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Carbonic anhydrase activity of rabbit lungs 总被引:6,自引:0,他引:6
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1-Phenylcyclopropylamine (1-PCPA) is shown to be a mechanism-based inactivator of mitochondrial monoamine oxidase (MAO). The strained cyclopropyl ring is important to inactivation since alpha,alpha-dimethylbenzylamine, the acyclic analogue of 1-PCPA, is neither an inactivator nor a substrate of MAO. Two different pathways occur during inactivation by 1-PCPA, both believed to be derived from a common intermediate. One pathway leads to irreversible inactivation of the enzyme and a 1:1 stoichiometry of radioactivity to the active site when 1-[phenyl-14C]PCPA is used as the inactivator; the other pathway results in a covalent reversible adduct. Three organic reactions are carried out on the irreversibly labeled enzyme in order to determine the structure of the active site adduct. Sodium boro[3H]hydride reduction results in the incorporation of 0.73 equiv of tritium, suggesting a carbonyl functionality. Baeyer-Villiger oxidation followed by saponification gives 0.8 equiv of phenol, indicating the presence of a phenyl ketone. Treatment of the labeled enzyme with hydroxide produces acrylophenone, as would be expected from the retro-Michael reaction of beta-X-propiophenone. The identity of X is determined in two ways. The optical spectrum of the flavin cofactor is reduced during inactivation; no reoxidation occurs upon denaturation. Pronase treatment of the radioactively labeled enzyme produces fragments that contain both the radioactivity and the flavin. The X group, therefore, is the flavin. The results of two tests designed to differentiate N5 from C4a attachment to the flavin suggest an N5 adduct. In addition to formation of this stable covalent adduct, another pathway occurs 7 times as often. This alternate reaction of 1-[phenyl-14C]PCPA with MAO produces 7 equiv of [14C]acrylophenone during the course of irreversible inactivation and is believed to arise from formation of the same type of adduct as described above except that X is something other than the N5-flavin (Y). Upon denaturation of this labeled enzyme, the flavin is completely oxidized when most of the radioactivity is still bound to the enzyme. This indicates that Y is not a C4a-flavin adduct and suggests attachment to an active site amino acid residue. More facile elimination of Y from this beta-substituted propiophenone adduct would give acrylophenone on the time scale of the inactivation. Treatment of the reversible adduct with sodium borohydride prior to denaturation prevents release of radioactivity.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
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Analytical errors in measuring radioactivity in cell proteins and their effect on estimates of protein turnover in L cells. 总被引:1,自引:1,他引:0 下载免费PDF全文
Previous studies from this laboratory on protein turnover in 3H-labelled L-cell cultures have shown recovery of total 3H at the end of a 3-day experiment to be always significantly in excess of the 3H recovered at the beginning of the experiment. In this study we have critically reviewed a number of possible sources for this error in measuring radioactivity in cell proteins. 3H-labelled proteins, when dissolved in 0.3 M-NaOH and counted for radioactivity in a liquid-scintillation spectrometer, showed losses of 30-40% of the radioactivity; neither external or internal standardization compensated for this loss. Hydrolysis of these proteins with either Pronase or concentrated HCl significantly increased the measured radioactivity. In addition, approx. 5-10% of the cell protein is left on the plastic culture dish when cells are recovered in phosphate-buffered saline. To aggravate this latter loss further, this surface-adherent protein, after pulse labelling, contains proteins of high radioactivity that turn over rapidly and make a major contribution to the accumulating radioactivity in the medium. These combined errors can account for up to 60% of the total radioactivity in the cell culture. Similar analytical errors have been found in studies of other cell cultures. The effect of these analytical errors on estimates of protein turnover in cell cultures is discussed. 相似文献
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Twelve cases of methanol poisoning are reviewed. The clinical presentation and biochemical features are described and the results of treatment with alkali, ethanol and dialysis reported. The outcome of methanol poisoning appears to be related more to the interval between the time of ingestion and the start of therapy and to the degree of acidosis than to the initial serum methanol level. Therefore, early and aggressive treatment with bicarbonate and ethanol and subsequent institution of hemodialysis are strongly recommended whenever methanol can be detected in the blood, especially when metabolic acidosis of the anion-gap type is present, when mental or visual disturbances are present, or when more than 30 ml of absolute methanol has been consumed. 相似文献
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After transfer into fresh medium, Escherichia coli cells containing ribosomal helices resume growth without a lag period. The helices disappear within 15 min after transfer, the number of 70S ribosomes decreases, and a steady-state ribosomal profile appears within one cell generation time. Subunits isolated from the helices support in vitro protein synthesis, but efficiency is optimal only when supplemented with an undetermined factor that is contained in the S-100 fraction of log-phase cells. The data suggest a possible role of helices as ribosomal reserve units. 相似文献
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Vibrio parahaemolyticus distinguishes between life in a liquid environment and life on a surface. Growth on a surface induces differentiation from a swimmer cell to a swarmer cell type. Each cell type is adapted for locomotion under different circumstances. Swimmer cells synthesize a single polar flagellum (Fla) for movement in a liquid medium, and swarmer cells produce an additional distinct flagellar system, the lateral flagella (Laf), for movement across a solid substratum, called swarming. Recognition of surfaces is necessary for swarmer cell differentiation and involves detection of physical signals peculiar to that circumstance and subsequent transduction of information to affect expression of swarmer cell genes (laf). The polar flagellum functions as a tactile sensor controlling swarmer cell differentiation by sensing forces that restrict its movement. Surface recognition also involves a second signal, i.e. nutritional limitation for iron. Studying surface-induced differentiation could reveal a novel mechanism of gene control and lead to an understanding of the processes of surface colonization by pathogens and other bacteria. 相似文献
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(Z)- and (E)-4-amino-2-(trifluoromethyl)-2-butenoic acid (4 and 5, respectively) were synthesized and investigated as potential mechanism-based inactivators of gamma-aminobutyric acid aminotransferase (GABA-AT) in a continuing effort to map the active site of this enzyme. The core alpha-trifluoromethyl-alpha,beta-unsaturated ester moiety was prepared via a Reformatsky/reductive elimination coupling of the key intermediates tert-butyl 2,2-dichloro-3,3,3-trifluoropropionate and N,N-bis(tert-butoxy-carbonyl)glycinal. Both 4 and 5 inhibited GABA-AT in a time-dependent manner, but displayed non-pseudo-first-order inactivation kinetics; initially, the inactivation rate increased with time. Further investigation demonstrated that the actual inactivator is generated enzymatically from 4 or 5. This inactivating species is released from the active site prior to inactivation, and as a result, 4 and 5 cannot be defined as mechanism-based inactivators. Furthermore, 4 and 5 are alternate substrates for GABA-AT, transaminated by the enzyme with Km values of 0.74 and 20.5 mM, respectively. Transamination occurs approximately 276 and 305 times per inactivation event for 4 and 5, respectively. The enzyme also catalyzes the elimination of the fluoride ion from 4 and 5. A mechanism to account for these observations is proposed. 相似文献