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121.
Use of permeabilised cells of Chara corallina provides a uniqueopportunity to study the electrical characteristics of the tonoplastwhilst being able to control ionic conditions on the outsideof the membrane. Current-voltage (I/V) analysis over wide voltagespans, and admittance measurements at 5 Hz showed that manypermeabilised cells had a similar conductance and capacitanceto the tonoplast of intact cells. Cells developed two regionsof negative-slope conductance upon addition of external Cl,which suggests the existence of potential-dependent Clchannels in the Chara tonoplast. With Cl concentrationssimilar to those expected in vivo, the resting potential wasmore sensitive to changes in external K+ than Cl; however,a decrease in external K+ did not significantly alter the shapeof the I/V relation.
1Present address: Biopysics Laboratory, School of BiologicalSciences, A12, University of Sydney, Sydney, N.S.W., 2006, Australia
2Permanent address: Department of botany, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan (Received May 6, 1987; Accepted September 21, 1987) 相似文献
122.
Characterisation of a new monoclonal antibody (mAb), designated LAS 41, directed against 124-kilodalton (kDa) etiolated-oat (Avena sativa L.) phytochrome, indicates that it recognises an epitope unique to the red-light-absorbing form, Pr. In a solid-phase enzyme-linked immunosorbent assay (ELISA), LAS 41 exhibits a seven- to eight-fold higher affinity for Pr than for the far-red-light-absorbing form of phytochrome, Pfr. In addition, in immunoprecipitation assays LAS 41 effectively precipitates 100% of phytochrome presented as Pr but only precipitates a maximum of 24.5% of phytochrome presented as Pfr. These values are indicative of binding exclusively to Pr. Peptide-mapping studies show that LAS 41 recognises and epitope located within a region 6–10 kDa from the aminoterminus of the phytochrome molecule. Since binding of LAS 41 to Pr induces alterations in the spectral properties of Pr, this indicates that at least part of the 4 kDa domain to which the antibody binds is essential for protein-chromophore interaction. Subsequent photoconversion of LAS 41-Pr complexes produces native Pfr spectra, with concomitant production of free antibody and antigen, as shown by a modified ELISA. The specificity of LAS 41 for Pr has facilitated the purification of Pfr which is free of contaminating Pr. This has enabled direct determination of the mole fraction of Pfr established by red light to be 0.874.Abbreviations ELISA
enzyme-linked immunsorbent assay
- kDa
kilodalton
- mAb
monoclonal antibody
- Pfr
far-red-absorbing form of phytochrome
- Pr
red-absorbing form of phytochrome
- SDS-PAGE
sodium dodecyl sulphate polyacrylamide gel electrophoresis
- (A)
difference in absorbance (A
665
Pr
–A
730
Pr
)-(A
665
Pfr
–A
730
Pfr
)
- Ar/Afr
spectral change ratio (SCR)
- max
mole fraction of Pfr following saturating red light 相似文献
123.
We found that extensive metabolic switching can be triggered in aromatization. Up to 20–35% of (19-3H3)-labeled substrate is diverted to non-estrogenic product, namely 1β- and 2β-hydroxy androgens, negating usefulness for [3H]water assays. The strikingly substrate-specific metabolite patterns observed reflect positioning at the active site, and the large kinetic isotope effects involved. This switching is unusual in that it involves: 1) tritium labeling, 2) a multistep enzymic process, and 3) the potential unraveling of an enzymic mechanism. 相似文献
124.
Stanley A. Vinores Mary M. Herman Lucien J. Rubinstein 《The Histochemical journal》1987,19(8):439-448
Summary Neuron-specific () enolase, a glycolytic enzyme used as a relatively specific marker for normal neurons and neuroendocrine cells, has recently been found in a variety of neoplastic cells and in reactive astrocytes. Its localization was investigated by immunohisto- and electron-immunocyto-chemistry, in the proliferating supportive Schwann cells of a peripheral ganglioneuroblastoma and in the neoplastic Schwann cells of four acoustic tumours. By light microscopy, the neoplastic Schwann cells showed moderate uneven diffuse immunopositivity for enolase. By electron-immunocy-tochemistry, both types of Schwann cells demonstrated immunopositivity discretely limited to their cell surface membranes. The neoplastic ganglion cells and axons of the ganglioneuroblastoma and the normal neurons and axons included in the schwannomas were, as expected, intensely immunopositive. The visualization of enolase on the cell surface membranes of both neoplastic and non-neoplastic proliferating Schwann cells suggests that increased glycolytic activity may occur on the surface of these proliferating cells irrespective of the nature of the proliferation. 相似文献
125.
Heat shock-induced changes in the structural stability of proteinaceous karyoskeletal elements in vitro and morphological effects in situ 总被引:15,自引:5,他引:10 下载免费PDF全文
Karyoskeletal protein fractions prepared from Drosophila melanogaster embryos contain morphologically identifiable remnants of nuclear pore complexes and peripheral lamina as well as what appears to be an internal nuclear "matrix" (Fisher, P. A., M. Berrios, and G. Blobel, 1982, J. Cell Biol., 92: 674-686). Structural stability of these proteinaceous assemblies is dependent on thermal incubation in vitro (37 degrees C, 15 min) before subfractionation of nuclei. In the absence of such incubation, greater than 90% of the total karyoskeletal protein including major polypeptide components of internal "matrix," pore complexes, and the peripheral lamina, is solubilized by 1 M NaCl. In vivo heat shock induces karyoskeletal stabilization resembling that resulting from thermal incubation in vitro. Immunocytochemical studies have been used to establish the effects of heat shock on the organization and distribution of major karyoskeletal marker proteins in situ. Taken together, these results are consistent with the notion that in vivo, regulation of karyoskeletal plasticity (and perhaps form) may be a functionally significant component of the Drosophila heat shock response. They also have broad practical implications for studies pertaining to the structure and function of karyoskeletal protein (nuclear "matrix") fractions isolated from higher eukaryotic cells. 相似文献
126.
Patrick Sullivan 《CMAJ》1987,137(11):1040-1041
127.
128.
129.
TRH receptors have been solubilized from GH4C1 cells using the plant glycoside digitonin. Solubilized receptors retain the principal binding characteristics exhibited by the TRH receptor in intact pituitary cells and their membranes. The binding of the methylhistidyl derivative of TRH [( 3H]MeTRH) attained equilibrium within 2-3 h at 4 C, and it was reversible, dissociating with a t1/2 of 7 h. Analysis of [3H]MeTRH binding to soluble receptors at 4 C yielded a dissociation constant (Kd) of 3.8 nM and a total binding capacity (Bmax) of 3.9 pmol/mg protein. Peptides known to interact with non-TRH receptors on GH cells failed to interfere with the binding of [3H]MeTRH, indicating that the TRH binding was specific. Chlordiazepoxide, a competitive antagonist for TRH action in GH cells, inhibited TRH binding to soluble receptors with an IC50 of 11 microM. When [3H]MeTRH was bound to membranes and the membrane proteins were then solubilized, we found enhanced dissociation of the prebound [3H]MeTRH from its solubilized receptor by guanyl nucleotides. Maximal enhancement of [3H]MeTRH dissociation by 10 microM GTP gamma S occurred within about 45 min at 22 C. GTP gamma S, GTP, GDP beta S, and GDP were all effectors of [3H]MeTRH dissociation, exhibiting EC50s in the range of 14-450 nM. The rank order of potency of the tested nucleotides was GTP gamma S greater than GTP congruent to GDP beta S greater than GDP much greater than ATP gamma S greater than GMP. We conclude that TRH receptors have been solubilized from GH cells with digitonin and retain the binding characteristics of TRH receptors in intact pituitary cells. Furthermore, prebinding [3H]MeTRH to GH4C1 cell membranes results in the solubilization of a complex in which the TRH receptor is linked functionally to a GTP binding protein. 相似文献
130.
Carbohydrate components (simple sugars and polysaccharides) of cell walls of pearl millet (Pennisetum americanum L., cv. Gahi) were studied as potential substrates for the root-associated diazotroph Azospirillum brasiliense Sp. 7. Simple sugars were utilized, but no evidence was obtained to support the suggestion that the polysaccharide components tested might serve as substrates for growth following hydrolysis by the associated azospirilla. 相似文献