Improved methods for determination of guanylyl cyclase activity and synthesis of [α-32P]GTP

A modification of the adenylyl cyclase assay method of Y. Salomon, C. Londos, and M. Rodbell (1974, Anal. Biochem. 48, 541–548) is described. It makes the method applicable to the determination of guanylyl cyclase in subcellular tissue fractions. The modification consists of performing the Dowex 50 chromatography at acid pH in a column that has bed volume that is three times as large. This modification results in low blanks and allows for reuse of both Dowex 50 and aluminum oxide columns. A modification of the method of Symons [1974, Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 29, pp. 105–115, Academic Press, New York] for synthesis of [α-³²P]nucleoside triphosphates is also presented. It allows all steps to be carried out within 5 h in a single reaction vessel. Synthesized NTPs are then purified by DEAE-Sephadex A-25 (HCO₃^?form) using a linear ammonium bicarbonate gradient. Its applicability to synthesis of [α-³²P]GTP, [α-³²P]dGTP, and [α-³²P]ATP having specific activities of up to 75 Ci/mmol is shown.     

A rapid method for the measurement of [γ-32P]ATP specific radioactivity in tissue extracts and its application to the study of 32Pi uptake in perfused rat heart

(i) A new, rapid method for the measurement of [γ-³²P]ATP specific radioactivity in tissue extracts in the presence of other ³²P-containing compounds is described. The deproteinized extract is incubated with phosphorylase b and phosphorylase kinase, and the incorporation of ³²P into protein from [γ-³²P]ATP is measured by precipitation on filter paper in trichloroacetic acid. No separation of ATP or other treatment of the extracts is required for the assay. (ii) ³²P_i uptake in perfused rat heart was found to be a relatively slow process, with a K_m of 0.084 mm, whereas equilibration between intracellular ³²P_i and [γ-³²P]ATP occurred rapidly.     

L7811鼠腹水肿瘤细胞 31P核磁共振的研究

用 ³¹P核磁共振技术(³¹P-NMR)研究了L₇₈₁₁鼠腹水肿瘤细胞和615系鼠胸腺细胞(正常对照细胞)。结果发现在肿瘤晚期阶段,L₇₈₁₁腹水肿瘤细胞的含磷化合物未进入完全不活跃状态。此外,腹水肿瘤细胞的磷脂组成与含量亦有明显改变。因此, ³¹P-NMR谱可做为观察肿瘤细胞内能量生成和某些磷脂合成宏观动态过程的一项参考指标。     

两个特用稻的营养供应水平与32P、14C分布和谷产量的关系

香稻(竹香粘)的分蘖数多,其分蘖期根系吸收³²P和³²P分布到分蘖,以高、中肥和高N,高K肥处理的为高;¹⁴C-葡萄糖同化物分布在分蘖比黑米稻(黑优粘)和常规稻“双桂36”的高,但灌浆期和黄熟期¹⁴C分布在稻穗较少.低肥处理的稻谷产量较高.而在高、中肥条件下结实率降低,实粒数减少,成穗率稍低,谷产量也比“黑优粘”和“双桂36”低. “黑优粘”在高肥、高N条件下的分蘖多,成穗率高、结实粒数也较多,稻谷产量比“竹香粘”高,在高、中肥、高 N高 P条件下,³²P分布于分蘖多,分蘖的¹⁴C分布则以高 N,高 K肥条件下的多;开花和灌浆用稻穗的¹⁴C分布皆以高 K肥水平的为高。“黑优粘”是耐肥和需钾肥品种,高肥、高K可提高其稻谷产量,但仍比“双桂36”低.     

A simple modification to an enzymic method for the preparation of[γ-32P]ATP free of salt and buffer

An existing enzymic method for preparing [γ-³²P]ATP from 32P_i has been modified toyield [γ-³²P]ATP free of salt and buffer. ³²P is incorporated into the γ-position of ATP by isotopic exchange in the presence of glyceraldehyde 3-phosphate dehydrogenase and 3-phosphoglycerate kinase. Unreacted ³²P_i is separated from [γ-³²P]ATP by column chromatography on Dowex 1 bicarbonate. [γ-³²P]ATP is eluted with 2 m triethylammonium bicarbonate, which is then completely removed by freeze-drying.     

[3H] BOC [NLE28,31]CCK27−33, a new highly labelle ligand for CCK receptors: Binding on brain and on pancreas

Preliminary results on the binding of [³H]Boc[Nle^28,31]CCK_27^?33, designated [³H]Boc[diNle]CCK₇, on mouse brain and rat pancreas membranes are presented. This new ligand for CCK receptors possesses a high specific activity (144 Ci/mmole), and binds in a saturable manner to mouse brain (Kd = 0.49 nM, Bmax = 49 fmoles/mg protein) and rat pancreas (Kd = 4.4 nM, Bmax = 696 fmoles/mg protein). Unlabelled Boc[diNle]CCK₇ displaces [¹²⁵I]CCK₈ from its binding sites on mouse brain membranes with a high affinity, slightly superior to that of CCK₈. The order of potencies to displace [³H]Boc[diNle]CCK₇ from its binding sites was the same on mouse brain and rat pancreas: $\text{[}{}^3\text{HBoc[diNle]CCK}{}_7\text{>}\text{CCK}{}_8\text{,}\text{Boc-CCK}{}_7\text{>}\text{non-sulfated CCK}{}_8$, the pancreas binding sites being more discriminative than the brain binding sites. Thus, [³H]Boc[diNle]CCK₇ is a very promising new probe for the characterization of CCK receptors and their interaction with different CCK fragments.     

杂交水稻生育后期不同水分管理对32P的吸收、分布和结实的影响

<正> 杂交水稻具有穗大、粒多、粒重的经济性状,且根系发达,吸收水、肥的能力较强,生育后期的生理需水明显地比一般品种多,植株从抽穗至成熟积累磷素亦多,这些生理特点可能与结实有关。然而,几年来关于环境条件影响杂交水稻结实的研究,着重于生育后期温度、光照条件的分析,并取得了许多成绩,对于生育后期水分管理支配磷素的吸收、     

Irreversible binding of [3H] (−) N-chloroethylnorapomorphine to mammalian brain tissue

A proposed dopamine (DA) receptor labeling agent, [³H] (?) N-chloroethylnorapomorphine (³H-NCA) underwent relatively little chemical change at 25°C and pH 6.4 up to an hour of incubation. At low (nM) concentrations it bound rapidly and avidly to a membrane preparation of calf caudate nucleus, but the binding did not saturate over two hours of incubation or at ligand concentrations between 0.2 nM and 10 μM. While similarly bound [³H]-(?) apomorphine was rapidly displaced by DA and other agents that interact with DA receptors, ³H-NCA could not be displaced by unlabeled DA, apomorphine and (+)butaclamol, nor by denaturation of the tissue with trichloracetic acid (TCA). There was no evidence of selectivity of binding of ³H-NCA in regions of rat brain, and binding occurred even to TCA-denatured caudate tissue. Catechol-aporphines prevented binding of ³H-NCA to calf caudate membranes by up to 30%, but this effect was not stereoselective and was lost at concentrations of ³H-NCA above 100 nM. In contrast, DA and ADTN as well as neuroleptics and adrenergic agonists had no such effect. The results suggest that while ³H-NCA may bind irreversibly, and possibly covalently, it does not have high selectivity for labeling dopamine D-3 or D-2 receptor sites, but may be partially selective for an aporphine binding site.     

Equilibration of [3H]glucosamine and [35S]sulfate with intracellular pools of UDP-N-acetylhexosamine and 3′-phosphoadenosine-5′-phosphosulfate (PAPS) in cultured fibroblasts

Nucleotides and sugar nucleotides were extracted from cultures of human fibroblasts with perchloric acid, separated by isotachophoresis, and quantified by uv absorption analysis at 254 nm. ATP (936 pmol/μg DNA) was, as expected, the dominating nucleotide pool. The energy charge was estimated to 0.9. The UDP-N-acetylhexosamine pool was also a very prominent compound (596 pmol/μg DNA). After incubation of fibroblasts with [³H]glucosamine, more than 95% of the acid-soluble radioactivity was found in the UDP-N-acetylhexosamine pool. Incubation with [³⁵S]sulfate resulted in the incorporation of [³⁵S]sulfate into 3′-phosphoadenosine-5′-phosphosulfate (PAPS). The latter could, however, only be measured as radioactivity, as the amount was too small to be quantified as total mass. Pulse-labeling of fibroblasts with [³⁵S]sulfate and [³H]glucosamine from 5 min to 16 h showed that [³⁵S]PAPS was equilibrated in less than 10 min, while [³H]glucosamine required a longer time, 2–4 h, to attain a steady state with UDP-N-acetylhexosamine. [¹⁴C]Glucose required approximately the same time as [³H]glucosamine to reach steady state with UDP-acetylhexosamine, which suggests that the reason for the long equilibration time is the slow turnover of this pool.     

Photoaffinity labeling of the (Ca+Mg)ATPase of skeletal and cardiac sarcoplasmic reticulum with [γ-32P]-8-azido-ATP

8-azido-ATP, when used in the 0.2–5 μM concentration range, fulfills the criteria for a specific photoaffinity label for the (Ca+Mg)ATPase of sarcoplasmic reticulum. It is a substrate for the enzyme. It is a mixed inhibitor of ATPase activity. When photolyzed at 0° it is an inhibitor of ATPase activity. The photoinduced binding of 8-azido-ATP to the (Ca+Mg)ATPase is promoted by Ca²⁺. The dependence of the labeling of the (Ca+Mg)ATPase on 8-azido-ATP, Ca²⁺ and Mg²⁺ concentrations strongly suggests that 2 classes of sites are labeled. When 10–60 μM 8-azido-ATP was used to label sarcoplasmic reticulum, proteins in addition to the (Ca+Mg)ATPase were labeled.     

Properties of [3H] prostaglandin F2α binding to dispersed bovine luteal cells

Intact viable bovine luteal cell suspensions prepared by collagenase digestion of luteal tissue were used in studying the selected properties of [³H] prostaglandin (PG) F_2α binding and compared with those observed in plasma membranes. [³H]PGF_2α specific binding to luteal cells was a rapid (K₁ = 8.4 × 10⁴M^?12αS^?1), reversible (K_?1 = 1.8 × 10^?4S^?1) and saturable process at 24°. There was a single class of receptors with an apparent dissociation constant of 10.6 nM and 1.8 × 10⁵ receptors per cell. The presence of increasing amounts of unlabeled PGs inhibited [³H]PGF_2α binding in a dose-dependent manner. The potency order for this inhibition was: (15S) 15-methyl-PGF_2α methyl ester > ICI-80,996 > PGF_2α > ICI-81,008 > PGF_1α > PGE₂, (15S) 15-methyl-PGE₂ methyl ester > PGF_2α metabolites > other PGs, PGF_1α metabolites and PGE metabolites. Other than the homegeneous nature of binding and a greater association rate in cells, the rest of the [³H]PGF_2α binding properties in cells were in good agreement with those observed in plasma membranes.