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941.
Measurements of cytoplasmic and vacuolar pH in Neurospora using nitrogen-15 nuclear magnetic resonance spectroscopy 总被引:3,自引:0,他引:3
The nitrogen-15 chemical shift of the N1 (tau)-nitrogen of 15N-labeled histidine and the half-height line widths of proton-coupled resonances of the delta- and omega,omega'-nitrogens of 15N-labeled arginine and of the alpha-nitrogens of 15N-labeled alanine and proline were measured in intact mycelia of Neurospora crassa to obtain to estimates of intracellular pH. For intracellular 15N-labeled histidine, the N1 (tau)-nitrogen chemical shift was 200.2 ppm. In vitro measurements showed that the chemical shift was slightly affected by the presence of phosphate, with which the basic amino acids may be associated in vivo. These considerations indicate a pH of 5.7-6.0 for the environment of intracellular histidine. The half-height line widths of the delta- and omega,omega'-nitrogens of [15N]arginine were 15 and 26 Hz, respectively. In vitro studies showed that these line widths also are influenced by the presence of phosphate, and, after suitable allowance for this, the line widths indicate pH 6.1-6.5 for intracellular arginine. The half-height line widths for intracellular alanine and proline were 17 and 12 Hz, respectively, which are consistent with an intracellular pH of 7.1-7.2. Pools of histidine and arginine are found principally in the vacuole of Neurospora, most likely in association with polyphosphates. Proline and alanine are cytoplasmic. The results reported here are consistent with these localizations and indicate that the vacuolar pH is 6.1 +/- 0.4 while the cytoplasmic pH is 7.15 +/- 0.10. Comparisons of these estimates with those obtained by other techniques and their implications for vacuolar function are discussed. 相似文献
942.
943.
Halothane inhibits the neurotoxin stimulated [14C]guanidinium influx through 'silent' sodium channels in rat glioma C6 cells 总被引:1,自引:0,他引:1
We have investigated the effect of pharmacological agents on [14C]guanidinium ion influx through sodium channels in C6 rat glioma and N18 mouse neuroblastoma cells. The sodium channels of the N18 cells can be activated by aconitine alone, indicating that they are voltage-dependent channels. In contrast, sodium channels in the C6 cells require the synergistic action of aconitine and scorpion toxin for activation and are therefore characterized as so-called silent channels. The general anesthetic halothane used at clinical concentrations, specifically inhibited the ion flux through the silent sodium channel of C6 rat glioma cells. The voltage-dependent channels of the N18 cells were insensitive to halothane at the concentrations tested. 相似文献
944.
The mechanism of allosteric regulation of rabbit muscle pyruvate kinase (PK) was examined in the presence of the allosteric inhibitor phenylalanine (Phe). Steady-state kinetic, equilibrium binding, and structural studies were conducted to provide a broad data base to establish a reasonable model for the interactions. Phe was shown to induce apparent cooperativity in the steady-state kinetic measurements at pH 7.5 and 23 degrees C. The apparent Km for phosphoenolpyruvate was shown to increase with increasing Phe concentrations. These results imply that Phe reduces the affinity of PK for phosphoenolpyruvate. This conclusion was substantiated by equilibrium binding studies which yielded association constants of phosphoenolpyruvate as a function of Phe concentration. The binding constant of Phe was also determined at pH 7.0 and 23 degrees C. The effect of ligands on the hydrodynamic properties of PK was monitored by difference sedimentation velocity, sedimentation velocity, and equilibrium experiments. The results showed that PK remains tetrameric both in the presence and in the absence of Phe. However, Phe induces a small decrease in the sedimentation coefficient of the enzyme; hence, it suggests a loosening of the protein structure. The accessibility of the sulfhydryl residues of the enzyme also increases in the presence of Phe. Furthermore, the Phe-induced conformational change was approximately 90% complete when only 25% of the binding sites were saturated. This result suggested that the regulatory behavior of PK might satisfactorily be described by the two-state model of Monod-Wyman-Changeux [Monod, J., Wyman, J., & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88-118]. 相似文献
945.
Enumeration of denitrifying microbial populations in turf 总被引:2,自引:0,他引:2
Summary Denitrifer populations of a silt and silt loam soil under a Kentucky bluegrass turf were enumerated using the most probable
number (MPN) procedure. The influence of soil texture, soil depth, soil moisture, and additions of nitrate fertilizer on denitrifier
populations were determined.
Saturated soil conditions increased denitrifier populations 87-fold in the silt soil and 121-fold in the silt loam soil. Denitrifier
populations did not differ significantly between soil depths and additions of fertilizer nitrate did not influence populations. 相似文献
946.
Dityrosine is a prominent component of the yeast ascospore wall. A proof of its structure 总被引:14,自引:0,他引:14
P Briza G Winkler H Kalchhauser M Breitenbach 《The Journal of biological chemistry》1986,261(9):4288-4294
The yeast ascospore wall consists of four morphologically distinct layers. The hydrophobic surface layers are biogenically derived from the prospore wall and appear dark after OsO4 staining. They seem to be responsible for the stability of the spores against attack by lytic enzymes. By amino acid analysis of acid hydrolysates of ascospore walls, two new peaks were detected, which were shown to be the racemic and meso form, respectively, of dityrosine. The identity of this hitherto unknown component of the yeast ascospore wall with standard dityrosine was proven by 1H NMR and by mass spectrometry. A 13C NMR spectroscopic investigation of the structure of dityrosine confirmed that, in natural dityrosine, the biphenyl linkage is located ortho, ortho to the hydroxyl groups. Following digestion of the inner layers of isolated ascospore walls it was shown that dityrosine is very probably located only in the surface layers. The same conclusion was reached independently by an investigation of spores of a strain homozygous for the mutation gcn1, which lack the outermost layers of the spore wall and were practically devoid of dityrosine. In sporulating yeast, L-tyrosine was readily incorporated into the dityrosine of the ascospore wall. Control experiments involving vegetative a/alpha cells and nonsporulating alpha/alpha cells under sporulation conditions showed that dityrosine is indeed sporulation-specific. 相似文献
947.
Diphasic ventilatory response to hypoxia in newborn lambs 总被引:2,自引:0,他引:2
948.
Cytochrome b561 spectral changes associated with electron transfer in chromaffin-vesicle ghosts 总被引:1,自引:0,他引:1
The involvement of cytochrome b561, an integral membrane protein, in electron transfer across chromaffin-vesicle membranes is confirmed by changes in its redox state observed as changes in the absorption spectrum occurring during electron transfer. In ascorbate-loaded chromaffin-vesicle ghosts, cytochrome b561 is nearly completely reduced and exhibits an absorption maximum at 561 nm. When ferricyanide is added to a suspension of these ghosts, the cytochrome becomes oxidized as indicated by the disappearance of the 561 nm absorption. If a small amount of ferricyanide is added, it becomes completely reduced by electron transfer from intravesicular ascorbate. When this happens, cytochrome b561 returns to its reduced state. If an excess of ferricyanide is added, the intravesicular ascorbate becomes exhausted and the cytochrome b561 remains oxidized. The spectrum of these absorbance changes correlates with the difference spectrum (reduced-oxidized) of cytochrome b561. Cytochrome b561 becomes transiently oxidized when ascorbate oxidase is added to a suspension of ascorbate-loaded ghosts. Since dehydroascorbate does not oxidize cytochrome b561, it is likely that oxidation is caused by semidehydroascorbate generated by ascorbate oxidase acting on free ascorbate. This suggests that cytochrome b561 can reduce semidehydroascorbate and supports the hypothesis that the function of cytochrome b561 in vivo is to transfer electrons into chromaffin vesicles to reduce internal semidehydroascorbate to ascorbate. 相似文献
949.
950.