Characterization of 3''-Phosphoadenosine 5''-Phospho[35S]Sulfate Transport Carrier from Rat Brain Microsomes

3'-Phosphoadenosine 5'-phospho[35S]sulfate [( 35S]PAPS) specific binding properties of rat brain tissue were studied. [35S]PAPS specific binding was optimal at pH 5.8 in either Tris-maleate or potassium phosphate buffers. Association was maximal at low temperature, reaching equilibrium in 20 min. Dissociation was rapid, with a dissociation time of 80 s. Scatchard analysis of [35S]PAPS specific binding was consistent with a single site having a KD of 0.46 +/- 0.06 microM and a Bmax of 20.8 +/- 2.0 pmol/mg of protein. Low concentrations of Triton X-100 (0.025%) were effective in increasing the number of binding sites to a Bmax of 44.5 +/- 4.6 pmol/mg of protein without affecting the affinity. [35S]PAPS specific binding was enriched in crude synaptic membranes (P2) and microsomes (P3). Regional distribution of [35S]PAPS specific binding was quite homogeneous in all brain structures studied. The pharmacological profile of [35S]PAPS specific binding in rat brain microsomes was consistent with a membrane protein having a high selectivity for the 3'-O-phosphoryl group substitution on the ribose moiety. Thus, 3'-phosphoadenosine 5'-phosphate was more potent than 2'-phosphoadenosine 5'-phosphate in competing for [35S]PAPS specific binding. Adenosine 5'-phosphosulfate was a good inhibitor of [35S]PAPS specific binding. ATP and ADP were also good displacers. Dipyridamole, a highly selective marker for adenosine uptake sites, was ineffective. 4,4-Diisothiocyanostilbene-2,2-disulfonic acid, the chloride transporter inhibitor, showed an IC50 of 36 +/- 5.1 microM for inhibition of [35S]PAPS specific binding. 2,6-Dichloro-4-nitrophenol had a low selectivity in competing for the [35S]PAPS binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

35S]-labeling of the Salmonella typhimurium glutathione pool to assess glutathione-mediated DNA binding by 1,2-dibromoethane

Biotransformation of drugs and environmental chemicals to reactive intermediates is often studied with the use of radiolabeled compounds that are synthesized by expensive and technically difficult procedures. In general, glutathione (GSH) conjugation serves as a detoxification mechanism, and conjugation of reactive intermediates with GSH is often a surrogate marker of reactive species formation. However, several halogenated alkanes can be bioactivated by GSH to yield highly reactive GSH conjugates, some of which are DNA-reactive (e.g. conjugates of 1,2-dibromoethane). The purpose of this study was to metabolically radiolabel the in vivo GSH pool of Salmonella typhimurium with a [35S]-label and to examine the GSH-mediated bioactivation of a model haloalkane, 1,2-dibromoethane, by measuring the binding of [35S]-label to DNA. The strain of Salmonella used in this study had been transformed previously with the gene that codes for rat glutathione transferase theta 1-1 (GSTT1-1), an enzyme that can catalyze formation of genotoxic GSH conjugates. Bacteria were grown to mid-log phase and then incubated with [35S]-L-cysteine in minimal medium (thio-free) until stationary phase of growth was reached. At this stage, the specific activity of Salmonella GSH was estimated to be 7.1 mCi/mmol by derivatization and subsequent HPLC analysis, and GSTT1-1 enzyme activity was still demonstrable in Salmonella cytosol following growth in a minimal medium. The [35S]-labeled bacteria were then exposed to 1,2-dibromoethane (1 mM), and the Salmonella DNA was subsequently purified to quantify [35S]-binding to DNA. The amount of [35S]-label that was covalently bound to DNA in the GSTT1-1-expressing Salmonella strain (33.2 nmol/mg DNA) was sevenfold greater than that of the control strain that does not express GSTT1-1. Neutral thermal hydrolysis of the DNA yielded a single [35S]-labeled adduct with a similar t(R) as S-[2-(N(7)-guanyl)ethyl]GSH, following HPLC analysis of the hydrolysate. This adduct accounted for 95% of the total [35S]-label bound to DNA. Thus, this [35S]-radiolabeling protocol may prove useful for studying the DNA reactivity of GSH conjugates of other halogenated alkanes in a cellular context that maintains GSH at normal physiological levels. This is also, to our knowledge, the first demonstration of de novo incorporation of [35S]-L-cysteine into the bacterial GSH pool.     

ABCG2 transports sulfated conjugates of steroids and xenobiotics

The mechanism for the cellular extrusion of sulfated conjugates is still unknown. In the present study, we investigated whether human wild type ABCG2 transports estrone 3-sulfate (E1S) using membrane vesicles from cDNA-transfected mouse lymphoma cell line (P388 cells). The uptake of [3H]E1S into ABCG2-expressing membrane vesicles was stimulated by ATP, and the Km value for [3H]E1S was determined to be 16.6 microm. The ABCG2-mediated transport of [3H]E1S was potently inhibited by SN-38 and many sulfate conjugates but not by glucuronide and glutathione conjugates or other anionic compounds. Other sulfate conjugates such as [3H]dehydroepiandrosterone sulfate (DHEAS) and [35S]4-methylumbelliferone sulfate (Km = 12.9 microm) and [35S]6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl)benzothiazole (E3040) sulfate (Km = 26.9 microm) were also transported by ABCG2. Although [3H]methotrexate, [3H]17beta-estradiol-17beta-D-glucuronide, [3H]2,4-dinitrophenyl-S-glutathione, and [14C]4-methylumbelliferone glucuronide were transported by ABCG2, this took place to a much lesser extent compared with [3H]E1S. It was suggested that ABCG2 preferentially transports sulfate conjugates and that E1S and DHEAS are the potential physiological substrates for this transporter.     

Direct photoaffinity labeling of proteins with adenosine 3'-[32P]phosphate 5'-phosphosulfate. Atractyloside inhibits labeling of a Mr = 34,000 protein in an adrenal medullary Golgi fraction

Direct photoaffinity labeling with radioactively labeled adenosine 3'-phosphate 5'-phosphosulfate (PAPS) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography was used to identify PAPS binding proteins in a Golgi membrane preparation of bovine adrenal medulla. [3'-32P]PAPS was synthesized from adenosine 5'-phosphosulfate (APS) and [gamma-32P]ATP using APS kinase prepared from yeast and was purified by reverse-phase ion pair high performance liquid chromatography. Upon irradiation with UV light, [3'-32P]PAPS, as well as [35S]PAPS under conditions which minimized sulfotransferase-catalyzed incorporation of 35SO4 from [35S]PAPS into proteins, bound selectively to a 34-kDa protein of the Golgi membrane preparation. PAPS binding to the 34-kDa protein was strongly inhibited by the presence of 50 microM atractyloside. The 34-kDa PAPS binding protein therefore appears to be similar to the mitochondrial ATP/ADP translocator with regard to both molecular weight and inhibition by atractyloside of adenine nucleotide binding. Photoaffinity labeling will be useful in the purification and functional identification of the 34-kDa protein.     

Kinetic and pharmacological analysis of L-[35S]cystine transport into rat brain synaptosomes

The synaptosomal transport of L-[35S]cystine occurs by three mechanisms that are distinguishable on the basis of their ionic dependence, kinetics of transport and the specificity of inhibitors. They are (a) low affinity sodium-dependent transport (Km 463 +/- 86 microM, Vmax 185 +/- 20 nmol mg protein-1 min-1), (b) high affinity sodium-independent transport (Km 6.90 +/- 2.1 microM, Vmax 0.485 +/- 0.060 nmol mg protein(-1) min(-1)) and (c) low affinity sodium-independent transport (Km 327 +/- 29 microM, Vmax 4.18 +/- 0.25 nmol mg protein(-1) min(-1)). The sodium-dependent transport of L-cystine was mediated by the X(AG)- family of glutamate transporters, and accounted for almost 90% of the total quantity of L-[35S]cystine accumulated into synaptosomes. L-glutamate (Ki 11.2 +/- 1.3 microM) was a non-competitive inhibitor of this transporter, and at 100 microM L-glutamate, the Vmax for L-[35S]cystine transport was reduced to 10% of control. L-cystine did not inhibit the high-affinity sodium-dependent transport of D-[3H]aspartate into synaptosomes. L-histidine and glutathione were the most potent inhibitors of the low affinity sodium-independent transport of L-[35S]cystine. L-homocysteate, L-cysteine sulphinate and L-homocysteine sulphinate were also effective inhibitors. 1 mM L-glutamate reduced the sodium-independent transport of L-cystine to 63% of control. These results suggest that the vast majority of the L-cystine transported into synaptosomes occurs by the high-affinity glutamate transporters, but that L-cystine may bind to a site that is distinct from that to which L-glutamate binds. The uptake of L-cystine by this mechanism is sensitive to inhibition by increased extracellular concentrations of L-glutamate. The importance of these results for understanding the mechanism of glutamate-mediated neurotoxicity is discussed.     

Nad-299 antagonises 5-HT-stimulated and spiperone-inhibited [35S]GTPgammaS binding in cloned 5-HT1A receptors

In Chinese Hamster Ovary (CHO) cells expressing cloned human 5-hydroxytryptamine1A A (5-HT1A) receptors, (R)-3-N,N-dicyclobutylamino-8-fluoro-[6-3H]-3,4-dihydro-2H-1-benzopyan-5-carboxamide ([3H]NAD-299) exhibited high affinity (Kd = 0.16 nM) and labeled 34% more receptors than 8-hydroxy-2-([2,3-3H]di-n-propylamino)tetralin ([3H]8-OH-DPAT). NAD-299 behaved as a silent antagonist in [35S]GTPgammaS binding similar to N-tert-butyl-3-(4-(2-methoxyphenyl)-piperazin-1-yl)-2-phenylpropanamide (WAY-100635) and (S)-5-fluoro-8-hydroxy-2-(di-n-propylamino)tetralin ((S)UH-301). 5-HT and 5-carboxamidotryptamine (5-CT) stimulated [35S]GTPgammaS binding 2.5-fold while spiperone and methiothepin inhibited [35S]GTPgammaS binding 1.4-fold. Furthermore, NAD-299 antagonised both the 5-HT stimulated and the spiperone inhibited [35S]GTPgammaS binding to basal levels. The KiL/KiH ratios for spiperone (0.66), methiothepin (0.39), WAY-100635 (0.32), (S)UH-301 (0.94), NAD-299 (1.29), NAN-190 (1.23), (S)pindolol (5.85), ipsapirone (13.1), buspirone (24.6), (+/-)8-OH-DPAT (47.3), flesinoxan (55.8), 5-HT (200) and 5-CT (389) correlated highly significantly with the intrinsic activity obtained with [35S] GTPgammaS (r = 0.97).     

Bidirectional mechanism of plasma membrane transport of reduced glutathione in intact rat hepatocytes and membrane vesicles.

We determined the trans effects of extracellular reduced glutathione (GSH) on the rate of efflux of endogenous labeled GSH from freshly isolated rat hepatocytes. The presence of GSH (10 mM) in the medium significantly stimulated the fractional rate of efflux of [35S]GSH from 5.2 to 12.6%/15 min (p < 0.01). This effect was concentration-dependent, had sigmoid type of kinetics (D50 of 0.32 mM), and was reversible upon removal of external GSH. trans-Stimulation (counter-transport) was also observed with 5 mM oxidized glutathione (GSSG) and ophthalmic acid (fractional [35S] GSH efflux: 13.4% +/- 4.1 and 8.8% +/- 2.3 in 15 min, respectively, compared with control: 4.7 +/- 2.5/15 min). Bromosulphthalein-glutathione (BSP-GSH, 5 mM) in Krebs buffer inhibited the fractional [35S]GSH efflux (1.1%/15 min), whereas in Cl(-)-free buffer, GSH efflux was stimulated (14.2%/15 min) compared with control. trans-Stimulation was independent of chloride. BSP-GSH cis-inhibited and trans-stimulated the initial rate of GSH transport in basolateral-enriched membrane vesicles (bLPM) but not in canalicular-enriched membrane vesicles (cLPM). gamma-Glutamyl compounds also cis-inhibited and trans-stimulated GSH transport in bLPM vesicles. GSH-depleted hepatocytes incubated with 10 mM [35S]GSH accumulated more GSH than repleted cells, but the initial rate of uptake of radioactivity was faster in repleted cells. In contrast, repleted hepatocytes incubated with tracer or 50 microM [35S]GSH did not take up GSH. Thus, the sinusoidal membrane GSH transporter exhibits low affinity kinetics with sigmoid features for both GSH uptake and trans-stimulation of efflux, explaining the lack of uptake of GSH at low physiologic extracellular concentrations. Therefore, our findings support and explain the widely held view that GSH transport is unidirectional under physiologic conditions. However, the efflux of GSH may also occur in exchange for the uptake of organic anions and gamma-glutamyl compounds.     

In Vitro Autoradiographic Visualization of Guanosine-5'-O-(3-[35S]Thio)triphosphate Binding Stimulated by Sphingosine 1-Phosphate and Lysophosphatidic Acid

Sphingosine 1-phosphate or lysophosphatidic acid activation of guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding to G proteins was studied by in vitro autoradiography in rat and guinea pig brain. The highest stimulation of [35S]GTPgammaS binding by sphingosine 1-phosphate was observed in the molecular layer of the cerebellum. Marked stimulation was observed in most forebrain areas, including neocortex and striatum. With the exception of the substantia gelatinosa and nucleus of the solitary tract, sphingosine 1-phosphate-enhanced binding was weaker in the brainstem and spinal cord. Lysophosphatidic acid-enhanced labeling was only observed in white matter areas. The G protein inhibitor 5'-p-fluorosulfonylbenzoyl guanosine completely inhibited lysophosphatidic acid-enhanced [35S]GTPgammaS binding but only partially sphingosine 1-phosphate-enhanced binding. N-Ethylmaleimide abolished binding stimulated by both agonists. Sphingosine 1-phosphate enhanced labeling by another GTP analogue (beta,gamma-imido[8-3H]guanosine-5'-triphosphate) similarly to that of [35S]GTPgammaS. Lysophosphatidic acid stimulated [35S]GTPgammaS binding in the olfactory bulb, glia limitans, and cortical subventricular zone of 1-day-old rats, whereas enhanced labeling was not observed in the latter area of 5-day-old rats. Sphingosine 1-phosphate stimulated binding in the cortical and striatal subventricular zones and olfactory bulb in 1- and 5-day-old rats. In the absence of radioligand for sphingosine 1-phosphate and lysophosphatidic acid receptors, [35S]GTPgammaS autoradiography provides a unique opportunity to study the spatial distribution, ontogeny, and coupling properties of these receptors.     

Photochemical labeling of membrane hydrophobic core of human erythrocytes using a new photoactivable reagent 2-[3H]diazofluorene

Photoactivable reagents have been useful for studying the structural aspects of membrane hydrophobic core. We have reported earlier (Anjaneyulu, P.S.R., and Lala, A. K. (1982) FEBS Lett. 146, 165-167) the use of diazofluorene as a probe for fluorescent photochemical labeling of hydrophobic core in artificial membranes. To quantitate and enhance the monitoring ability of this probe, we have synthesized 2-[3H]diazofluorene of high specific activity. This reagent rapidly partitions into phosphatidylcholine vesicles and selectively labels the fatty acyl chains of phosphatidylcholine. The insertion yield (13%) is not affected by the presence of scavengers like reduced glutathione. 2-[3H]Diazofluorene also readily partitions into erythrocyte membranes and on photolysis labels the membrane. The overall insertion was 48% with 9.7% in protein fraction and the rest in lipids. The distribution of radioactivity in labeled protein fraction was restricted to integral membrane proteins with Band 3 being the major protein labeled. There is little or no labeling associated with extrinsic proteins like spectrin. Further analysis of labeled Band 3 by treatment with chymotrypsin indicated that the labeling was restricted to the membrane spanning CH-17 and CH-35 fragments. No labeling of the cytoplasmic fragment of Band 3 could be observed. 2-[3H]Diazofluorene should prove useful for studying integral membrane proteins and their membrane-spanning regions.     

Characterization of a sulfotransferase responsible for the 4-O-sulfation of terminal beta-N-acetyl-D-galactosamine on asparagine-linked oligosaccharides of glycoprotein hormones

The Asn-linked oligosaccharides on the glycoprotein hormones lutropin (LH) and thyrotropin terminate with the sequence SO4-4GalNAc beta 1-4GlcNAc beta 1-2 Man alpha-. Using a chemically synthesized trisaccharide GalNAc beta 1-4GlcNAc beta 1-2Man alpha 1-O(CH2)8COOCH3 (GGnM-MCO), we have developed a sensitive assay for the sulfotransferase responsible for the 4-O-sulfation of the terminal beta-D-GalNAc. GGnM-MCO is incubated with a bovine pituitary membrane extract and [35S]3'-phosphoadenosine 5'-phosphosulfate ([35S]PAPS). The sulfated product [35S]SGGnM-MCO is separated from [35S]PAPS, PAPS degradation products and endogenous sulfated products by a two-step procedure utilizing an Ecteola cellulose column and a Sep-Pak (C18) cartridge. Characterization of the [35S]SGGnM-MCO produced in the assay indicates that sulfate is incorporated exclusively on the 4-position of GalNAc. Linear incorporation of sulfate into GGnM-MCO can be maintained for greater than 10 h. GGnM-4-sulfotransferase has a pH optimum of 7.2, requires the presence of a reducing agent, and is stimulated by, but does not require, divalent cations. Initial velocity studies indicate an apparent Km (Henri-Michaelis-Menten equilibrium constant) for PAPS of 4 microM and for GGnM-MCO of 9 microM. Incorporation of sulfate into the trisaccharide is stimulated 3-fold by the presence of basic proteins including deglycosylated LH. The stimulation by deglycosylated LH suggests that the protein component of glycoproteins that bear oligosaccharides terminating with GalNAc-GlcNAc-Man- may modulate GGnM-4-sulfotransferase.     

delta-Opioid receptors exhibit high efficiency when activating trimeric G proteins in membrane domains

Low-density membrane fragments (domains) were separated from the bulk of plasma membranes of human embryonic kidney (HEK)293 cells expressing a delta-opioid (DOP) receptor-Gi1alpha fusion protein by drastic homogenization and flotation on equilibrium sucrose density gradients. The functional activity of trimeric G proteins and capacity of the DOP receptor to stimulate both the fusion protein-linked Gi1alpha and endogenous pertussis-toxin sensitive G proteins was measured as d-Ala2, d-Leu5-enkephalin stimulated high-affinity GTPase or guanosine-5'-[gamma-35S]triphosphate ([35S]GTPgammaS) binding. The maximum d-Ala2-d-Leu5 enkephalin (DADLE)-stimulated GTPase was two times higher in low-density membrane fragments than in bulk of plasma membranes; 58 and 27 pmol/mg/min, respectively. The same difference was obtained for [35S]GTPgammaS binding. Contrarily, the low-density domains contained no more than half the DOP receptor binding sites (Bmax = 6.6 pmol/mg versus 13.6 pmol/mg). Thus, when corrected for expression levels of the receptor, low-density domains exhibited four times higher agonist-stimulated GTPase and [35S]GTPgammaS binding than the bulk plasma membranes. The regulator of G protein signaling RGS1, enhanced further the G protein functional activity but did not remove the difference between domain-bound and plasma membrane pools of G protein. The potency of the agonist in functional studies and the affinity of specific [3H]DADLE binding to the receptor were, however, the same in both types of membranes - EC50 = 4.5 +/- 0.1 x 10(-8) and 3.2 +/- 1.4 x 10(-8) m for GTPase; Kd = 1.2 +/- 0.1 and 1.3 +/- 0.1 nm for [3H]DADLE radioligand binding assay. Similar results were obtained when sodium bicarbonate was used for alkaline isolation of membrane domains. By contrast, detergent-insensitive membrane domains isolated following treatment of cells with Triton X100 exhibited no DADLE-stimulated GTPase or GTPgammaS binding. Functional coupling between the DOP receptor and cognate G proteins was also blocked by high-energy ultrasound and repeated freezing-thawing. Our data indicate, for the first time, that membrane domains isolated using 'detergent-free' procedures exhibit higher efficiency of coupling between a G protein-coupled receptor and its corresponding G protein(s) than bulk plasma membranes. Detergent-extraction diminishes these interactions, even when the receptor and G proteins are physically tethered together.     

Ligand-induced formation of the leukotriene B4 receptor-G protein complex of human polymorphonuclear leukocytes.

The components of the polymorphonuclear leukocyte (PMNL) receptor for leukotriene B4 (LTB4) were examined by Sephacryl S-300 exclusion chromatography of PMNL membrane proteins, which were solubilized before and after the binding of [3H] LTB4. When the PMNL membranes were solubilized in 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and filtered on Sephacryl S-300 prior to addition of [3H] LTB4, the binding activity was associated with a 65 kD protein. In contrast, the radioactivity of [3H] LTB4 bound to PMNL membranes prior to solubilization was recovered predominantly with a 140 kD protein. When PMNL membranes had been pretreated with pertussis toxin, but not cholera toxin, before the addition of LTB4 and subsequent solubilization, radioactivity was recovered predominantly with the 65 kD protein. The addition of guanylylimidodiphosphate (GMP-PNP), a nonhydrolyzable derivative of guanosine triphosphate (GTP), to PMNL membrane receptors bearing [3H] LTB4 either prior to or after CHAPS solubilization reduced the yield of the 140 kD presumed LTB4 receptor protein-G protein complex. That the maximum specific binding of [35S] guanosine-5'-0-3-thiotriphosphate (GTP-gammaS) to LTB4-binding proteins in the Sephacryl S-300 effluent corresponded to the 140 kD protein supported the presence of a G protein in the LTB4 receptor complex.     

Evidence that guanosine 5''-[gamma-thio]triphosphate stimulates plasma membrane Ca2+ inflow when introduced into hepatocytes.

1. Slowly hydrolysable analogues of GTP were introduced into hepatocytes by incubating the cells in the absence of Mg2+ and in the presence of ATP4-. Experiments using guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S])indicated that about 50% of the GTP[S] loaded into the cells was subsequently hydrolysed. 2. In cells loaded with GTP[S] and incubated in the absence of added extracellular Ca2+ (Ca2+o), the rate of activation of glycogen phosphorylase observed after addition of 1.3 mM-Ca2+o was 250% greater than the rate observed in unloaded cells. Smaller effects (130%) were observed in cells loaded with either guanyl-5'-yl imidodiphosphate or guanosine 5-[beta-thio]diphosphate (GDP[S]). Cells loaded with adenosine 5'-[gamma-thio]triphosphate showed no increase in glycogen phosphorylase activity on addition of Ca2+o. 3. The effect of a submaximal concentration of GTP[S] on the Ca2+-induced activation of glycogen phosphorylase was additive with that of a half-maximally effective concentration of vasopressin. GTP[S] did not increase the effect of a maximally effective concentration of the hormone. 4. Cells loaded with GTP[S] exhibited an increased initial rate of 45Ca2+ exchange measured at 1.3 mM-Ca2+o. 5. GTP[S] did not affect the amount of 45Ca2+ exchanged by cells incubated at 0.1 mM-Ca2+o or the ability of vasopressin to release 45Ca2+ from these cells. 6. It is concluded that the introduction of slowly hydrolysable analogues of GTP to the liver cell cytoplasmic space stimulates the inflow of Ca2+ across the plasma membrane through a channel similar to that activated by vasopressin.     

Preparation and quantification of 3'-phosphoadenosine 5'-phospho[35S]sulfate with high specific activity

The synthesis and quantitation of the sulfate donor 3'-phosphoadenosine 5'-phospho[35S]sulfate (PAP35S), prepared from inorganic [35S]sulfate and ATP, were studied. An enzymatic transfer method based upon the quantitative transfer of [35S]sulfate from PAP35S to 2-naphthol and 4-methylumbelliferone by the action of phenolsulfotransferase activity from rat brain cytosol was also developed. The 2-naphthyl[35S]sulfate or 35S-methylumbelliferone sulfate formed was isolated by polystyrene bead chromatography. This method allows the detection of between 0.1 pmol and 1 nmol/ml of PAP35S. PAP35S of high specific activity (75 Ci/mmol) was prepared by incubating ATP and carrier-free Na2 35SO4 with a 100,000g supernatant fraction from rat spleen. The product was purified by ion-exchange chromatography. The specific activity and purity of PAP35S were estimated by examining the ratios of Km values for PAP35S of the tyrosyl protein sulfotransferase present in microsomes from rat cerebral cortex. The advantage and applications of these methods for the detection of femtomole amounts, and the synthesis of large scale quantities of PAP35S with high specific activity are discussed.     

[35S]Thiosulphate oxidation by rat liver mitochondria in the presence of glutathione

1. Rat liver mitochondria incubated in oxygen with glutathione and [(35)S]-thiosulphate produced labelled sulphate. 2. Inner-labelled thiosulphate (S.(35)SO(3))(2-) was converted into [(35)S]sulphate more rapidly than outer-labelled thiosulphate ((35)S.SO(3))(2-). 3. Thiosulphate labelled in both sulphur atoms was formed during ((35)S.SO(3))(2-) oxidation; the outer sulphur atom before oxidation to sulphate was incorporated into the inner position. 4. A thiosulphate cycle in the metabolic pathway of sulphate formation in animal tissues is discussed.     

Interleukin-1 signal transduction. Increased GTP binding and hydrolysis in membranes of a murine thymoma line (EL4)

The post-receptor events which follow the binding of interleukin 1 (IL1) to cells are unclear. The present studies provide evidence for the activation of a guanine nucleotide binding protein (G protein) by IL1 in the membranes of an IL1 receptor-rich strain (NOB-1) of the EL4 murine thymoma line. IL1 alpha and beta increased the binding of the GTP analogue [35S]guanosine 5'-[gamma-thiol]trisphosphate (GTP gamma S) to membranes prepared from these cells. By 1 min after addition of IL1 there was a 2-fold enhancement in binding which was dose dependent in the range 0.1-100 ng/ml. A qualitatively similar result was obtained with IL1 beta although it was 10 times less potent. Specific neutralizing antisera to IL1 alpha and IL1 beta abolished the response. Experiments in which the concentration of [35S]GTP gamma S was varied revealed that IL1 increased the affinity of the binding sites for [35S]GTP gamma S and not their number. IL1 alpha was shown to stimulate GTPase activity in the membranes, the time and concentration dependence of this was similar to that observed for increased [35S]GTP gamma S binding. Half-maximal enhancement of [35S]GTP gamma S binding by IL1 alpha, measured after 4 min, occurred at 5% IL1 receptor occupancy. Maximal stimulation was achieved when 30% of receptors were occupied. Experiments with pertussis and cholera toxins revealed that pretreating membranes with pertussis toxin (100 ng/ml) inhibited by 50% the IL1-induced [35S]GTP gamma S binding and [gamma-32P]GTP hydrolysis. Cholera toxin (100 ng/ml) was without effect. However, both pertussis and cholera toxins at concentrations of 100 ng/ml inhibited IL1-induced IL2 secretion in EL4 NOB-1 cells. These results show that the IL1 receptor of a responsive thymoma line activates, and may be coupled to, a G protein(s). This is a possible mechanism of IL1 signal transduction.     

The epidermal growth factor receptor is coupled to a pertussis toxin-sensitive guanine nucleotide regulatory protein in rat hepatocytes.

Activation of epidermal growth factor (EGF) receptors stimulates inositol phosphate production in rat hepatocytes via a pertussis toxin-sensitive mechanism, suggesting the involvement of a G protein in the process. Since the first event after receptor-G protein interaction is exchange of GTP for GDP on the G protein, the effect of EGF was measured on the initial rates of guanosine 5'-O-(3-[35S]thiotriphosphate) [( 35S]GTP gamma S) association and [alpha-32P]GDP dissociation in rat hepatocyte membranes. The initial rate of [35S]GTP gamma S binding was stimulated by EGF, with a maximal effect observed at 8 nM EGF. EGF also increased the initial rate of [alpha-32P]GDP dissociation. The effect of EGF on [35S]GTP gamma S association was blocked by boiling the peptide for 5 min in 5 mM dithiothreitol or by incubation of the membranes with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S). EGF-stimulated [35S]GTP gamma S binding was completely abolished in hepatocyte membranes prepared from pertussis toxin-treated rats and was inhibited in hepatocyte membranes that were treated directly with the resolved A-subunit of pertussis toxin. The amount of guanine nucleotide binding affected by occupation of the EGF receptor was approximately 6 pmol/mg of membrane protein. Occupation of angiotensin II receptors, which are known to couple to G proteins in hepatic membranes, also stimulated [35S]GTP gamma S association with and [alpha-32P]GDP dissociation from the membranes. The effect of angiotensin II on [alpha-32P]GDP dissociation was blocked by the angiotensin II receptor antagonist [Sar1,Ile8]angiotensin II, demonstrating that the guanine nucleotide binding was receptor-mediated. In A431 human epidermoid carcinoma cells, EGF stimulates inositol lipid breakdown, but the effect is not blocked by treatment of the cells with pertussis toxin. In these cells, EGF had no effect on [35S]GTP gamma S binding. Occupation of the beta-adrenergic receptor in A431 cell membranes with isoproterenol did stimulate [35S] GTP gamma S binding, and the effect could be completely blocked by l-propranolol. These results support the concept that in hepatocyte membranes, EGF receptors interact with a pertussis toxin-sensitive G protein via a mechanism similar to other hormone receptor-G protein interactions, but that in A431 human epidermoid carcinoma cells, EGF may activate phospholipase C via different mechanisms.     

Guanine nucleotide-sensitive interaction of a radiolabeled agonist with a phospholipase C-linked P2y-purinergic receptor

Analogs of ATP and ADP produce a guanine nucleotide-dependent activation of phospholipase C in turkey erythrocyte membranes with pharmacological properties consistent with those of a P2y-purinergic receptor (Boyer, J. L., Downes, C. P., and Harden, T.K. (1989) J. Biol. Chem. 264, 884-890). This study describes the interaction of adenosine-5'-O-2-thio[35S] diphosphate ([35S]ADP beta S) with this putative P2y-purinergic receptor on purified plasma membranes prepared from turkey erythrocytes. In binding assays performed at 30 degrees C, the association rate constant of [35S] was 1.1 x 10(7) M-1 min-1 and the dissociation rate constant was 3.8 x 10(-2) min-1. [35S]ADP beta S bound with high affinity (Kd = 6-10 nM) to an apparently homogeneous population of sites (Bmax = 2-4 pmol/mg protein). ATP and ADP analogs (2-methylthio ATP, ADP beta S, ATP, ADP, 5'-adenylyl imidodiphosphate, alpha, beta-methylene adenosine-5'-triphosphate, and beta, gamma-methylene adenosine 5'-triphosphate) inhibited the binding of [35S]ADP beta S with properties consistent with ligand interaction by simple law of mass action kinetics at a single site. The rank order of potency for inhibition of [35S]ADP beta S binding was identical to the potency order observed for these same agonists for stimulation of phospholipase C in turkey erythrocyte ghosts. Guanine nucleotides inhibited [35S]ADP beta S binding in a noncompetitive manner with the following potency order: guanosine 5'-O-(3-thiotriphosphate) greater than 5'-guanylyl imidodiphosphate greater than GTP = GDP greater than guanosine 5'-O-2-(thiodiphosphate). The data are consistent with the idea that [35S]ADP beta S may be used to radiolabel the P2y-purinergic receptor linked to activation of phospholipase C in turkey erythrocyte membranes. In addition, interaction of radiolabeled agonist with the receptor is modified by guanine nucleotides, providing evidence that an agonist-induced receptor/guanine nucleotide regulatory protein complex may be involved in P2y-receptor action.