Solubilization and Characterization of the Nitrendipine Receptor in Rat Brain

The nitrendipine receptor associated with the voltage-dependent calcium channel in rat brain was solubilized by detergent extraction and sonication. The detergent solution used for extraction consisted of 10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 0.25% (wt/vol) polyoxyethylene 20 cetyl ether (Brij 58), and 0.025% (wt/vol) polyoxyethylene 17 cetyl stearyl ether (Lubrol WX) in the presence of 30% (wt/vol) glycerol as a stabilizer. The molecular weight of the receptor was estimated to be 1,800K by Sephacryl S-500 gel filtration and 800K by sucrose density gradient sedimentation. The equilibrium dissociation constant of [3H]nitrendipine to the solubilized receptors was 5.6 nM, which is approximately 10 times that of the membrane-bound receptor. The binding of nitrendipine to the receptor was inhibited noncompetitively by the structurally unrelated calcium channel inhibitors verapamil and prenylamine; their concentrations for 50% inhibition were both 1.0 X 10(-7) M, and they caused maximal inhibitions of 70 and 100%, respectively.     

白兰瓜果实输入~(14)C-葡萄糖的转化与蔗糖合成

~(14)C 追踪试验结果表明,白兰瓜幼果中输入的~(14)C-葡萄糖,50%以上转化为稀酸水解和稀酸不水解的结构物质;果实发育后期,输入后48小时,在果肉和种子中分别只有18%和32%的~(14)C 参入结构物质。根据醇溶性糖的纸层析鉴定,幼果薄片渗入的~(14)C-葡萄糖仅转化为果糖,而发育后期果实则更多转化为蔗糖。显然,幼果的代谢模式是使物质和能量导向结构物质的形成;而后期果实生长已基本停止,物质代谢的方向又转向蔗糖合成的轨道上来。蔗糖合成底物试验结果表明,供给幼果不同底物都只有很低的蔗糖合成活性;发育后期果实供给UDPG+F-6-P 底物时可测出较高的蔗糖合成活性,初步推测白兰瓜中蔗糖合成主要是通过蔗糖磷酸酯合成酶来实现的。     

Sucrose transport into the phloem of Ricinus communis L. seedlings as measured by the analysis of sieve-tube sap

Careful cutting of the hypocotyl of Ricinus communis L. seedlings led to the exudation of pure sieve-tube sap for 2–3 h. This offered the possibility of testing the phloem-loading system qualitatively and quantitatively by incubating the cotyledons with different solutes of various concentrations to determine whether or not these solutes were loaded into the sieve tubes. The concentration which was achieved by loading and the time course could also be documented. This study concentrated on the loading of sucrose because it is the major naturally translocated sieve-tube compound. The sucrose concentration of sieve-tube sap was approx. 300 mM when the cotyledons were buried in the endosperm. When the cotyledons were excised from the endosperm and incubated in buffer, the sucrose concentration decreased gradually to 80–100 mM. This sucrose level was maintained for several hours by starch breakdown. Incubation of the excised cotyledons in sucrose caused the sucrose concentration in the sieve tubes to rise from 80 to 400 mM, depending on the sucrose concentration in the medium. Thus the sucrose concentration in the sieve tubes could be manipulated over a wide range. The transfer of labelled sucrose to the sieve-tube sap took 10 min; full isotope equilibration was finally reached after 2 h. An increase of K⁺ in the medium or in the sieve tubes did not change the sucrose concentration in the sievetube sap. Similarly the experimentally induced change of sucrose concentration in the sieve tubes did not affect the K⁺ concentration in the exudate. High concentrations of K⁺, however, strongly reduced the flow rate of exudation. Similar results were obtained with Na⁺ (data not shown). The minimum translocation speed in the sieve tubes in vivo was calculated from the growth increment of the seedling to be 1.03 m·h^-1, a value, which on average was also obtained for the exudation system with the endosperm attached. This comparison of the in-vivo rate of phloem transport and the exudation rate from cut hypocotyls indicates that sink control of phloem transport in the seedlings of that particular age was small, if there was any at all, and that the results from the experimental exudation system were probably not falsified by removal of the sink tissues.Abbreviations PTS 3-hydroxy-5,8, 10-pyrenetrisulfonate     

Altered tissue specificity of the revertant shrunken allele upon Dissociation (Ds) excision is associated with loss of expression and molecular rearrangement at the corresponding non-allelic isozyme locus in maize

Summary Transposable element Activator (Ac) induced wild-type stable revertants, derived from McClintock's Dissociation (Ds) insertion shrunken (sh) mutant sh-m5933, have been examined for sucrose synthases, SS1 and SS2, encoded by the revertant (Sh) locus and the non-allelic gene Sus (previously designated as Ss2), respectively. A structurally normal Sh locus has been previously described in these revertants. Immuno-blot (Western) and Southern hybridization analyses reported here identify one of the nine alleles, Sh-r5, as unique for several features. It showed altered tissue specificity, as the SS1 protein encoded by the Sh-r5 allele was readily detectable in the immature embryo which is otherwise characterized by the Sus expression only. The level of Sh-r5 expression at the protein and enzyme level was marked by endosperm specific SS1 abundance and a significant down-regulation in the embryo similar to the standard Sh and Sus loci in endosperm and embryo, respectively. We infer that tissue specific levels of gene expression among maize Ss genes is significantly determined by trans-regulatory factors present in these two tissues. The Sh-r5 strain also exhibited a complete loss of the Sus expression in all tissues tested in the plant. Lack of any detectable phenotypic abnormality in the Sh-r5 strain due to the loss of SS2 protein indicated that either the SS2 protein is nonessential or that the two SS isozymes are functionally compensatory. Genomic filter hybridizations with the Sus cDNA clone indicated that the Sus locus in the Sh-r5 strain was not deleted and was, in fact, unique among these revertants. Together, these data provide an unusual insight into the regulation and function of the two SS isozymes in the maize plant.     

Sucrose transport in tonoplast vesicles of red beet roots is linked to ATP hydrolysis

Sucrose uptake into tonoplast vesicles, which were prepared from red beet (Beta vulgaris L.) vacuoles isolated by two different methods, was stimulated by MgATP. Using the same medium as for osmotic disruption of vacuoles, membrane vesicles were prepared from tissue homogenates of dormant red beet roots and separated by high-speed centrifugation through a discontinuous dextran gradient. A low-density microsomal fraction highly enriched in tonoplast vesicles could be further purified from contaminating ER vesicles by inclusion of 5 mM MgCl₂ in the homogenization medium. These vesicles were able to transport sucrose in an ATP-dependent manner against a concentration gradient, whereas vesicles from regions of other densities lacked this feature, indicating that ATP stimulation of sucrose uptake took place only at the tonoplast membrane. Sucrose uptake was optimal at pH 7 in the presence of MgATP and could be stimulated by superimposed pH gradients (vesicle interior acidic) in the absence of MgATP, which is consistent with the operation of a sucrose/H⁺-antiporter at the tonoplast. Tonoplast vesicles, obtained in high yield from tissue homogenates of red beet roots, exhibited sugar-uptake characteristics comparable to those of intact vacuoles; these characteristics included similarities in K _m (1.7 mM), sensitivity to inhibitors and specificity for sucrose.Many experiments were carried out at the Experiment Station of the HSPA, Aiea, Hawaii and financed by an NSF grant to Dr. Maretzki and Mrs. M. Thom.     

Evidence for the involvement of a UDP-glucose-dependent group translocator in sucrose uptake into vacuoles of storage roots of red beet

Vacuoles isolated from the storage roots of red beet (Beta vulgaris L.) accumulate sucrose via two different mechanisms. One mechanism transports sucrose directly, and its rate is increased by the addition of MgATP. The other mechanism utilizes uridine diphosphate glucose (UDP-glucose) to synthesize and simultaneously transport sucrose phosphate and sucrose into the vacuole. This group translocation mechanism has also been found in sugarcane vacuoles. As in sugarcane, the beet group translocator does not require fructose 6-phosphate, nor is the latter substance transported into the vacuole. The uptake of UDP[¹⁴C]glucose in inhibited by high concentrations of osmoticum.Abbreviations EDTA ethylenediaminetetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - UDP uridine 5-diphosphate