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51.
Rat kidney 5-oxo-L-prolinase catalyzes the endergonic hydrolysis of 5-oxo-L-proline (L-pyroglutamate, L-2-pyrrolidone-5-carboxylate) to form L-glutamate; the reaction is driven by and dependent on the stoichiometric concomitant hydrolysis of ATP to ADP and inorganic phosphate. The present studies are concerned with the mechanism by which the free energy of ATP hydrolysis is conserved and made available for 5-oxoproline hydrolysis. Studies with 18O-labeled substrates showed that (a) all three oxygen atoms of 5-oxoproline are recovered in the product glutamate, and (b) the two water molecules consumed in the reaction contribute one oxygen atom to inorganic phosphate and one oxygen atom to the gamma-carboxyl group of glutamate. It was shown that the enzyme also catalyzes the intrinsically exergonic hydrolysis of alpha-hydroxyglutarate lactone, a reaction that is ATP-dependent. Intermediates in the 5-oxoprolinase reaction were not detected by exchange experiments with radioactive ADP and phosphate, nor were they trapped by adding hydroxylamine. In the presence of very high glutamate concentrations, a slow reversal of the 5-oxoprolinase reaction was demonstrated by measuring ATP formation. The findings are consistent with a mechanism in which 5-oxo-L-proline is phosphorylated by ATP on the amide carbonyl oxygen and the resulting intermediate is subsequently hydrolyzed to yield gamma-glutamyl phosphate; the latter is hydrolyzed to glutamate and inorganic phosphate. 相似文献
52.
The apparent glutathione oxidase activity of gamma-glutamyl transpeptidase. Chemical mechanism 总被引:3,自引:0,他引:3
The apparent glutathione oxidase activity of gamma-glutamyl transpeptidase is due to nonenzymatic oxidation and transhydrogenation reactions of cysteinylglycine, an enzymatic product formed from glutathione by hydrolysis or autotranspeptidation. Since cysteinylglycine reacts with oxygen more rapidly than does glutathione, the rate of disulfide formation is increased and either cystinyl-bis-glycine or the mixed disulfide of cysteinylglycine and glutathione forms as an intermediate product. Nonenzymatic transhydrogenation reactions of these disulfides with glutathione yield glutathione disulfide and thus account for the apparent glutathione oxidase activity of gamma-glutamyl transpeptidase. A sensitive assay for glutathione oxidation is described, and it is shown that covalent inhibitors of gamma-glutamyl transpeptidase abolish the oxidase activity of the purified enzyme and of crude homogenates of mouse and rat kidney. 相似文献
53.
Cultured chick embryo fibroblasts derived from skin and skeletal muscle exhibit hyaluronidase activity both associated with the cell layer and secreted into the medium. Although both forms of the enzyme have a number of similar characteristics (R.W. Orkin and B.P. Toole, 1980, J. Biol. CHem. 255), they differ in thermal stability at neutral pH and in behavior on ion-exchange chromatography. Both forms of the enzyme are equally stable at acidic pH for long intervals, but the cell-associated hyaluronidase is significantly less stable than the secreted froms at neutral pH and at temperatures more than or equal to 30 degrees C. Neither the presence of proteases nor inhibitors of hyaluronidase appear to be involved in the cell-asspcoated enzyme. Chromatography of the two forms of hyaluronidase on carboxymethyl cellulose reveals that most (60-90 percent) of the secreted form of the enzyme elutes at a lower ionic strength than the cell- associated enzyme. Treatment of the secreted form of hyaluronidase with neuraminidase shifts its elution profile on carboxymethyl cellulose toward that of the cell-associated form, and also decreases its thermal stability at neutral pH. In contrast, treatment of the secreted form of hyaluronidase with alkaline phosphatase has no detectable effect. These data suggest that the secreted hyaluronidase differs from the cellular form in possessing additional sialic acid residues which endow the former with increased stability in the extracellular milieu. 相似文献
54.
55.
Quantum yield and image contrast of bacteriochlorophyll monolayers in photoelectron microscopy. 总被引:2,自引:0,他引:2 下载免费PDF全文
The photoelectron quantum yield spectrum of bacteriochlorophyll aGg (Bchl a ) from Rhodospirillum rubrum was determined in order to evaluate the possibility of mapping photoreceptor distribution and organization in bacterial chromatophores. The quantum yield is on the order of 1 X 10(-3) electrons/incident photon at 180 nm and decreases to 2.5 X 10(-5) electrons/incident photon at 230 nm. Photoelectron micrographs confirm the high contrast predicted between monolayers of Bchl a against a lipid background (calcium arachidate). A significant contrast difference is found between the two monolayer orientations, demonstrating that photoelectron microscopy is a sensitive detector of asymmetry in Bch1 a monolayers. 相似文献
56.
J K Griffith 《Developmental biology》1978,62(1):229-246
The rates of total RNA synthesis and accumulation have been measured in the polytenic salivary gland cells of the blowfly, Calliphora erythrocephala, by three methods: (1) injecting larvae with [2-3H]adenosine and determining its flow into the cellular ATP pool and RNA, (2) measuring the increase in glandular RNA optically, and (3) measuring the rate of flow of ATP out of the cellular pool. The size of the steady-state pool of rapidly turning over RNA and its half-life, were calculated from these kinetic data and, also, by an independent measurement of the steady-state content of nuclear RNA. These parameters were compared at a number of developmental stages which differed in degree of chromosomal polytenization. The results indicate that these polytenic cells synthesize RNA at a rate approximately 103 times those of other diploid eukaryotic cells. This rate is independent of the increase in chromosomal polyteny that accompanies larval development. Approximately 67% of the newly synthesized salivary gland RNA is an unstable component with an average first-order half-life of 20–25 min. The remainder is a long-lived species with an estimated average first-order half-life of about 30 hr. 相似文献
57.
C. C. Lin M. M. Gedeon P. Griffith W. K. Smink D. R. Newton L. Wilkie L. M. Sewell 《Human genetics》1976,31(3):315-328
Summary Chromosome analysing using quinacrine fluorescence was performed on 930 consecutive newborn infants. The total incidence of major chromosome aberrations including numerical changes of the sex chromosomes, and structural changes of autosomes, was 0.54%. Incidences of XYY (0.4%) and XXY (0.2%) were relatively higher as compared to other studies. About 0.75% of the newborn infants were found to have a variable bright fluorescent band located on the proximal area of the short arm (p11) rather than on the proximal long arm (q11) of chromosome No. 3. Attempts were also made to record the variable fluorescent regions on 7 autosomes and the Y chromosome. 相似文献
58.
Properties and origins of infectious rhinovirus type 14 particles of different buoyant densities. 总被引:4,自引:4,他引:0 下载免费PDF全文
Isopycnic centrifugation of rhinovirus type 14 (RV14), purified from infected HeLa or KB cell cultures, into CsCl gradients resolved two bands of infectious virus particles with buoyant density values of 1.409 +/- 0.007 (H virus) and 1.386 +/- 0.004 (L virus) g/ml. Only H virus was detected by incorporation of radiolabeled uridine into viral RNA, and H virus accounted for the majority of infectivity in gradients. H and L virus could not be differentiated by plaque morphology, extent of neutralization by RV14-specific antiserum, or particle size. Electron microscope studies showed that most L-virus particles were associated with an amorphous material. Treatment of L virus with proteolytic enzymes or rebanding L virus in CsCl gradients resulted in recovery of the majority of infectivity as H virus. Virus purified from cell-free fluids from infected HeLa or KB cell cultures banded only as H virus. HeLa cell cultures challenged with purified H virus and harvested at 3 h postinoculation for virus purification yielded only infectious H virus. Both H and L viruses were detected in cell cultures that had been challenged with purified H virus and harvested at 12 h postinoculation. The data suggest that H virus represents progeny virus, whereas L virus represents sequestered infectious virus particles which become associated with an amorphous material and do not enter into viral replicative processes. 相似文献
59.
60.
Zoe S. Davies Richard J. Gilbert Roger J. Merry Douglas B. Kell Michael K. Theodorou Gareth W. Griffith 《Applied microbiology》2000,66(4):1435-1443
The enormous variety of substances which may be added to forage in order to manipulate and improve the ensilage process presents an empirical, combinatorial optimization problem of great complexity. To investigate the utility of genetic algorithms for designing effective silage additive combinations, a series of small-scale proof of principle silage experiments were performed with fresh ryegrass. Having established that significant biochemical changes occur over an ensilage period as short as 2 days, we performed a series of experiments in which we used 50 silage additive combinations (prepared by using eight bacterial and other additives, each of which was added at six different levels, including zero [i.e., no additive]). The decrease in pH, the increase in lactate concentration, and the free amino acid concentration were measured after 2 days and used to calculate a “fitness” value that indicated the quality of the silage (compared to a control silage made without additives). This analysis also included a “cost” element to account for different total additive levels. In the initial experiment additive levels were selected randomly, but subsequently a genetic algorithm program was used to suggest new additive combinations based on the fitness values determined in the preceding experiments. The result was very efficient selection for silages in which large decreases in pH and high levels of lactate occurred along with low levels of free amino acids. During the series of five experiments, each of which comprised 50 treatments, there was a steady increase in the amount of lactate that accumulated; the best treatment combination was that used in the last experiment, which produced 4.6 times more lactate than the untreated silage. The additive combinations that were found to yield the highest fitness values in the final (fifth) experiment were assessed to determine a range of biochemical and microbiological quality parameters during full-term silage fermentation. We found that these combinations compared favorably both with uninoculated silage and with a commercial silage additive. The evolutionary computing methods described here are a convenient and efficient approach for designing silage additives. 相似文献