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1.
The intracellular localisation of phytochrome and ubiquitin in irradiated oat coleoptiles was analysed by electron microscopy. We applied indirect immunolabeling with polyclonal antibodies against phytochrome from etiolated oat seedlings or polyclonal antibodies against ubiquitin from rabbit reticulocytes, together with a goldcoupled second antibody, on serial ultrathin sections of resin-embedded material. Immediately after a 5-min pulse of red light-converting phytochrome from the red-absorbing (Pr) to the far-redabsorbing (Pfr) form-the label for phytochrome was found to be sequestered in electron-dense areas. For up to 2 h after irradiation, the size of these areas increased with increasing dark periods. The ubiquitin label was found in the same electrondense areas only after a dark period of 30 min. A 5 min pulse of far-red light, which reverts Pfr to Pr, given immediately after the red light did not cause the electron-dense structures to disappear; moreover, they contained the phytochrome label immediately after the far-red pulse. In contrast, after the reverting far-red light pulse, ubiquitin could only be visualised in the electron-dense areas after prolonged dark periods (i.e. 60 min). The relevance of these data to light-induced phytochrome pelletability and to the destruction of both Pr and Pfr is discussed.Abbreviations FR
far-red light; Pfr
- Pr
far-red-absorbing and red-absorbing forms of phytochrome, respectively
- R
red light 相似文献
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F Wunderlich M Helwig G Schillinger V Speth M F Wiser 《European journal of cell biology》1988,47(2):157-164
Erythrocytes infected with the malaria parasite Plasmodium chabaudi contain the neo-protein Pc90 in their plasma membrane. We investigate origin, membrane disposition, and intraerythrocytic traffic of this Pc90. Metabolic labeling of P.-infected erythrocytes, combined with cell fractionation as well as Western blot analysis and immunoprecipitation using a Pc90-recognizing monoclonal antibody, show that Pc90 is synthesized by early to mid trophozoites and is transported without any apparent processing steps to the erythrocyte membrane. Based upon the inaccessibility of Pc90 from the outside in intact erythrocytes and the water solubility of membrane-associated Pc90, it is concluded that Pc90 is localized on the cytoplasmic face of the host erythrocyte membrane. Immunoelectron microscopy using a Pc90-specific monoclonal antibody and the occurrence of soluble Pc90 in host cell cytosol indicate that the Pc90 is transported in both a 'vesicle-bound' and a 'free' form through the erythrocyte cytoplasm. 相似文献
4.
Auxin-mediated elongation growth of isolated subapical coleoptile segments of maize (Zea mays L.) is controlled by the extensibility of the outer cell wall of the outer epidermis (Kutschera et al., 1987). Here we investigate the hypothesis that auxin controls the extensibility of this wall by changing the orientation of newly deposited microfibrils through a corresponding change in the orientation of cortical microtubules. On the basis of electron micrographs it is shown that cessation of growth after removal of the endogenous source of auxin is correlated with a relative increase of longitudinally orientated microfibrils and microtubules at the inner wall surface. Conversely, reinduction of growth by exogenous auxin is correlated with a relative increase of transversely orientated microfibrils and microtubules at the inner wall surface. These changes can be detected 30–60 min after the removal and addition of auxin, respectively. The functional significance of directional changes of newly desposited wall microfibrils for the control of elongation growth is discussed. 相似文献
5.
Rabbit skeletal muscle protein phosphatases C-I and C-II have been previously isolated as two proteins of Mr = approximately 35,000. Both enzymes display broad substrate specificities but have distinct enzymatic properties in regard to their susceptibility to heat-stable protein inhibitor-2 and their response to divalent cations. Monoclonal antibodies against both protein phosphatase C-I and C-II were produced by fusion of spleen cells of immunized BALB/c mice with SP2/0-Ag14 mouse myeloma cells. The products of the hybrid cells were screened by solid phase radioimmunoassay for the production of antibodies to protein phosphatase C-I and C-II. Positive cells were cloned and injected into mice to produce ascitic fluids. Ten monoclonal antibodies against phosphatase C-I and eight monoclonal antibodies against phosphatase C-II were obtained. These antibodies were characterized with regard to their relative binding affinities to the two protein phosphatases and their abilities to inhibit the phosphorylase phosphatase activities of the two enzymes. All ten of the phosphatase C-I monoclonal antibodies inhibited the phosphorylase phosphatase activity of phosphatase C-I, and three of these also inhibited phosphatase C-II. Only one of the eight antibodies to phosphatase C-II was inhibitory and inhibited the activities of both phosphatase C-I and C-II. Examination of the binding of these monoclonal antibodies by a solid phase radioimmunoassay showed that eight of the ten phosphatase C-I antibodies cross-reacted with phosphatase C-II, while all eight of the phosphatase C-II antibodies cross-reacted with phosphatase C-I. These findings show that phosphatases C-I and C-II possess common antigenic determinant(s) and may, therefore, be structurally related proteins. 相似文献
6.
We have analysed the intracellular localisation of phytochrome in oat coleoptile cells by electron microscopy and confirm and extend light-microscopical findings of previous authors. We used indirect immuno-labeling with polyclonal antibodies against 60-KDa phytochrome from etiolated oat seedlings, and a gold-coupled second antibody, on ultrathin sections of LR-white-embedded material. In dark-grown seedlings, phytochrome-labeling is distributed diffusely throughout the cytoplasm. Organelles and membranes are not labeled. After photoconversion of the red-absorbing form of phytochrome to the far-red absorbing form (Pfr) (5-min red light; 660 nm), the label is sequestered uniquely in electron-dense areas within the cytoplasm. These areas are irregularly shaped, are often located in the vicinity of the vacuole, are not surrounded by a membrane, exclude cellular organelles and ribosomes and are not found in dark-grown material; an immediate 5-min farred light pulse after the red light does not cause these structures to disappear. After a dark period of 3–4 h following red-light irradiation, these electron-dense structures disappear together with any specific labeling. We suggest a Pfr-induced aggregation of an unknown, phytochrome-binding protein or proteins.Abbreviations
Pr and Pfr
phytochrome in its red and far-red absorbing form, respectively 相似文献
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Autoradiographic Localization of Angiotensin II Receptor Binding Sites on Noradrenergic Neurons of the Locus Coeruleus of the Rat 总被引:2,自引:2,他引:0
The locus coeruleus (LC) of the rat was lesioned by microinjection of selective neurotoxins into the brainstem. 6-Hydroxydopamine (6-OHDA), 3 micrograms/microliter, given unilaterally at two sites 0.6 mm apart on the rostro-caudal axis of the LC, was used to lesion catecholamine-containing neuronal elements. Ibotenic acid, 2.5 micrograms/0.5 microliters, administered similarly was used to lesion nerve cell bodies. Two weeks after administration of the neurotoxin, lesion efficacy was determined based on the norepinephrine content of the cerebral cortex ipsi- and contralateral to the lesion. 6-OHDA lesions of the LC caused a 46% reduction in ipsilateral cortical norepinephrine and a 60% reduction in specific 125I-[Sar1, Ile8]-angiotensin II (125I-SIAII) binding in the LC. Ibotenic acid lesions of the LC caused a 73% reduction in ipsilateral cortical norepinephrine and a 81% reduction in specific 125I-SIAII binding in the LC. These results indicate that AII receptor binding sites in the LC are localized on noradrenergic nerve cell bodies or their dendritic and axonal ramifications within the LC. 相似文献
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10.
In vitro autoradiographic localization of [I]-angiotensin II binding sites in the rat and dog kidney
Light microscopic autoradiographic techniques have been utilized to demonstrate specific regions of the rat and dog kidney where angiotensin II receptors exist. Slide mounted tissue sections were labeled with [125I]-angiotensin II using conditions which provided for highly specific binding. These angiotensin II binding sites were localized to several distinct renal structures. In the renal cortex, angiotensin II binding sites were found concentrated in all parts of the glomeruli including the vascular components, the macula densa and the juxtaglomerular apparatus. Angiotensin II binding in the medulla was more diffusely associated with the vasa recta, and to a lesser extent, the thick ascending segment of the loop of Henle. Binding sites specific for angiotensin II were also found in the smooth muscle laminae of the ureter. Scatchard analysis of the binding kinetics allowed the demonstration of two subpopulations of binding sites which differ slightly in their affinities for [125I]-angiotensin II. These subpopulations appear to be associated with distinct components of the renal structure. 相似文献