Guanosine diphosphate binding,metabolism and regulation of follitropin-sensitive adenylate cyclase activity in Sertoli cell membranes

Nucleotides such as GTP and GDP appear to be involved in signal transduction via G protein modulation of adenylate cyclase activity. Studies on direct binding of [³H]GDP to membranes prepared from cultured immature rat Sertoli cells indicated that this process was reversible, approached steady state within 10 min, had a K_a of 4.5 ·10⁶M⁻¹ and was specific for guanine nucleotides. The non-hydrolyzable analog, guanosine 5′-O-[3-thio]triphosphate (GPPP[S]), was most effective as an inhibitor of [³H]GDP binding (ED₅₀ = 4.8·10⁻⁸M), whereas guanosine 5′-O-[2-thio]diphosphate (Gpp[S]) was less potent (ED₅₀ = 3.4·10⁻⁷M). Release of bound GDP was enhanced by follitropin (FSH) in the presence of Gppp[S], although not by FSH alone. Sertoli cell membranes possess guanine nucleotide hydrolase activity, where 95% of added nucleotide was rapidly degraded to guanosine. Binding kinetics were significantly influenced by nucleotide metabolism, which was prevented by controlling the Mg²⁺ concentration with EDTA and including App[NH]p to reduce nonspecific hydrolysis. Kinetic studies indicated that Gpp[S] inhibited (P < 0.05) Gppp[S]-stimulated adenylate cyclase activity (K_i = 1.8·10⁻⁷M), whereas basal activity remained unaffected. Addition of Gpp[S] to pre-activated enzyme (FSH plus GTP) resulted in a time-dependent decay of adenylate cyclase activity with a K_off value of 6 ± 1·min⁻¹. Using a two-stage pre-inculbation technique, adenylate cyclase activity was demonstrated to be sensitive to the nucleotide bound. When FSH was included, catalytic activity was not altered by the order of pre-incubation with the nucleotides. This suggested that the exchange of bound Gpp[S] for Gppp[S] was enhance by FSH. Activation and attenuation of FSH-sensitive adenylate cyclase activity is dependent on a nucleotide exchange mechanism which is driven by (1) the higher affinity of G for GTP than GDP, (2) enhanced release of GD when FSH is present and (3) GTP hydrolysis coupled to rapid metabolism of guanine nucleotides.     

Purine and pyrimidine nucleotides potentiate activation of NADPH oxidase and degranulation by chemotactic peptides and induce aggregation of human neutrophils via G proteins

Whereas the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), induced NADPH-oxidase-catalyzed superoxide (O2-) formation in human neutrophils, purine and pyrimidine nucleotides per se did not stimulate NADPH oxidase but enhanced O2- formation induced by submaximally and maximally stimulatory concentrations of fMet-Leu-Phe up to fivefold. On the other hand, FMet-Leu-Phe primed neutrophils to generate O2- upon exposure to nucleotides. At a concentration of 100 microM, purine nucleotides enhanced O2- formation in the effectiveness order adenosine 5'-O-[3-thio]triphosphate (ATP[gamma S]) greater than ITP greater than guanosine 5'-O-[3-thio]triphosphate (GTP[gamma S]) greater than ATP = adenosine 5'-O-[2-thio]triphosphate (Sp-diastereomer) = GTP = guanosine 5'-O-[2-thio]diphosphate (GDP[beta S] = ADP greater than adenosine 5'-[beta, gamma-imido]triphosphate = adenosine 5'-O-[2-thio]triphosphate] (Rp-diastereomer). Pyrimidine nucleotides stimulated fMet-Leu-Phe-induced O2- formation in the effectiveness order uridine 5'-O-[3-thio]triphosphate (UTP[gamma S]) = UTP greater than CTP. Uracil (UDP[beta S]) = uridine 5'-O[2-thio]triphosphate (Rp-diastereomer) (Rp)-UTP[beta S]) = UTP greater than CTP. Uracil nucleotides were similarly effective potentiators of O2- formation as the corresponding adenine nucleotides. GDP[beta S] and UDP[beta S] synergistically enhanced the stimulatory effects of ATP[gamma S], GTP[gamma S] and UTP[gamma S]. Purine and pyrimidine nucleotides did not induce degranulation in neutrophils but potentiated fMet-Leu-Phe-induced release of beta-glucuronidase with similar nucleotide specificities as for O2- formation. In contrast, nucleotides per se induced aggregation of neutrophils. Treatment with pertussis toxin prevented aggregation induced by both nucleotides and fMet-Leu-Phe. Our results suggest that purine and pyrimidine nucleotides act via nucleotide receptors, the nucleotide specificity of which is different from nucleotide receptors in other cell types. Neutrophil nucleotide receptors are coupled to guanine-nucleotide-binding proteins. As nucleotides are released from cells under physiological and pathological conditions, they may play roles as intercellular signal molecules in neutrophil activation.     

Intracellular signalling by nucleotide receptors in PC12 pheochromocytoma cells

The effect of extracellular ATP was studied in PC12 cells, a neurosecretory line that releases ATP. The addition of micromolar concentrations of ATP to PC12 cells evoked a transient increase in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i), as measured with the Ca²⁺-dye fura 2. AMP and adenosine were without effect, ruling out the involvement of P₁ receptors in mediating this response. The increase in [Ca²⁺]_i was reduced in calcium-free media and virtually eliminated by the addition of EGTA, suggesting that calcium influx was the primary response initiated by extracellular ATP. Nucleotide triphosphates such as UTP and, to a lesser degree, ITP also evoked an increase in [Ca²⁺]_i while GTP and CTP had little effect. In order to identify the receptor subtype mediating this response, the efficacy of ATP and ATP cogeners was assessed. The rank order potency was ATP > adenosine 5′-[γ-thio]triphosphate > ADP > 2-methylthioadenosine triphosphate (2-MeSATP) ～ adenosine 5′-[β-thio]diphosphate ? adenosine 5′-[αβ-methylene] triphosphate, adenosine 5′-[βγ-imido]triphosphate. This profile is not characteristic of either the P_2X or the conventional P_2Y receptors. The Ca²⁺ response exhibited desensitization to ATP that was dependent on the extracellular metabolism of ATP. UTP was equally effective in desensitizing the response. ATP, UTP, ITP, and to a much lesser extent 2MeSATP increased inositol phosphate production in a dose-dependent manner, suggesting receptor coupling to phosphatidylinositol-specific phospholipase C. These data are consistent with the view that PC12 cells express a class of non-P_2Y nucleotide receptors (P_2N) that mediate calcium influx and the accumulation of inositol phosphates. © 1993 Wiley-Liss, Inc.     

Ca2+-Guanine Nucleotide Interactions in Brain Membranes. II. Characteristics of [3H]Guanosine Triphosphate and [3H]β, γ-Imidoguanosine 5'-Triphosphate Binding and Catabolism in the Rat Hippocampus and Striatum

Abstract: With [³H]guanosine triphosphate ([³H]GTP) and [³H]β, γ -imidoguanosine 5′-triphosphate ([³H]GppNHp) as the labelled substrates, both the binding and the catabolism of guanine nucleotides have been studied in various brain membrane preparations. Both labelled nucleotides bound to a single class of noninteracting sites (K_D= 0.1-0.5 μm ) in membranes from various brain regions (hippocampus, striatum, cerebral cortex). Unlabelled GTP, GppNHp, and guanosine diphosphate (GDP) but not guanosine monophosphate (GMP) and guanosine competitively inhibited the specific binding of [³H]guanine nucleotides. Calcium (0.1–5 mm ) partially prevented the binding of [³H]GTP and [³H]GppNHp to hippocampal and striatal membranes. This resulted from both an increased catabolism of [³H]GTP (into [³H]guanosine) and the likely formation of Ca-guanine nucleotide^2- complexes. The blockade of guanine nucleotide catabolism was responsible for the enhanced binding of [³H]GTP to hippocampal membranes in the presence of 0.1 mm -ATP or 0.1 mm -GMP. Striatal lesions with kainic acid produced both a 50% reduction of the number of specific guanine nucleotide binding sites and an acceleration of [³H]GTP and [³H]GppNHp catabolism (into [³H]guanosine) in membranes from the lesioned striatum. This suggests that guanine nucleotide binding sites were associated (at least in part) with intrinsic neurones whereas the catabolising enzyme(s) would be (mainly) located to glial cells (which proliferate after kainic acid lesion). The characteristics of the [³H]guanine nucleotide binding sites strongly suggest that they may correspond to the GTP subunits regulating neurotransmitter receptors including those labelled with [³H]5-hydroxytryptamine ([³H]5-HT) in the rat brain.     

Guanine nucleotide- and muscarinic agonist-dependent phosphoinositide metabolism in synaptoneurosomes from cerebral cortex of immature rats

Guanine nucleotide-, neurotransmitter-, and fluoride-stimulated accumulation of [³H]inositol phosphates ([³H]InsPs) was measured in [³H]inositol-labeled synaptoneurosomes from cerebral cortex of immature (7-day-old) and adult rats, in order to clarify the role of GTP-binding proteins (G-proteins) in modulating phosphoinositide (PtdIns) metabolism during brain development. GTP(S) [Guanosine 5-O-(3-thio)triphosphate] time- and concentration-dependently stimulated PtdIns hydrolysis. Its effect was potentiated by full (carbachol, metacholine) and partial (oxotremorine) cholinergic agonists through activation of muscarinic receptors. The presence of deoxycholate was required to demonstrate agonist protentiation of the guanine nucleotide effect. The response to GTP(S) was higher in adult than in immature rats, while the effect of cholinergic agonists was similar at the two ages examined. At both ages, histamine potentiated the effect of GTP(S), while norepinephrine was ineffective. At both ages, guanosine 5-O-(2-thio)diphosphate [GDP(S)] and pertussis toxin significantly decreased GTP(S)-induced [³H]InsPs formation. The phorbol ester phorbol 12-myristate 13-acetate (PMA), on the other hand, did not inhibit the guanine nucleotide response in synaptoneurosomes from immature rats. NaF mimicked the action of GTP(S) in stimulating PtdIns hydrolysis. Its effect was not affected by carbachol and was highly synergistic with that of AlCl₃, according to the concept that fluoroaluminate (AlF₄ ^–) is the active stimulatory species. No quantitative differences were found in the response to these salts between immature and adult animals. These results provide evidence that, in both the immature and adult rat brain, neuroreceptor activation is coupled to PtdIns hydrolysis through modulatory G-proteins.     

The synthesis of guanosine 5′-diphosphate-l-galactose by extracts of Chlorella pyrenoidosa

Extracts of the green alga Chlorella pyrenoidosa have been shown to catalyze the epimerization of guanosine 5′-diphosphate-d-mannose to guanosine 5′-diphosphate-l-galactose. The equilibrium is about 0.1 in the direction of the l-galactosyl nucleotide and is independent of temperature. The K_m for guanosine 5′-diphosphate-d-mannose was determined to be about 1.2 × 10^?4m. Guanosine 5′-diphosphate-l-fucose (6-deoxy-l-galactose) also serves as a substrate for the enzyme, and the product of that reaction appears to be guanosine 5′-diphosphate-d-rhamnose (6-deoxy-d-mannose).     

Mouse embryos fail to synthesize detectable quantities of guanosine 5'-diphosphate 3'-diphosphate.

The unusual highly phosphorylated nucleotide, guanosine 5′-diphosphate 3′-diphosphate, has been implicated in the control of development of the mouse (Irr, J. D., et al. (1974) Cell3, 249). We have been unable, however, to detect guanosine 5′-diphosphate 3′-diphosphate synthesis either in preimplantation and postimplantation mouse embryos cultured in the presence of [³²P]orthophosphate or in assays using ribosomes isolated from 10- to 13-day mouse embryos. Three unidentified phosphorous-containing compounds were detected in blastocyst stage mouse embryos.     

The Proline-rich N-terminal Domain of G18 Exhibits a Novel G Protein Regulatory Function

The protein G18 (also known as AGS4 or GPSM3) contains three conserved GoLoco/GPR domains in its central and C-terminal regions that bind to inactive Gα_i, whereas the N-terminal region has not been previously characterized. We investigated whether this domain might itself regulate G protein activity by assessing the abilities of G18 and mutants thereof to modulate the nucleotide binding and hydrolytic properties of Gα_i1 and Gα_o. Surprisingly, in the presence of fluoroaluminate (AlF₄⁻) both G proteins bound strongly to full-length G18 (G18wt) and to its isolated N-terminal domain (G18ΔC) but not to its GoLoco region (ΔNG18). Thus, it appears that its N-terminal domain promotes G18 binding to fluoroaluminate-activated Gα_i/o. Neither G18wt nor any G18 mutant affected the GTPase activity of Gα_i1 or Gα_o. In contrast, complex effects were noted with respect to nucleotide binding. As inferred by the binding of [³⁵S]GTPγS (guanosine 5′-O-[γ-thio]triphosphate) to Gα_i1, the isolated GoLoco region as expected acted as a guanine nucleotide dissociation inhibitor, whereas the N-terminal region exhibited a previously unknown guanine nucleotide exchange factor effect on this G protein. On the other hand, the N terminus inhibited [³⁵S]GTPγS binding to Gα_o, albeit to a lesser extent than the effect of the GoLoco region on Gα_i1. Taken together, our results identify the N-terminal region of G18 as a novel G protein-interacting domain that may have distinct regulatory effects within the G_i/o subfamily, and thus, it could potentially play a role in differentiating signals between these related G proteins.     

Use of 5′-[<Emphasis Type="Italic">p</Emphasis>-(Fluorosulfonyl)benzoyl] Guanosine as an Affinity Probe for the Guanine Nucleotide-Binding Site of Transducin

Transducin (T) mediates vision in retinal rods by transmitting light signals detected by rhodopsin to a cGMP phosphodiesterase. The flow of information relies on a subunit association/dissociation cycle of T regulated by a guanine nucleotide exchange/hydrolysis reaction. 5′-[p-(Fluorosulfonyl)benzoyl] guanosine (FSBG) was synthesized and examined here as an affinity label for the guanine nucleotide binding site of T. Although the relative binding affinity of FSBG to T was much lower than for GTP and β,γ-imido-guanosine 5′-triphosphate (GMPPNP), the incorporation of FSBG to T inhibited its light-dependent [³H] GMPPNP binding activity in a concentration dependent manner. Additionally, GDP, GTP and GTP analogs hindered the binding of [³H] FSBG to T. These results demonstrated that FSBG could be used to specifically modify the active site of T. In addition, FSBG was not capable of dissociating T from T:photoactivated rhodopsin complexes, suggesting that in this case FSBG is acting as a GDP analog.     

The Biosynthesis of Transfer Ribonucleic Acid in the Developing Rat Brain and in Cultured Glial Cells

Abstract: The biosynthesis of tRNA was investigated in cultured astroglial cells and the 3-day-old rat brain in vivo. In the culture system astrocytes were grown for 19 days and were then exposed to [³H]guanosine for 1.5–7.5 h; 3-day-old rats were injected with [³H]guanosine and were killed 5–45 min later. [³H]tRNA was extracted, partially purified, and hydrolyzed to yield [³H]-guanine and [³H]methyl guanines. The latter were separated from the former by high performance liquid chromatography and their radioactivity determined as a function of the time of exposure to [³H]guanosine. The findings indicate that labeling of astrocyte tRNA continued for 7.5 h and was maximal, relative to total RNA labeling, at 3 h, while in the immature brain tRNAs were maximally labeled at 20 min after [³H]guanosine administration. The labeling pattern of the individual methyl guanines differed considerably between astrocyte and brain tRNAs. Thus, [³H]1-methylguanine represented up to 35% of the total [³H]methyl guanine radioactivity in astrocyte [³H]tRNA, while it became only negligibly labeled in brain [³H]tRNA. Conversely, brain [³H]tRNA contained more [³H]N²-methylguanine than did astrocyte [³H]tRNA. Approximately equal proportions of [³H]7-methylguanine were found in the [³H]tRNAs of both neural systems. The [³H]methylguanine composition of brain [³H]tRNA was followed through several stages of tRNA purification, including benzoylated DEAE-cellulose and reverse phase chromatography (RPC-5), and differences were found between the [³H]methylguanine composition of RPC-5 fractions containing, respectively, tRNA^lys and tRNA^phe. The overall results of this study suggest that developing brain cells biosynthesize their particular complement of tRNAs actively and in a cell-specific manner, as attested by the significant differences in the labeling rates of their methylated guanines. The notion is advanced that cell-specific tRNA modifications may be a prerequisite for the successful synthesis of cell-specific neural proteins.     

Guanosine negatively modulates the gastric motor function in mouse

The aim of the present study was to evaluate if guanine-based purines may affect the gastric motor function in mouse. Thus, the influence of guanosine on the gastric emptying rate in vivo was determined and its effects on spontaneous gastric mechanical activity, detected as changes of the intraluminal pressure, were analyzed in vitro before and after different treatments. Gastric gavage of guanosine (1.75–10 mg/kg) delayed the gastric emptying. Guanosine (30 μM–1 mM) induced a concentration-dependent relaxation of isolated stomach, which was not affected by the inhibition of the purine nucleoside phosphorylase enzyme by 4′-deaza-1′-aza-2′-deoxy-1′-(9-methylene)-immucillin-H. The inhibitory response was antagonized by S-(4-nitrobenzyl)-6-thioinosine, a membrane nucleoside transporter inhibitor, but not affected by 9-chloro-2-(2-furanyl)-[1,2,4]triazolo[1,5-c]quinazolin-5-amine, a nonselective adenosine receptor antagonist, or by tetrodotoxin, a blocker of neuronal voltage-dependent Na⁺ channels. Moreover, guanosine-induced effects persisted in the presence of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylyl cyclase or tetraethylammonium, a nonselective potassium channel blocker, but they were progressively reduced by increasing concentrations of 2′5′dideoxyadenosine, an adenylyl cyclase inhibitor. Lastly, the levels of cyclic adenosine monophosphate (cAMP), measured by ELISA, in gastric full thickness preparations were increased by guanosine. In conclusion, our data indicate that, in mouse, guanosine is able to modulate negatively the gastric motor function, reducing gastric emptying and inducing muscular relaxation. The latter is dependent by its cellular uptake and involves adenylyl cyclase activation and increase in cAMP intracellular levels, while it is independent on neural action potentials, adenosine receptors, and K⁺ channel activation.     

Two inward K+ channels in the xylem parenchyma cells of barley roots are regulated by G-protein modulators through a membrane-delimited pathway

In order to study the mechanism and regulation of K⁺ resorption from the xylem by the cells that border the xylem vessels (the xylem parenchyma cells), K⁺ inward-rectifying channels (KIRCs) in the plasma membrane of xylem parenchyma cells from Hordeum vulgare L. cv. Apex were studied using the patch-clamp technique. In the inside-out configuration, three different types of K⁺ channel and a further K⁺ conductance could be identified. Two of these channels, named KIRC1 and KIRC2, were activated by guanosine 5′-[β,γ-imido]triphosphate (Gpp(NH)p; 150 μM), a non-hydrolyzable derivative of GTP, indicating that channel activity was up-regulated by G-proteins; modulation of channel activity occurred via a membrane-delimited pathway, since the effect could be demonstrated in cell-free patches. At 100 mM external K⁺, KIRC1 had a conductance of 8 pS. There was no effect of ATP on channel activity. Likewise, addition of 150 μM guanosine 5′-[β-thio]diphosphate (GDPβS) or adenosine 5′-[γ-thio]triphosphate (ATPγS) failed to activate KIRC1, indicating nucleotide specificity of the effect. A second K⁺ channel, activated by Gpp(NH)p (KIRC2) with gating properties clearly different from the first one was less frequently observed. Four different substates could be identified; the main level had a conductance of about 2 pS. Gating below the Nernst potential of K⁺ (E_K) was voltage-independent. The channel closed at potentials more positive than E_K. A third, hyperpolarization-activated K⁺ channel, KIRC3, with a low open probability was encountered in inside-out patches. It had a conductance of 45 pS in 100 mM K⁺. Channel activity was not affected by the addition of G-protein modulators. Moreover, slowly activating inward currents carried by K⁺ were recorded in several patches that are ascribed to a `subpicosiemens conductance'. Neither GDPβS nor Gpp(NH)p appeared to have an effect on the currents. Whole-cell measurements with these G-protein modulators included in the pipette solution were in general agreement with the results obtained on cell-free patches. A statistical evaluation revealed that time-dependent inward currents were larger when the G-protein activator Gpp(NH)p was included in the pipette medium compared to measurements with the inhibitor GDPβS. With the GTP analogue, an additional instantaneous component was elicited that was ascribed to KIRC2 activity. Data are discussed with respect to the putative role of G-proteins in conveying hormonal signals. Regulation by G-protein may either serve to fine-tune K⁺ uptake by xylem parenchyma cells or to initiate depolarization, followed by salt-efflux through depolarization-activated cation and anion channels. Received 11 October 1996 / Accepted: 21 April 1997     

Nucleic Acid Precursor Incorporation by Eimeria nieschulzi (Protozoa: Apicomplexa) and Jejunal Villus Epithelium*

Autoradiography was used to investigate incorporation of tritiated adenine, adenosine, guanosine and thymidine by Eimeria nieschulzi and rat jejunal villus epithelial cells. At 2 1/2 days postinoculation, parasitized and control tissues were incubated for 20 min in oxygenated Tyrode's solution (37 C, pH 7.5) containing 30 μCi/ml of each nucleic acid precursor. Treatment of tissues with ribonuclease revealed that E. nieschulzi incorporated label from [³H]adenine primarily into RNA while that from [³H]adenosine and [³H]guanosine was present mainly in DNA. Label from [³H]thymidine was not utilized by parasites. Host villus epithelial cells incorporated label from [³H]purines primarily into RNA. Labeled cytoplasmic RNA was significantly increased in parasitized cells after incubation in [³H]adenine. Tritiated nuclear RNA and cytoplasmic RNA were significantly decreased in parasitized cells after incubation in [³H]adenosine. Incorporation of label from [³H]guanosine was similar for parasitized and control cells. A small quantity of label from each [³H]precursor was incorporated into DNA of villus epithelial cell nuclei.     

Differential Sensitivity of Basal and Opioid-Stimulated Low Km GTPase to Guanine Nucleotide Analogs

In membranes derived from NG108-15 cells, the opioid peptide [D-Ala2,D-Leu5]enkephalin (DADLE) stimulates a low Km GTPase. The nucleotide analogs guanosine 5'-O-(2-thio)diphosphate (GDP beta S), guanosine 5'-(beta,gamma-imido)triphosphate [Gpp(NH)p] and guanosine 5'-O-(3-thio)-triphosphate (GTP gamma S) inhibit the basal enzymatic activity with the order of potency GTP gamma S greater than Gpp (NH)p greater than GDP beta S. In the presence of DADLE, the inhibition isotherms of GDP beta S and Gpp(NH)p are shifted to the right five- and fourfold, respectively, compared to the inhibition observed in the absence of DADLE. In contrast, the IC50 of GTP gamma S for inhibiting the enzyme is reduced by 55% in the presence of the opioid. Both Gpp(NH)p and GTP gamma S produce a concentration-dependent increase in the Km(app) of GTPase, without affecting its Vmax, indicating a competitive inhibition. However, the replots of Km(app) versus inhibitor concentration are hyperbolic, suggesting a partial type of inhibition. Both Gpp(NH)p and GTP gamma S, but not GTP, induce an increase in the EC50 of DADLE for stimulating GTPase. These findings indicate that the basal and the opioid-stimulated low Km GTPase differ in their respective sensitivities to inhibition by guanine nucleotide analogs.     

Kinetics and thermodynamics of activation by pretreatment with guanosine 5′-[β-imido]triphosphate of smooth-muscle adenylate cyclase

Catalytic subunits (C) of uterine smooth-muscle adenylate cyclase were activated (C*) by incubating the enzyme with the GTP analogue guanosine 5′-[βγ-imido]triphosphate (p[NH]ppG), followed by treatment with GTP and washing at 2°C. Activation (C→C*) proceeded in a time- and temperature-dependent manner as disclosed by subsequent assay of the pretreated particles at 37°C. The properties of the activated subunits were a function of the pretreatment temperature and not those of the enzyme assay performed at 37°C. Over the range 6–24°C, activation by pretreatment with p[NH]ppG followed simple Michaelis–Menten kinetics, and increase in temperature increased the concentration of catalytic subunits in the C* state and decreased K_m for the guanosine nucleotide. Characterization of the temperature-dependent effects of pretreatment with p[NH]ppG suggested that activation of the catalytic subunit at the temperature in situ (37°C) was moderately endergonic (ΔH⁰ ~8kJ·mol⁻¹) and accompanied by an increase in entropy (ΔS⁰ ~146J·mol⁻¹·K⁻¹). The β-adrenergic catecholamine receptor, reflected by isoproterenol's effect on activation by pretreatment with p[NH]ppG, increased the concentration of catalytic subunits in the C* state but had an insignificant (P>0.05) effect on the K_m at every temperature. This result suggested that formation of the receptor–hormone complex produced an increase in the first-order rate constant without an appreciable effect on the actual catalytic-subunit activation step. The primary function of the β-adrenergic catecholamine receptor under these conditions appeared to be regulation of the concentration of activation sites available for binding of p[NH]ppG.     

The use of nucleotide phosphorothioate diastereomers to define the structure of metal-nucleotide bound to GTP-AMP and ATP-AMP phosphotransferases from beef-heart mitochondria

The diastereomers of adenosine 5'-O-[1-thio]triphosphate (ATP[alpha S]) and adenosine 5'-O-[2-thio]triphosphate (ATP[beta S]) were utilized to seek unambiguous assignment of Mg2+ coordination to ATP when bound to ATP-AMP phosphotransferase from beef heart mitochondria (AK2). Similarly, the diastereomers of guanosine 5'-O-[thio]triphosphate (GTP[alpha S]) and guanosine 5'-O-[2-thio]triphosphate (GTP[beta S]) were utilized to seek unambiguous assignment of Mg2+ coordination to GTP when bound to GTP-AMP phosphotransferase from beef heart mitochondria (AK3). Furthermore the diastereomers of guanosine 5'-O-[1-thio]diphosphate (GDP-[alpha S]) have been used to assign Mg2+ coordination to GDP when bound to AK3. The ratios (V for isomer Sp)/(V for isomer Rp) obtained in the presence of Mg2+ and Cd2+ are compared to those already published for ATP-AMP phosphotransferases from pig muscle (AK1) [Kalbitzer et al. (1983) Eur. J. Biochem. 133, 221-227] and from baker's yeast (AKy) [Tomasselli and Noda (1983) Eur. J. Biochem. 132, 109-115]. In all cases, coordination of Mg2+ to the beta-phosphate via the pro-R oxygen is present, as shown by reversal of specificity for the diastereomers of ATP [beta S] or GTP [beta S] respectively on changing the metal ion. In contrast, there is no reversal of specificity for the diastereomers of ATP [alpha S] or GTP[alpha S], or for GDP[alpha S] in the case of AK3 for the reverse reaction, indicating that there is no interaction of the metal with the alpha-phosphate group. The observed stereospecificity for the alpha-thiophosphate is consistent with the assumption of an interaction of the pro-R oxygen of the alpha-phosphate group with the enzyme.     

Characterization of3H-labelled platelet activating factor receptor complex solubilized from rabbit platelet membranes

Rabbit platelet membranes, preincubated with³H-labeled platelet activating factor ([³H]PAF), were solubilized with 2% digitonin. Sedimentation of the detergent extract in a sucrose density gradient revealed a major labeled component with a sedimentation coefficient (s_20,ω) of 10.5 S, which was substantially diminished when an excess of unlabeled PAF or L-652,731, (trans-2,5-bis(3,4,5-trimethoxyphenyl)tetrahydrofuran), (PAF antagonist) was present in the preincubation mixture, suggesting that the 10.5 S component is a specific receptor-bound [³H]PAF complex. Gel filtration of the [³H]PAF-receptor complex on Sephacryl S-300 revealed a single radiolabeled fraction with an apparent Stokes' radius of 4.9 nm. The apparent molecular weight and the frictional ratio of the agonist-receptor complex were computed to be 220 000 and 1.13, respectively. Dissociation of [³H]PAF from the radioligand-receptor complex was facilitated by Na⁺ and Li⁺, whereas K⁺ and Cs⁺ were ineffective. The guanine nucleotide, GTP, was also found to promote the dissociation in a manner that is additive with the effect of Na⁺, suggestive of the coupling of a guanine nucleotide binding protein to the solubilized PAF-receptor complex.     

Metabolic fate of guanosine in higher plants

The aim of the present study was to investigate the metabolic fate of guanine nucleotides in higher plants. The rate of uptake of [8-¹⁴C]guanosine by suspension-cultured Catharanthus roseus cells was more than 20 times higher than that of [8-¹⁴C]guanine. The rate of uptake of [8-¹⁴C]guanosine increased with the age of the culture. Pulse-chase experiments with [8-¹⁴C]guanosine revealed that some of the guanosine that had been taken up by the cells was converted to guanine nucleotides and incorporated into nucleic acids. A significant amount of [8-¹⁴C]guanosine was degraded directly to xanthine, allantoin and allantoic acid, with the generation of ¹⁴CO₂ as the final product. The rate of salvage of [8-¹⁴C]guanosine for the synthesis of nucleic acids was highest in young cells, while the rate of degradation increased with the age of the cells. In segments of roots from Vigna mungo seedlings, nearly 50% of the [8-¹⁴C]guanosine that had been absorbed over the course of 15 min was recovered in guanine nucleotides. A significant amount of the radioactivity in nucleotides became associated with nucleic acids and ureides during ‘chase’ periods. In segments of young leaves of Camellia sinensis, [8-¹⁴C]guanosine was initially incorporated into guanine nucleotides, nucleic acids, theobromine and ureides, and the radioactivity in these compounds was transferred to caffeine and CO₂ during a 24-h incubation. Our results suggest that guanosine is an intermediate in the catabolism of guanine nucleotides and that it is re-utilised for nucleotide synthesis by ‘salvage’ reactions. Guanosine was catabolised by the conventional degradation pathway via xanthine and allantoin. In some plants, guanosine is also utilised for the formation of ureide or the biosynthesis of caffeine.     

Guanine Nucleotide-Binding Protein Regulation of Microsomal Phospholipase D Activity of Canine Cerebral Cortex

The hydrolytic activity of microsomal phospholipase D from canine cerebral cortex was measured by a radiochemical assay using 1,2-dipalmitoyl-sn-glycerol-3-phosphoryl[3H]choline and 1-palmitoyl-2-[9,10(n)-3H]palmitoyl-sn-glycerol-3-phosphorylcholine as the exogenous substrates. Of several detergents tested, Triton X-100 was found to be the most effective in allowing expression of phospholipase D hydrolytic activity. The microsomal phospholipase D does not require any metal ion for its hydrolytic activity. Calcium and magnesium were slightly inhibitory between concentrations of 1 and 4 mM, but zinc was greatly inhibitory, causing a loss of greater than 90% activity at the 4 mM concentration. Non-hydrolyzable guanine nucleotide analogues such as guanosine 5'-(3-O-thio)triphosphate and guanyl-5'-yl-(beta, gamma-methylene)diphosphonate but not guanosine 5'-(2-thio)diphosphate were able persistently to stimulate phospholipase D hydrolytic activity at micromolar concentrations. Guanosine 5'-(2-thio)diphosphate was capable of partially blocking guanosine 5'-(3-O-thio)triphosphate stimulation of phospholipase D. Aluminum fluoride was able to cause a two- to threefold increase in hydrolytic activity of the phospholipase D. Cholera toxin had a stimulatory effect on the hydrolytic activity of phospholipase D, whereas islet-activating protein pertussis toxin had no effect. These results indicate that regulation of microsomal phosphatidylcholine phospholipase D activity by the guanine nucleotide-binding protein(s) in canine cerebral cortex may play an important role in signal transduction processes as well as in brain choline metabolism.     

Changes in plasma hydrolase activities in hereditary and streptozotocin-induced diabetes.

The characteristics of the hydrolysis of 5′-adenylylimidodiphosphate [AMP-P(NH)P] by partially purified plasma membranes from rat liver are described. Hydrolysis was less with membranes from fat cells and was poor with a detergent-dispersed preparation from rat cerebellum. The Chromatographic behavior of the principal degradation products suggests that AMP-P(NH)P is first hydrolyzed to 5'?AMP, which is then hydrolyzed further to adenosine. The adenosine is shown to inhibit adenylate cyclase noncompetitively with respect to substrate and in a cation-dependent manner. Sensitivity to inhibition by adenosine was markedly enhanced by agents that stimulated adenylate cyclase. The characteristics of the initial hydrolysis of AMP-P(NH)P fit best those of nucleotide pyrophosphatase and support the conclusion that several of the various phosphatase activities present in membranes may be due to the same enzyme. Under conditions shown to be linear with respect to time and membrane protein concentration, hydrolysis of AMP-P(NH)P exhibited a pH optimum between 9.5 and 10. At pH 9.5, hydrolysis occurred with a K_m of about 20 μm and a V of about 220 nmol (min) ¹ (mg of protein)^?1. The initial hydrolysis of AMP-P(NH)P was inhibited in a linear-competitive manner by ATP, ADP, 5′-AMP, GTP, 5′-guanylylimidodiphosphate, NAD⁺, and p-nitrophenyl-dTMP and in a noncompetitive manner by UDP-glucose. Adenosine 3′:5?cyclic phosphate and guanosine 3′:5′-cyclic phosphate were not inhibitory at concentrations up to 1 mm. ATP, GTP, and 5′-guanylylimidodiphosphate were also hydrolyzed in a manner comparable to that for AMP-P(NH)P. Hydrolysis of AMP-P(NH)P did not require the presence of added metal, and some metals were inhibitory. Activity was inhibited by dithiothreitol (50% at <1 mm) and by EDTA (50% at about 10 mm). Following pretreatment with EDTA or dithiothreitol, the readdition of certain metals, especially Zn or Co, caused some restoration of hydrolytic activity. The evidence suggests that hydrolytic activity involves the participation of bound metal and that the enzyme is a metallo-protein.