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991.
Molecular cloning of cDNA for rat L-type pyruvate kinase and aldolase B 总被引:13,自引:0,他引:13
M P Simon C Besmond D Cottreau A Weber P Chaumet-Riffaud J C Dreyfus J S Trépat J Marie A Kahn 《The Journal of biological chemistry》1983,258(23):14576-14584
Two double-stranded cDNA recombinant pBR322 plasmid libraries were constructed starting from high carbohydrate diet rat liver poly(A)+ mRNA, either fractionated by denaturing sucrose gradient centrifugation for the cloning of L-type pyruvate kinase cDNA, or nonfractionated for aldolase B. Both libraries were screened with single-stranded cDNA probes reverse transcribed from fasted or high carbohydrate diet rat liver mRNAs. mRNAs from fasted animals were also fractionated by sucrose gradient centrifugation and mRNAs from the fed animals were, in addition, further purified by high performance liquid gel filtration chromatography. Those clones hybridizing with the "positive" probe (from animals fed the high carbohydrate diet) and not with the "negative" one (from fasted animals) were preselected and their plasmid DNA was purified and analyzed by positive hybridization-selection. Thirty of 4500 bacteria colonies transformed by recombinant plasmids were preselected by differential screening for pyruvate kinase, and 8 of 864 colonies for aldolase B. Twenty-two recombinant plasmids for pyruvate kinase and two for aldolase B were shown to contain specific cDNA inserts by positive hybridization-selection. Plasmids DNAs of some pyruvate kinase and aldolase B clones (whose inserts ranged from 700 to 1050 bases in length) were labeled by nick translation and used as probes for Northern blot hybridization. The pyruvate kinase cDNA probes recognized mainly a 3400-base RNA species which was detected in high carbohydrate diet rat liver, but not in fasted rat liver and in tissues which do not synthesize L-type pyruvate kinase. In addition, some pyruvate kinase probes hybridized with minor RNA species of about 2000 bases in length, only observed after carbohydrate diet. For aldolase B, the recombinant plasmid DNA hybridized with a single RNA species of 1750 bases. This RNA, detected in kidney, small intestine and liver, was induced by a high carbohydrate diet and increased with liver development. The rat probe cross-hybridized with human aldolase B messenger RNA. 相似文献
992.
Interaction of medroxyprogesterone acetate with cytosol androgen receptors in the rat hypothalamus and pituitary 总被引:1,自引:0,他引:1
G Pérez-Palacios B Chávez F Vilchis N Escobar F Larrea A E Pérez 《Journal of steroid biochemistry》1983,19(6):1729-1735
The binding of medroxyprogesterone acetate (MPA) with cytosol androgen receptors from rat pituitary and hypothalamus was studied. The pituitary and hypothalamic cytosol androgen receptors from adult castrated female rats were in vitro labeled using 3H natural (testosterone (T) and 5 alpha-dihydrotestosterone (DHT] and [3H]synthetic (methyltrienolone) androgens as radioligands. The [3H]androgen-receptor complexes sedimented with a coefficient of 8S in linear sucrose gradients. When incubated with an excess of radioinert MPA, specific binding was abolished indicating interaction of MPA with androgen receptors. Furthermore specific [3H]MPA-androgen cytosol receptor complexes could be identified in these neuroendocrine tissues when a post-gradient receptor labeling technique was used in the absence or presence of radioinert MPA, DHT, and triamcinolone acetonide. A study of binding kinetics disclosed that the equilibrium dissociation constant and saturation binding capacity for the MPA binder, were similar to those exhibited by DHT binding to androgen receptors in both studied tissues under identical experimental conditions. The overall results were interpreted as demonstrating that MPA interacts with cytosol steroid receptors other than those of progesterone in the rat hypothalamus and anterior pituitary. The data are consistent with MPA binding to androgen receptors. 相似文献
993.
G. Le Fur M.L. Perrier N. Vaucher F. Imbault A. Flamier J. Benavides A. Uzan C. Renault M.C. Dubroeucq C. Guérémy 《Life sciences》1983,32(16):1839-1847
[3H] R05-4864 binding sites have been characterized in kidney, heart, brain, adrenals and platelets in the rat. In all these organs the following order of potency in the R05-4864 displacement was found : R05-4864 > diazepam > clonazepam indicating that they correspond to the “peripheral type” of benzodiazepine binding sites. PK 11195, an isoquinoline carboxamide derivative, displaces [3H] R05-4864 from its binding sites in all the organs. PK 11195 was as potent as R05-4864 in the platelets, heart, adrenals, kidney and several brain regions (midbrain, hypothalamus, medulla + pons and hippocampus. However it was 5 to 10 times more effective in cortex and striatum. In conclusion PK 11195 might represent a new tool to elucidate the physiological relevance of “peripheral type” benzodiazepine binding sites and might help to discriminate the hypothetical subclasses of these binding sites. 相似文献
994.
G Le Fur E Zarifian T Phan H Cuche A Flamier F Bouchami M C Burgevin H Loo A Gérard A Uzan 《Life sciences》1983,32(3):249-255
[3H]spiroperidol binding to lymphocytes was measured in untreated paranoid or disorganized and treated paranoid schizophrenic patients. An increase in the Bmax was detected in untreated paranoid patients but a decrease was found in the disorganized patients. No difference was detected in the KD value. Neuroleptic treatment produced a decrease in the Bmax without affecting the KD value. Such results did not comply with the down regulation but might be explained by a change in membrane viscosity as [3H]spiroperidol binding sites on lymphocytes were coupled to phospholipid methylation. 相似文献
995.
996.
In-vitro-grown cells of Mucuna pruriens, immobilized in calcium-alginate gels, were able to transform the precursor L-tyrosine into L-dihydroxyphenylalanine (L-DOPA). After the immobilization in alginate the plant cells released 90% of the produced L-DOPA into the medium; supplementation of the medium with calcium inhibited both the transformation of L-tyrosine into L-DOPA and the release of L-DOPA into the medium. Continuous illumination of the beads had a slight beneficial effect on the synthesis of L-DOPA. A simple production medium for the transformation of L-tyrosine into L-DOPA was designed. This medium contained only sucrose and sodium chloride as osmotic stabilizers, a low concentration of calcium chloride for stabilization of the alginate beads, and L-tyrosine as the precursor. 相似文献
997.
Calli derived from leaves and radicles of B. ternifolia were grown on Murashige and Skoog (MS) basal medium, and the effects of different nitrogen sources on the rate of callus growth and on the enzymes related to nitrogen assimilation were studied. Ammonium alone did not support callus growth unless a Krebs-cycle intermediate was added to the medium. The activities of glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 1.4.7.1), and glutamate dehydrogenase (EC 1.4.1.2) were measured in homogenates of callus grown on media supplied with different nitrogen sources. The results indicate that leaf and root calli have similar levels of these enzymes when grown on MS medium (Murashige and Skoog 1962. Physiol. Plant. 15, 473–497). However, when the calli were supplied with glutamine as the sole nitrogen source, the activity of glutamate synthase increased in leaf callus but was almost completely inhibited in root callus. The results indicate that calli originated from different B. ternifolia tissues do not have the same biochemical dedifferentiated state. 相似文献
998.
The kinetic properties of active Ca2+ transport into mitochondria and microsomal membrane vesicles prepared from coleoptiles of dark-and light-grown corn seedlings have been studied. The apparent values for K m and V max for Ca2+ of the mitochondrial transport system from dark-grown plants are about one order of magnitude higher than those from the microsomal transport system. Calmodulin has no effect on the Ca2+ accumulation into mitochondria whereas the apparent maximum transport velocity and affinity for Ca2+ of the microsomal Ca2+-transport system are both increased by calmodulin. When intact corn seedlings are irradiated with far-red light, the calmodulin-induced increase of the apparent maximum transport velocity and affinity for Ca2+ can no longer be observed. From these data it can be concluded that the low cytoplasmic Ca2+ concentration in the cytoplasm of coleoptile cells from dark-grown corn is maintained by a calmodulin-regulated Ca2+ pump. Irradiation with photomorphogenically active far-red light lowers the Ca2+-transport activity and thus causes an increase of the cytoplasmic, free-Ca2+ concentration. The physiological implications will be discussed. 相似文献
999.
The specific activity of bisphosphoglycerate synthase (EC 2.7.5.4) is significantly higher in rat mature erythrocytes than in reticulocytes from phenylhydrazine treated animals, while 2,3-bisphosphoglycerate levels do not differ between them. The enzyme specific activity and the concentration of 2,3-bisphosphoglycerate are both negligible in the erythroid cell pool from the bone marrow of anaemic animals. Thus, a cellular specialization in the last stages of erythropoiesis is confirmed. Human bisphosphoglycerate synthase shows a lower specific activity than that of rat without a parallel decrease in 2,3-bisphosphoglycerate levels. 相似文献
1000.