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11.
ATP as an alternative inhibitor of bacterial and endogenous nucleases and its effect on native chromatin compaction 总被引:1,自引:0,他引:1
Nancy Lynn Rosenberg 《Molecular and cellular biochemistry》1987,76(2):113-121
The studies reported here demonstrate that ATP may be used in lieu of EDTA to inhibit nuclease digestion of DNA and chromatin. Because ATP is a milder chelator than EDTA and is a biochemical common to the cellular microenvironment in vivo, critical studies of cellular processes that require native structure to be maintained are more feasible without the presence of strong chelators. During the digestion of chromatin into its components by nuclease treatment, ATP assures the retention of nucleoprotein compaction, particularly for large to intermediate-sized oligosomes (2400bp–1000bp in length). ATP used at a concentration of 3.3 mM appears to be somewhat better than EDTA, 1.0 mM, for minimizing degradation of nuclease-treated chromatin. However, termination of nuclease digestion of chromatin and minimization of further degradation by the addition of ATP to a concentration of 1.0 mM was almost equivalent to the addition of EDTA to a concentration of 1.0 mM. Slightly more degradation was observed for the latter condition. In addition, ATP can be used to inhibit endogenous nuclease activity when specific restriction enzymes are needed. Standard low ionic strength DNP, deoxyribonucleoprotein, and DNA electrophoresis of proteinized and deproteinized chromatin oligomers, respectively, indicated that ATP effectively inhibits staphylococcal nuclease. Low ionic strength nucleoprotein electrophoresis to resolve staphylococcal nuclease-digested chromatin indicates that as little as 10–4 M EDTA can promote structural unfolding resulting in changes in apparent mobilities for chromatin oligomers 250 and 600 by in length. Comparative digestion of chromatin with staphlococcal nuclease followed by reaction termination by ATP or EDTA showed that this observation was not merely the result of degradation due to inefficiency of ATP enzyme inhibition. 相似文献
12.
Changes in glycerol production and two parameters related to energy metabolism i. e. the heat production rate and the ATP pool, were assayed during growth of Saccharomyces cerevisiae and Debaryomyces hansenii in 4 mM and 1.35 M NaCl media. For both of the yeasts, the specific ATP pool changed during the growth cycle and reached maximum values around 10 nmol per mg dry weight in both types of media. The levels of glycerol were markedly enhanced by high salinity. In the presence of 1.35 M NaCl, D. hansenii retained most of its glycerol produced intracellularly, while S. cerevisiae extruded most of the glycerol to the environment. The intracellular glycerol level of S. cerevisiae equalled or exceeded that of D. hansenii, however, with values never lower than 3 mol per mg dry weight at all phases of growth. When D. hansenii was grown at this high salinity the intracellular level of glycerol was found to correlate with the specific heat production rate. No such correlation was found for S. cerevisiae. We concluded that during salt stress, D. hansenii possesses the capacity to regulate the metabolism of glycerol to optimize growth, while S. cerevisiae may not be able to regulate when exposed to different demands on the glycerol metabolism. 相似文献
13.
S. Kauffer R. Schmid K. Steffens G. Deckers-Hebestreit K. Altendorf 《Archives of microbiology》1987,148(3):187-192
The ATP synthase complex of Klebsiella pneumoniae (KF1F0) has been purified and characterized. SDS-gel electrophoresis of the purified F1F0 complexes revealed an identical subunit pattern for E. coli (EF1F0) and K. pneumoniae. Antibodies raised against EF1 complex and purified EF0 subunits recognized the corresponding polypeptides of EF1F0 and KF1F0 in immunoblot analysis. Protease digestion of the individual subunits generated an identical cleavage pattern for subunits , , , , a, and c of both enzymes. Only for subunit different cleavage products were obtained. The isolated subunit c of both organisms showed only a slight deviation in the amino acid composition. These data suggest that extensive homologies exist in primary and secondary structure of both ATP synthase complexes reflecting a close phylogenetic relationship between the two enterobacteric tribes.Abbreviations ACMA
9-amino-6-chloro-2-methoxyacridine
- DCCD
N,N-dicyclohexylcarbodiimide
- FITC
fluorescein isothiocyanate
- SDS
sodium dodecyl sulfate
- TTFB
4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole 相似文献
14.
15.
External ATP enhanced stomatal opening of Commelina communis L. differently from EDTA. ATP was more effective in opening stomata than EDTA, when both were applied in amounts yielding equivalent free Ca2+ concentration. The stimulation by ATP depended upon its de-phosphorylation and was not due to the P1 released. Hence an energetical contribution of external ATP appears possible. Increase in CO2 concentration increased the stimulation of stomatal opening by ATP and diminished the internal ATP level, ATP/(ADP+AMP) ratio and respiration rate. 相似文献
16.
Jackie R. Vandenheede Sigrid Staquet Wilfried Merlevede 《Molecular and cellular biochemistry》1989,87(1):31-39
Summary Fractionation of rabbit skeletal muscle cytosol on Aminohexyl-Sepharose has resulted in the identification of a latent ATP, Mg-dependent protein phosphatase whose catalytic subunit is in the active conformation, but is inhibited by the presence of more than one modulator unit. The partially purified enzyme is converted to an inactive, kinase FA-dependent form upon incubation at 30°C unless modulator-specific polyclonal antibodies are added to the preparation. The immunoglobulins also relieve the inhibition which is responsible for the low basal phosphatase activity of the enzyme, and they counteract all of the heat-stable inhibitor activity present in the preparation. Addition of free catalytic subunit abolishes the inhibition of the latent enzyme in a dose-dependent way, but cannot prevent the inactivation process. The inactivated phosphatase and the original latent enzyme exhibit the same apparent M
r in sucrose density-gradient centrifugation (70 000) and in gel filtration (110 000).Abbreviations PMSF
Phenylmethanesulphonyl Fluoride
- TLCK
L-l-chloro-3-(4-tosylamido)-7-amino2-heptanone-hydrochloride
- TPCK
L-l-chloro-3-(4-tosvlamido)-4-phenyl-2-butanone 相似文献
17.
Th. Wurm C. Albers 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1989,159(3):255-261
Summary The interaction of allosteric effectors (CO2, ATP, H+) with respect to the oxygen affinity of carp hemoglobin was analyzed by determining oxygen binding curves spectrophotometrically in dilute solutions of stripped hemoglobin at 20°C. The pH range studied was 6.8–8.2.P
CO2 was 0, 10 and 70 mmHg (0, 1.33 and 9.3 kPa). ATP/Hb4 was 0, 8 and 24. In the presence of either CO2 or ATP, the effects of the cofactors onP
50 were as expected over the whole pH range. In contrast to other published data, each cofactor also had a significant effect onP
50 in the presence of the other cofactor. Evidence was obtained that oxylabile carbamate is formed by carp hemoglobin and that the formation of carbamate persists at a lower level in the presence of ATP. The results support the view that the binding of ATP to carp hemoglobin requires only one terminal amino group, leaving the other N-terminal of the -chain free to react with CO2. 相似文献
18.
Vesna A. Eterović Lian Li Andrew Palma Mark G. McNamee 《Cellular and molecular neurobiology》1990,10(3):423-433
1. Nicotinic acetylcholine receptors (nAChR)4 from BC3H1 cells (which express a skeletal muscle-type receptor) and from Torpedo californica electric organ were expressed in Xenopus laevis oocytes and studied with a voltage-clamp technique. 2. We found that bath application of ATP in the micromolar to millimolar range increased the ACh-elicited current in both muscle and electrocyte receptors. The effect of ATP increased with successive applications. This "use-dependent" increase in potentiation was Ca2+ dependent, while the potentiation itself was not. 3. Four other nucleotides were tested on muscle nAChR: ADP, AMP, adenosine, and GTP. Of these, only ADP was a potentiator, but its effect was not use dependent. Neither ATP nor ADP affected the resting potential of the oocyte membrane. 4. ADP potentiated the response to suberyldicholine and nicotine, as well as ACh. 5. Finally, ADP reversed the phencyclidine-induced block of ACh currents in oocytes expressing muscle nAChR. 相似文献
19.
Robert M. Levin Penelope A. Longhurst Sheila S. Levin Niels Haugaard Alan J. Wein 《Molecular and cellular biochemistry》1990,97(2):153-159
The urinary bladder depends on intracellular ATP for the support of a number of essential intracellular processes including contraction. The concentration of ATP is maintained constant primarily via the rapid transfer of a phosphate from creatine phosphate (CP) to ADP catalyzed by the enzyme creatine kinase (CK). Since muscular pathologies associated with diabetes are in part related to intracellular alterations in metabolism, we have characterized the CK activity in both skeletal muscle and urinary bladder from control and streptozotocin-diabetic rats.The following is a summary of the results: 1) Bladder tissue from control rats showed linear kinetics with a Vmax = 390 nmoles/mg protein/min, and a Km = 275 µM. 2) Urinary bladder tissue isolated from diabetic rats displayed biphasic kinetics with Vmax = 65 and 324 nmoles/mg protein/min, and Km's = 10 µM and 190 µM respectively. 3) Skeletal muscle isolated from control rats showed linear kinetics with an approximate Vmax of 800 nmoles/mg protein/min and a Km of 280 µM CP. 4) Homogenates of skeletal muscle from diabetic rats showed complex kinetics not separable into distict component forms. 5) The Km for ADP for both skeletal muscle and bladder was approximately 10 µM.These studies demonstrate that whereas bladders isolated from both control and diabetic rats possess a low-affinity isomer(s) of CK with similar maximum enzymatic activity, there is a high affinity isomer present within the urinary bladder muscle of diabetic rats that is not present in bladder tissue isolated from control rats. Skeletal muscle isolated from both diabetic and control rats exhibited a maximal activity 2 to 3 times higher than that of the bladder. 相似文献
20.
Energy metabolism in glutamatergic neurons,GABAergic neurons and astrocytes in primary cultures 总被引:9,自引:0,他引:9
Several aspects of energy metabolism (glucose utilization, lactate production,14CO2 production from labeled glucose, glutamate or pyruvate, oxygen consumption and contents of ATP and phosphocreatine) were measured in cerebellar granule cells (glutamatergic) in primary cultures and compared with corresponding data for cerebral cortical neurons (mainly GABA-ergic) and astrocytes. Cerebellar granule cells and astrocytes were metabolically more active than cerebral cortical neurons. Glutamate which is utilized as a major metabolic fuel as astrocytes and, to a lesser extent, in cerebral cortical neurons, was virtually not oxidized in cerebellar granule cells.Special Issue dedicated to Prof. Holger Hydén. 相似文献