.10) in a process that is remarkable for its unusual degree of time dependence. The binding of bestatin by both Aeromonas aminopeptidase and cytosolic leucine aminopeptidase (EC 3.4.11.1) is slow and tight, with Ki values (determined from rate constants) of 1.8 X 10(-8) and 5.8 X 10(-10) M, respectively. In contrast, microsomal aminopeptidase (EC 3.4.11.2) binds bestatin in a rapidly reversible process with a Ki value of 1.4 X 10(-6) M. Kinetic analysis of the slow inhibition observed is facilitated by the use of a variety of experimental treatments, primarily measurements made during pre-equilibrium; however, careful selection of conditions permits use also of steady state observations. When titrated with bestatin, 1 mol of cytosolic leucine aminopeptidase (containing 6 g atoms each of zinc and manganese) is rendered 80% inactive by 1 mol of inhibitor, thus suggesting that enzymatic activity depends on one active site/hexamer; titration of Aeromonas aminopeptidase by bestatin reveals a 1:1 stoichiometry. Amastatin inhibits all three aminopeptidases through the mechanism of slow, tight binding with Ki values ranging from 3.0 X 10(-8) to 2.5 X 10(-10) M. This behavior of microsomal aminopeptidase contrasts sharply with its rapidly reversible inhibition by bestatin. The slow, tight binding observed with five of the six aminopeptidase-inhibitor pairs investigated suggests the formation of a transition state analog complex between the enzyme and inhibitor. Physical evidence consistent with this possibility was provided by the observation that both bestatin and amastatin perturb the absorption spectrum of cobalt Aeromonas aminopeptidase. 相似文献
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Christopher E. Taylor Philip W. Stashak George Caldes Benjamin Prescott B.J. Fowlkes Phillip J. Baker 《Cellular immunology》1984,83(1):26-33
Several lectins were tested for their capacity to alter the antibody response to type III pneumococcal polysaccharide (SSS-III). The antibody response was enhanced by concanavalin A (Con A), phytohemagglutinin (PHA), as well as lectins from Phytolacca americana (Pa-2), Pisum sativum (PSA), and Lens culinaris (LCH), when these lectins were given 2 days after immunization with SSS-III; however, suppression was obtained when Con A and Pa-2 were given at the time of immunization. By contrast the lectins from Vicia villosa (VVL) and Bauhinia purpurea (BPA) did not alter the antibody response. Since the lectins PSA and LCH bind to the same monosaccharide as Con A, whereas the other lectins bind to different monosaccharides, these findings indicate that there is no relationship between nominal monosaccharide specificity and the capacity to modulate the antibody response. Substantial increases in the magnitude of the IgG1 antibody response was noted after the administration of Con A whereas profound enhancement of IgG2a antibody response was noted after PHA was given. 相似文献
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Organization of gene and non-gene sequences in micronuclear DNA of Oxytricha nova. 总被引:8,自引:0,他引:8 下载免费PDF全文
In order to study the derivation of the macronuclear genome from the micronuclear genome in Oxytricha nova micronuclear DNA was partially digested with EcoRI, size fractionated, and then cloned in the lambda phage Charon 8. Clones were selected a) at random b) by hybridization with macronuclear DNA or c) by hybridization with clones of macronuclear DNA. One group of these clones contains only unique sequence DNA, and all of these had sequences that were homologous to macronuclear sequences. The number of macronuclear genes with sequences homologous to these micronuclear clones indicates that macronuclear sequences are clustered in the micronuclear genome. Many micronuclear clones contain repetitive DNA sequences and hybridize to numerous EcoRI fragments of total micronuclear DNA, yielding similar but non-identical patterns. Some micronuclear clones containing these repetitive sequences also contained unique sequence DNA that hybridized to a macronuclear sequence. These clones define a major interspersed repetitive sequence family in the micronuclear genome that is eliminated during formation of the macronuclear genome. 相似文献
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The timing of DNA synthesis in Amoeb proteus 总被引:1,自引:0,他引:1
19.
Use of Erythrocytes Sensitized with Purified Pneumococcal Polysaccharides for the Assay of Antibody and Antibody-producing Cells 总被引:53,自引:2,他引:51 下载免费PDF全文
A method was described for the sensitization of erythrocytes with purified type-specific pneumococcal polysaccharide antigens using chromium chloride as a coupling agent. Erythrocytes so sensitized can be used in routine passive hemagglutination and hemolysis tests as well as in the technique of localized hemolysis-in-gel for the detection of specific antibody and specific antibody-producing cells, respectively. 相似文献
20.
Lysis was induced in seven strains of Clostridium tetani by exposure to mitomycin C. The search for a suitable indicator strain to detect bacteriophage in lysates has, so far, been unsuccessful. Inhibition studies on macromolecular synthesis during induction have shown that deoxyribonucleic acid, ribonucleic acid, and protein syntheses are all involved in the lysis induced by mitomycin C. In experiments comparing toxin and protein content in induced and uninduced cells of C. tetani, the toxin-protein ratio proved to be the same in both systems up to the point of lysis. Several possible hypotheses deduced from these results are discussed. 相似文献