Properties of the 3′-phosphoadenosine-5′-phosphosulfate (PAPS) synthesizing systems of brain and liver

Chromatography of brain and liver 100,000g supernatants over HPLC molecular sieve columns revealed striking differences in the molecular weight distribution of ATP-sulfurylase and APS-kinase of the two tissues, pointing to different enzymic species for both enzymes in brain and liver. This was further substantiated by kinetic characterization of the two enzymes of both tissues. APS-kinase of liver is allosterically activated by ATP, while the brain enzyme is not. ATP-sulfurylase of brain is activated at high, but still physiological concentrations of ATP. Brain ATP-sulfurylase is inhibited by phenylalanine.     

Chemoenzymatic synthesis of 3′-phosphoadenosine-5′-phosphosulfate coupling with an ATP regeneration system

Applied Microbiology and Biotechnology - 3′-Phosphoadenosine-5′-phosphosulfate (PAPS) is the obligate cosubstrate and source of the sulfonate group in the chemoenzymatic synthesis of...     

A factor-dependent sulfotransferase specific for 3′-phosphoadenosine-5′-phosphosulfate (PAPS) in the Cyanobacterium Synechococcus 6301

A sulfotransferase isolated from the Cyanobacterium Synechococcus 6301 was found to be specific for 3-phosphoadenosine-5-phosphosulfate (PAPS). The molecular weight of this transferase has been estimated on a Sephadex-G-100 column to be about 58,000. The K _m for PAPS was determined to be 20 M. The pH optimum was 8.0. The thiol dithioerythritol was needed for activity; other thiols such as glutathione, cysteine, or mercaptoethanol did not catalyze this reaction. The transferase, however, could not react directly with the thiol. A heat-stable factor was needed in this reaction. This factor was purified by conventional techniques and its molecular weight was determined on a Sephadex-G-50 column to be about 11,500. The factor showed normal Michaelis-Menten behavior toward the PAPS-sulfotransferase. It has been identified as thioredoxin. The tranferase was inhibited by 3-5-ADP and 2–5-ADP; all other adenine-containing nucleotides such as 2-AMP, 3-AMP, 5-AMP, ADP, and c-AMP did not influence this reaction.Abbreviation PAPS 3-phosphoadenosine-5-phosphosulfate     

Effect of 17β estradiol on [3H] progesterone binding in rat brain

A progesterone-binding component is reported in the cerebral hemispheres of immature female rat. [³H] progesterone binding in the brain cytosol is increased following two weeks of estradiol administration. The [³H] progesterone binding by this component can be reduced by pretreatment with unlabeled steroid. In addition, the binder from both control and estradiol-treated groups shows inter-action with ATP immobilized on columns of ATP-Sepharose.     

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.     

Quantitative autoradiography of α1 adrenoceptors with [3H]tamsulosin in human hypertrophied prostate using computerized image analysis

Summary Fourteen specimens of human hypertrophied prostate were evaluated for the distribution of ₁ adrenoceptors using autoradiography with a computerized image analysis system. The hypertrophied prostatic specimens, obtained at open prostatectomy, were dissected vertically to the urethra, and sectioned at 10 m. They were immersed in 1 nM of specific ₁ ligand, [³H]tamsulosin chloride ([³H]tamsulosin) and exposed to autoradiographic film. The images were analysed by a computerized image analysis system. The total binding of [³H]tamsulosin in the whole section (n = 14) was 0.82 ± 0.21 (mean ± se) nCi mg^–1. The autographic data were correlated with data obtained in a membrane-binding assay. The prostatic tissue studied was divided into urethral, glandular and stromal zones, the latter two zones being further divided into the inner and outer areas. The total binding of [³H]tamsulosin in the urethral zone (n = 7) was 0.65 ± 0.32 nCi mg^–1. The glandular zone contained significantly more abundant ₁ adrenoceptors than the stromal zone and their densities (glandular vs stromal) were 1.15 ± 0.19 nCi mg^–1 (n = 14) vs 0.72 ± 0.15 nCi mg^–1 (n = 14), respectively (p < 0.05). The data from the whole section were not affected by prostatic weight. This method described enabled the distribution of the receptors in different sites to be evaluated both morphologically and quantitatively.     

Does high affinity [3H] imipramine binding label serotonin reuptake sites in brain and platelet?

Recent studies have demonstrated the presence of high affinity binding sites for [³H] imipramine in membrane preparations derived from rat brain, human platelet, and human brain. Although initial reports concluded that there was no relationship between these binding sites and the reuptake sites for biogenic amines, subsequent studies in our laboratory suggested a close relationship between the high affinity imipramine binding site and the serotonin uptake or transport site (cf. ref. 9). To further establish whether these binding sites are associated with either platelet or neuronal uptake of serotonin, the relative potencies of a series of tricyclic antidepressants in inhibiting [³H] imipramine binding and serotonin uptake were determined under identical assay conditions. A close correlation between inhibition of serotonin uptake and [³H] imipramine binding was observed (r = 0.99, p < 0.001). In addition, electrolytic lesions of the midbrain raphe produced a decrease in [³H] imipramine binding in hypothalamic synaptosomes that paralleled the decrease in [³H] serotonin uptake. Finally incubation of synaptosomal membranes with 2,8-dinitroimipramine, an irreversible inhibitor of [³H] imipramine binding, produced a dose-dependent decrease in serotonin uptake, without altering the uptake of nonrepinephrine or dopamine. Taken together our results strongly suggest that high affinity binding of [³]] imipramine selectively labels serotonin uptake sites in brain and platelet.     

Properties of [3H] ß-carboline-3-carboxylate ethyl ester binding to the benzodiazepine receptor

β-Carbolines are inhibitors of [³H] diazepam binding with the most potent inhibitor being β-carboline-3-carboxylate ethyl ester (β-CCE). In this report the binding of [³H] β-CCE to extensively washed rat forebrain membranes is characterized. [³H] ß-CCE binds with high affinity (K_D = 1.4 nM) to an apparently homogenous population of benzodiazepine receptor. The rank order of potency for inhibition of [³H] ß-CCE binding by different benzodiazepines is clonazepam > diazepam > chlordiazepoxide, which is similar to that observed for inhibition of [³H] diazepam binding. In marked contrast to [³H] diazepam, the binding of [³H] ß-CCE is not modulated by GABA since concentrations of GABA as high as 10^?3 M had no effect. [³H] ß-CCE is also less potent than [³H] diazepam in its interaction with the peripheral type kidney benzodiazepine receptor indicating that this ligand has a higher degree of specificity for the central brain type benzodiazepine receptor.     

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.     

Effects of transforming growth factor β and interleukin-1β on [3H]thymidine incorporation by human articular chondrocytes in vitro

This is a study of the regulation of human articular chondrocyte proliferation by transforming growth factor β (TGFβ) and interleukin-1β (IL-1β) in vitro. Human articular chondrocytes were cultured at different cell densities on plastic and on a collagen substratum, in the presence and absence of serum. The effects TGFβ amd IL-1β on proliferation of chondrocytes, as determined by [³H]thymidine incorporation, under these conditions of culture were examined. TGFβ was found to have both stimulatory and inhibitory effects on chondrocytes in vitro. Interactions between TGFβ and growth factors present in serum influence the modulation of chondrocyte proliferation by TGFβ. IL-1β caused a significant reduction of the TGFβ-stimulated increase in chondrocyte proliferation. The complex inter-relationships between TGFβ and IL-1β on chondrocytes have implications for cartilage repair.     

[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.     

Synthesis and in vitro and in vivo anticancer activity of novel 3-methyl-5H-isoxazolo[5′,4′:5,6]pyrido[2,3-b]indoles

A series of novel isoxazolo[5′,4′:5,6]pyrido[2,3-b]indoles 7a–h were synthesized and tested for their in vitro and in vivo anticancer activities. The analogs 7d and 7g have shown potential anticancer activity as compared with the reference compound Cisplatin.     

Chemical synthesis of [1β-3H]1α,25-dihydroxyvitamin D3 and [1α-3H]1β,25-dihydroxyvitamin D3: Biological activity of 1β,25-dihydroxyvitamin D3

The simple three-step preparation of [1β-³H]1α,25-dihydroxyvitamin D₃ and [1α-³H]1β,25-dihydroxyvitamin D₃ from 1α,25-dihydroxyvitamin D₃ is described. In the rat, 1β,25-dihydroxyvitamin D₃, when compared with its α-epimer, did not stimulate intestinal calcium transport or bone calcium mobilization at doses 1000-fold higher than the doses of the natural hormone, 1α,25-dihydroxyvitamin D₃.     

Studies on degradation of adenosine-5′-phosphosulphate (APS) and adenosine-3′-phosphate-5′-phosphosulphate (PAPS) in extracts of Anabaena cylindrica

[³⁵S]Adenosine-5′-phosphosulphate ([³⁵S]APS) and [³⁵S]adenosine-3′-phosphate-5′-phosphasulphate ([³⁵S]PAPS) were rapidly degraded by extracts of Anabaena cylindrica. The loss of radioactivity from these sulphur nucleotides resulted in a corresponding increase of free ³⁵SO₄^ in the incubation mixture. The soluble fraction of the broken cells (S₇₅) hydrolysed both PAPS and APS, whereas the pellet fractions (P₂₀ and P₇₅) hydrolysed PAPS only. The degradation of [³⁵S]PAPS was almost completely suppressed by various 5′-adenine nucleotides, 3′-AMP, nucleotide triphosphates or pyrophosphate, while glucose-6-phosphate, phosphate ions and sodium sulphite were less effective. The hydrolysis of [³⁵S]APS was prevented by sodium fluoride and 5′-AMP, but 3′-AMP was ineffective.