The effects of Ca2+ and guanylnucleotides on isoprenaline-stimulated cyclic AMP formation in rat reticulocyte ghosts

We have studied β-adrenergic stimulation of cyclic AMP formation in fragmented membranes and in unsealed or resealed ghosts prepared from rat reticulocytes. The maximal rate of isoprenaline-stimulated cyclic AMP formation with saturating MgATP concentrations and in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine was 5–8 nmol/min per ml ghosts are remained constant for at least 15 min. Transition from resealed ghosts to fragmented membranes was associated with a shift of the activation constant (K_a) for (±)-isoprenaline from 0.1 to 0.6 μM. The apparent dissociation constant for propranolol (0.01 μM) remained unchanged. The K_a values for isoprenaline in native reticulocytes and in resealed ghosts were identi The stimulating effect of NaF on cyclic AMP formation in resealed ghosts reached 15% of maximal β-adrenergic stimulation. Cyclic AMP formation, both in fragmented membranes and in ghosts, was half-maximally inhibited with Ca²⁺ concentrations ranging between 0.1 and 1 μM. GTP stimulated iosprenaline-dependent cyclic AMP formation in unsealed ghosts and in fragmented reticulocyte membranes by a factor of 3–5 but did not change the K_a value for isoprenaline. K_a values for the guanylnucleotides in different experiments varied between 0.3 and 2 μM. Ca²⁺ concentrations up to 4.6 μM reduced the maximal activation by GTP and Gpp(NH)p but did not affect their K_a values. Compared to GTP, maximal activation by Gpp(NH)p was higher in fragmented membranes, but much lower in ghosts. Our results suggest that the native β-receptor adenylate cyclase system of reticulocytes is more closely approximated in the ghost model than in fragmented membrane preparations. Membrane properties seem to modulate the actions of guanylnucleotides on isoprenaline-dependent cyclic AMP formation in ghosts. Some of these effects are not observed in isolated membranes.     

The effects of Ca2+ and guanylnucleotides on isoprenaline-stimulated cyclic AMP formation in rat reticulocyte ghosts

We have studied beta-adrenergic stimulation of cyclic AMP formation in fragmented membranes and in unsealed or resealed ghosts prepared from rat reticulocytes. The maximal rate of isoprenaline-stimulated cyclic AMP formation with saturating MgATP concentrations and in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine was 5-8 nmol/min per ml ghosts and remained constant for at least 15 min. Transition from resealed ghosts to fragmented membranes was associated with a shift of the activation constant (Ka) for (+/-)-isoprenaline from 0.1 to 0.6 microM. THe apparent dissociation constant for propranolol (0.01 microM) remained unchanged. The Ka values for isoprenaline in native reticulocytes and in resealed ghosts were identical. The stimulating effect of NaF on cyclic AMP formation in resealed ghosts reached 15% of maximal beta-adrenergic stimulation. Cyclic AMP formation, both in fragmented membranes and in ghosts, was half-maximally inhibited with Ca2+ concentrations ranging between 0.1 and 1 microM. GTP stimulated isoprenaline-dependent cyclic AMP formation in unsealed ghosts and in fragmented reticulocyte membranes by a factor of 3-5 but did not change the Ka value for isoprenaline. Ka values for the guanylnucleotides in different experiments varied between 0.3 and 2 microM. Ca2+ concentrations up to 4.6 microM reduced the maximal activation by GTP and Gpp(NH)p but did not affect their Ka values. Compared to GTP, maximal activation by Gpp(NH)p was higher in fragmented membranes, but much lower in ghosts. Our results suggest that the native beta-receptor adenylate cyclase system of reticulocytes is more closely approximated in the ghost model than in fragmented membrane preparations. Membrane properties seem to modulate the actions of guanylnucleotides on isoprenaline-dependent cyclic AMP formation in ghosts. Some of these effects are not observed in isolated membranes.     

Ca-stimulated, Mg-independent ATP hydrolysis and the high affinity Ca-pumping ATPase. Two different activities in rat kidney basolateral membranes

The Mg²⁺-dependency of Ca²⁺-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg²⁺- and Ca²⁺-buffering ligands. ATP hydrolysis is strongly stimulated by Mg²⁺ with a K_m of 13 μ M in the absence or presence of 1 μ M free Ca²⁺. At free Mg²⁺ concentrations of 1 μ M and lower, ATP hydrolysis is Mg²⁺ -independent, but is strongly stimulated by submicromolar Ca²⁺ concentrations K_m = 0.25 μM, V_max = 24 μmol P_i/h per mg protein). The Ca²⁺-stimulated ATP hydrolysis strongly decreases at higher Mg²⁺ concentrations. The Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg²⁺ concentrations calmodulin and trifluoperazine affect the high affinity Ca²⁺-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca²⁺-pumping in renal basolaterals and on Ca²⁺-ATPase in erythrocyte ghosts suggest that the Ca²⁺-pumping enzyme requires Mg²⁺. In contrast, a role of the Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis in active Ca²⁺ transport across basolateral membranes is rather unlikely.     

Prostaglandin F2a inhibition of epinephrine stimulated cyclic AMP and progesterone production by rat corpora lutea of various ages

Epinephrine can mimic the stimulatory effects of LH in vitro on cyclic AMP (cAMP) and progesterone production by isolated rat corpora lutea. The aim of the present study was to test whether the effects of epinephrine in vitro on the rat corpus luteum, as with LH, can be inhibited by prostaglandin F_2a (PGF_2a. The stimulatory effect of epinephrine on tissue levels of cAMP in 1-day-old corpora lutea was not inhibited by PGF₂. A dose-dependent inhibition by PGF_2a (0.5–50 μM) was seen for 3-day-old corpora lutea and this inhibition could not be overcome by higher concentrations of epinephrine (0.165–165 μM). The stimulation by epinephrine on progesterone production was inhibited by PGF_2a (5 μM) in 3- and 5-day-old, but not in 1-day-old corpora lutea. Thus, PGF_2a can inhibit the stimulatory effect of epinephrine in 3- and 5-day-old corpora lutea, but not in the newly formed corpora lutea (1-day-old) and PGF_2a shows in this respect the same agedependent inhibitory pattern as in relation to LH stimulation.     

Ultrastructure of hemoglobin-depleted human erythrocyte resealed ghosts

Both negative-stain and freeze-fracture electron microscopic techniques revealed that the ultrastructure of resealed white ghosts prepared at high dilution during the hemolysis step is very different from that of resealed ghosts prepared at low or moderate dilution (pink ghosts). The negative-stained resealed white ghosts showed light halo substructures on membrane surfaces and protrusions at the edge of the ghosts. Freeze-fracturing of these ghosts showed that membrane blebbing had occurred and that fragments of the membranes resealed to form small right-side-out vesicles ranging from 0.1 to 0.3 μm in diameter.     

Studies on (NA + K)-activated ATPase XLV. Magnesium induces two low-affinity non-phosphorylating nucleotide binding sites per molecule

ATP and adenylylimidodiphosphate (AdoPP[NH]P) bind to (Na⁺ + K⁺)-ATPase in the absence of Mg²⁺ (EDTA present) with a homogeneous but 15-fold different affinity, the K_d values being 0.13 μM and 1.9 μM, respectively. The binding capacities of the two nucleotides are nearly equal and amount to 3.9 and 4 nmol/mg protein or 1.7 and 1.8 mol/mol (Na⁺ + K⁺)-ATPase, respectively. The K_d value for ATP is equal to the K_m for phosphorylation by ATP (0.05–0.25 μM) and the binding capacity is equivalent to the phosphorylation capacity of 1.8 mol/mol (Na⁺ + K⁺)-ATPase. Hence, the enzyme contains two high-affinity nucleotide binding and phosphorylating sites per molecule, or one per α-subunit. Additional low-affinity nucleotide binding sites are elicited in the presence of Mg²⁺, as shown by binding studies with the non-phosphorylating (AdoPP[NH]P). The K_d and binding capacity for AdoPP[NH]P at these sites is dependent on the Mg²⁺ concentration. The K_d increases from 0.06 mM at 0.5 mM Mg²⁺ to a maximum of 0.26 mM at 2 mM Mg²⁺ and the binding capacity from 1.5 nmol/mg protein at 0.5 mM Mg²⁺ to 3.3 nmol/mg protein at 4 mM Mg²⁺. Extrapolation of a double reciprocal plot of binding capacity vs. total Mg²⁺ concentration yields a maximal binding capacity at infinite Mg²⁺ concentration of 3.8 nmol/mg protein or 1.7 mol/mol (Na⁺ + K⁺)-ATPase. The K_d for Mg²⁺ at the sites, where it exerts this effect, is 0.8 mM. The K_d for the high-affinity sites increases from 1.5–1.9 μM in the absence of Mg²⁺ to a maximum of 4.2 μM at 2 mM Mg²⁺ concentration. The binding capacity of these sites (1.8 mol/mol enzyme) is independent of the Mg²⁺ concentration. Hence, Mg²⁺ induces two low-affinity non-phosphorylating nucleotide binding sites per molecule (Na⁺ + K⁺)-ATPase in addition to the two high-affinity, phosphorylating nucleotide binding sites.     

Effect of thyroid status on α- and β-catecholamine responsiveness of hamster adipocytes

It has been suggested that part of the increased β-catecholamine responsiveness in hyperthyroid animals is due to a decrease in α-catecholamine action. The present results indicate that neither hyperthyroidism nor hypothyroidism altered the α₂-adrenergic inhibition of adenylate cyclase or the α₁-adrenergic stimulation of phosphatidylinositol turnover in adipocytes from the white adipose tissue of hamster. No effect of hyperthyroidism was found on the K_d of [³H]dihydroegocryptine or the number of binding sites in membranes prepared from hamster adipocyte tissue. The stimulation of cyclic AMP due to β-catecholamines was enhanced in adipocytes from hyperthyroid hamster, as was lipolysis. However, in adipocytes from hyperthyroid hamster the maximal stimulation of cyclic AMP due to isoproterenol, ACTH or epinephrine plus yohimbine, as seen in the presence of adenosine deaminase and theophylline, was less than in adipocytes from euthyroid hamsters. The activation of adenylate cyclase by isoproterenol was the same in membranes from hyperthyroid as compared to those from euthyroid hamsters in the absence or presence of guanine nucleotides. These data suggest that thyroid status has little effect on α-catecholamine action but enhances the activation of lipolysis by β-catecholamine agonists.     

PGJ2 and ΔPGJ2 inhibit cell growth accompanied with inhibition of phosphoinositide turnover in human astrocytoma cells

PGJ₂ and Δ¹²PGJ₂ (1 μM to 30 μm) inhibited the growth of human astrocytoma cells (1321N1) in a time-dependent manner within 48 hrs, determined by [³H]thymidine incorporation into acid-insoluble fraction or amounts of protein. The EC₅₀ values for PGJ₂ and Δ¹²PGJ₂ were approximately 8 μM and 6 μM, respectively. [³H]Thymidine incorporation to acid insoluble fraction was inhibited by these PGs within 1 hr, indicating that these PGs rapidly affect cell functions. Although it has been reported that an increase in cyclic AMP inhibits cell growth, PGJ₂ and Δ¹²PGJ₂, but not PGE₁, reduced isoproterenol (10 μM)-induced accumulation of cyclic AMP, suggesting that PGJ₂ and Δ¹²PGJ₂ may disturb adenylate cyclase system, which might be independent on cell growth. On the other hand, these PGs inhibited the incorporation of [³H]inositol into phospholipid fraction within 6 hrs. Furthermore, PGJ₂ and Δ¹²PGJ₂ inhibited carbachol- and/or histamine-induced accumulation of inositol phosphates with a similar dose-dependency to their inhibitions of cell growth. In membrane preparations, however, PGJ₂ and Δ¹²PGJ₂ failed to inhibit GTPγS (10 μM)- nor Ca²⁺ (1mM)-induced accumulation of inositol phosphate. The site of PGJ₂ or Δ¹²PGJ₂ in inhibition of inositol phosphate accumulation would not be phospholipase C nor a putative GTP binding protein involved in activation of phospholipase C. The present results indicate that PGJ₂ and Δ¹²PGJ₂ inhibit cell growth in human astrocytoma cells and the inhibition of phosphoinositide turnover by these PGs might be involved in the inhibition of cell growth.     

Metabolic control of the K+ channel of human red cells

Summary The effects of cAMP, ATP and GTP on the Ca²⁺-dependent K⁺ channel of fresh (1–2 days) or cold-stored (28–36 days) human red cells were studied using atomic absorption flame photometry of Ca²⁺-EGTA loaded ghosts which had been resealed to monovalent cations in dextran solutions. When high-K⁺ ghosts were incubated in an isotonic Na⁺ medium, the rate constant of Ca²⁺-dependent K⁺ efflux was reduced by a half on increasing the theophylline concentration to 40mm. This effect was observed in ghosts from both fresh and stored cells, but only if they were previously loaded with ATP. The inhibition was more marked when Mg²⁺ was added together with ATP, and it was abolished by raising free Ca²⁺ to the micromolar level. Like theophylline, isobutyl methylxanthine (10mm) also affected K⁺ efflux. cAMP (0.2–0.5mm), added both internally and externally (as free salt, dibutyryl or bromide derivatives), had no significant effect on K⁺ loss when the ghost free-Ca²⁺ level was below 1 m, but it was slightly inhibitory at higher concentrations. The combined presence of cAMP (0.2mm) plus either theophylline (10mm), or isobutyl methylxanthine (0.5mm), was more effective than cAMP alone. This inhibition showed a strict requirement for ATP plus Mg²⁺ and it, was not overcome by raising internal Ca²⁺. Ghosts from stored cells seemed more sensitive than those from fresh cells, to the combined action of cAMP and methylxanthines. Loading ATP into ghosts from fresh or stored cells markedly decreased K⁺ loss. Although this effect was observed in the absence of added Mg²⁺ (0.5mm EDTA present), it was potentiated upon adding 2mm Mg²⁺. The K⁺ efflux from ATP-loaded ghosts was not altered by dithio-bis-nitrobenzoic acid (10mm) or acridine orange (100 m), while it was increased two-to fourfold by incubating with MgF₂ (10mm), or MgF₂ (10mm)+theophylline (40mm), respectively. By contrast, a marked efflux reduction was obtained by incorporating 0.5mm GTP into ATP-containing ghosts. The degree of phosphorylation obtained by incubating membranes with (-³²P)ATP under various conditions affecting K⁺ channel activity, was in direct correspondence to their effect on K⁺ efflux. The results suggest that the K⁺ channel of red cells is under complex metabolic control, via cAMP-mediated and nonmediated mechanisms, some which require ATP and presumably, involve phosphorylation of the channel proteins.     

LHRH-receptor-regulated Ca2+-ATPase activity in murine pituitary gland

Addition of luteinizing hormone releasing hormone (LHRH) in vitro (10^–5–5×10^–9 M) to murine pituitary membranes resulted in a dose-related decrease in Ca²⁺-ATPase activity within 15 min. Inhibitory effects of LHRH (10^–7 M) occurred after 90 sec, and appeared maximal by 120 sec. Eadie-Hofstee analysis at 10^–7 M LHRH, at varying [Ca²⁺]_free, resulted in aK _m=0.89±0.06 M and aV _max=18.8±0.71 nmol/mg per 2 min, compared to aK _m=0.69±0.06 M and aV _max=32.8±1.21 nmol/mg per 2 min for controls. Pre-incubation for 5 min with LHRH antagonist (10^–8 M) significantly attenuated (50%) the inhibitory effects of 10^–7 M LHRH on pituitary Ca²⁺ ATPase activity with aK _m=0.97±0.24 M and aV _max=28.1±2.8 nmol/mg per 2 min. The addition of LHRH (10^–7 M) to pituitary homogenates significantly increased luteinizing hormone (LH) release already at 10 and up to 40 sec compared to basal LH release. Systemic administration of 50 ng LHRH (i.p.), significantly (P<0.05) reduced pituitary Ca²⁺-ATPase after 30, 60 and 90 min, with a return to control levels by 120 min. Pituitary LH content was reduced slightly at 15 min, but was increased significantly at 90 and 120 min post-treatment. Plasma LH levels were elevated by 5 min, reached a peak by 15 min and returned to control within 60 min. The present findings indicate that LHRH receptor activation may influence cytosolic Ca²⁺ transport through effects on membrane Ca²⁺-ATPase activity. These actions may regulate LHRH-induced synthesis, storage and release of LH from pituitary gonadotropes.     

Termination of translation in eukaryotes is mediated by the quaternary eRF1*eRF3*GTP*Mg2+ complex. The biological roles of eRF3 and prokaryotic RF3 are profoundly distinct

GTP hydrolysis catalyzed in the ribosome by a complex of two polypeptide release factors, eRF1 and eRF3, is required for fast and efficient termination of translation in eukaryotes. Here, isothermal titration calorimetry is used for the quantitative thermodynamic characterization of eRF3 interactions with guanine nucleotides, eRF1 and Mg²⁺. We show that (i) eRF3 binds GDP (K_d = 1.9 μM) and this interaction depends only minimally on the Mg²⁺ concentration; (ii) GTP binds to eRF3 (K_d = 0.5 μM) only in the presence of eRF1 and this interaction depends on the Mg²⁺ concentration; (iii) GTP displaces GDP from the eRF1•eRF3•GDP complex, and vice versa; (iv) eRF3 in the GDP-bound form improves its ability to bind eRF1; (v) the eRF1•eRF3 complex binds GDP as efficiently as free eRF3; (vi) the eRF1•eRF3 complex is efficiently formed in the absence of GDP/GTP but requires the presence of the C-terminus of eRF1 for complex formation. Our results show that eRF1 mediates GDP/GTP displacement on eRF3. We suggest that after formation of eRF1•eRF3•GTP•Mg²⁺, this quaternary complex binds to the ribosomal pretermination complex containing P-site-bound peptidyl-tRNA and the A-site-bound stop codon. The guanine nucleotide binding properties of eRF3 and of the eRF3•eRF1 complex profoundly differ from those of prokaryotic RF3.     

Characterization of particulate cyclic nucleotide phosphodiesterases of rat kidney.

Particulate cyclic nucleotide phosphodiesterases of rat kidney display some distinct kinetic and regulatory properties. Only a small portion (5–10%) of the total homogenate low K_m cyclic AMP phosphodiesterase activity (measured with concentrations of cyclic AMP less than l μm) is tightly associated with kidney membranes. Cyclic GMP phosphodiesterase activity (measured with 0.25–200 μm cyclic GMP) is readily detectable in these fractionated and washed membranes. Low concentrations of cyclic GMP stimulated the hydrolysis of cyclic AMP (K_a ～- 0.5 μM), an effect not noted in most other membrane systems. High concentrations of cyclic GMP (K_i ～- 450 μM) and cyclic AMP (K_i ～- 150 μM) inhibited the hydrolysis of each other noncompetitively. Solubilization of membrane bound activities by sonication or Sarkosyl L markedly alters enzyme kinetic properties and the responses to cyclic nucleotides and sulfhydryl reagents. Incubation of membrane fractions with dithiothreitol (5 mm) or storage of the membranes at 4 °C results in a change in extrapolated kinetic constants for cyclic AMP hydrolysis and an increase in the rate of denaturation at 45 °C. Our findings raise the possibility that regulation of membrane-bound cyclic nucleotide phosphodiesterase activity involves interactions with cyclic nucleotides themselves, as well as oxidation and reduction of disulfide bonds and membrane-enzyme interactions.     

Development and exploitation of CK2 inhibitors

A number of quite specific and fairly potent inhibitors of protein kinase CK2, belonging to the classes of condensed polyphenolic compounds, tetrabromobenzimidazole/triazole derivatives and indoloquinazolines are available to date. The structural basis for their selectivity is provided by a hydrophobic pocket adjacent to the ATP/GTP binding site, which in CK2 is smaller than in the majority of other protein kinases due to the presence of a number of residues whose bulky side chains are generally replaced by smaller ones. Consequently a doubly substituted CK2 mutant V66A,I174A is much less sensitive than CK2 wild type to these classes of inhibitors. The most efficient inhibitors both in terms of potency and selectivity are 4,5,6,7-tetrabromo-1H-benzotriazole, TBB (K_i = 0.4 μM), the TBB derivative 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, DMAT (K_i = 0.040 μM), the emodin related coumarinic compound 8-hydroxy-4-methyl-9-nitrobenzo[g]chromen-2-one, NBC (K_i = 0.22 μM) and the indoloquinazoline derivative ([5-oxo-5,6-dihydroindolo-(1,2a)quinazolin-7-yl]acetic acid), IQA (K_i = 0.17 μM). These inhibitors are cell permeable as judged from ability to block CK2 in living cells and they have been successfully employed, either alone or in combination with CK2 mutants refractory to inhibition, to dissect signaling pathways affected by CK2 and to identify the endogenous substrates of this pleitropic kinase. By blocking CK2 these inhibitors display a remarkable pro-apoptotic efficacy on a number of tumor derived cell lines, a property which can be exploited in perspective to develop antineoplastic drugs.     

Functional expression and sites of gene transcription of a novel α subunit of the GABAA receptor in rat brain

Two α subunits of the gabaa receptor in rat brain have been identified by molecular cloning. The deduced polypeptide sequences share major characteristics with other chemically gated ion channel proteins. One polypeptide represents the rat homologue of the α3 subunit previously cloned from bovine brain [14], while the other polypeptide is a yet unknown subunit, termed α5. When coexpressed with the β1 subunit in Xenopus oocytes the receptors containing the α5 subunit revealed a higher sensitivity to GABA than receptors expressed from α1 + β1 subunits or α3 + β1 subunits (K_a = 1 μM, 13 μM and 14 μM, respectively). The α5 subunit was expressed only in a few brain areas such as cerebral cortex, hippocampal formation and olfactory bulb granular layer as shown by in situ hybridization histochemistry. Since the mRNA of the α5 subunit was colocalized with the αl and α3 subunits only in cerebral cortex and in the hippocampal formation the α5 subunit may be part of distinct GABA_A receptors in neuronal populations within the olfactory bulb.     

Effects of sulfonylureas on membrane-bound low Km cyclic AMP phosphodiesterase in rat fat cells

The effects of sulfonylureas and a biguanide on membrane-bound low K_m cyclic AMP phosphodiesterase and lipolysis were examined in rat fat cells. Pharmacologically active sulfonylureas, such as tolbutamide (10 mM), acetohexamide (10 mM) and glibenclamide (200 μM) activated the phosphodiesterase when incubated with fat cells and suppressed lipolysis induced by isoproterenol. However, neither of these actions was observed in the presence of a pharmacologically inactive sulfonylurea, carboxytolbutamide (10 mM) and a biguanide, buformin (500 μM). Tolbutamide (0.5–10 mM) activated the enzyme, concentration dependently, and this manner of activation appears to coincide with that of the suppressive effect on the lipolysis. The time course of the enzyme activation was similar to that seen with insulin. K_m, optimal pH and sensitivity to temperature of the enzyme from tolbutamide-treated cells were the same as those of the enzyme from control and insulin-treated cells. Direct incubation of the enzyme from control cells with tolbutamide did not affect the activity, while as little as 10 μM 3-isobutyl-1-methylxanthine markedly inhibited the enzyme. Tolbutamide continued to activate the enzyme in cells in which insulin receptor had been destroyed by trypsin-pretreatment. These results are compatible with the idea that the enzyme activated by sulfonylurea and that activated by insulin may be the same species of phosphodiesterase and that the antilipolytic action of sulfonylurea may be mediated by the activation of the enzyme which does not occur through the insulin receptor.     

Calmodulin-dependent high-affinity cyclic AMP phosphodiesterase in liver membranes

Plasma membranes from hamster liver were prepared by differential and continuous sucrose gradient centrifugation. The membranes contained a low K_m cyclic AMP phosphodiesterase (EC 3.5. lc) and calmodulin. The activity of the membrane phospho-diesterase was reduced with EGTA and LaCl₃. The membrane low K_m cyclic AMP phosphodiesterase was solubilized with Triton X-100 and then chromatographed on DEAE-cellulose to remove calmodulin. After elution, phosphodiesterase was stimulated with exogenous calmodulin; this activation was blocked with EGTA. Thus a low K_m cyclic AMP phosphodiesterase has been shown to be dependent on calmodulin for “maximal” activity.     

Purification and kinetic characterization of γ-aminobutyraldehyde dehydrogenase from rat liver

Oxidative deamination of putrescine, the precursor of polyamines, gives rise to γ-aminobutyraldehyde (ABAL). In this study an aldehyde dehydrogenase, active on ABAL, has been purified to electrophoretic homogeneity from rat liver cytoplasm and its kinetic behaviour investigated. The enzyme is a dimer with a subunit molecular weight of 51,000. It is NAD⁺-dependent, active only in the presence of sulphhydryl compounds and has a pH optimum in the range 7.3–8.4. Temperatures higher than 28°C promote slow activation and the process is favoured by the presence of at least one substrate. K_m for aliphatic aldehydes decreases from 110 μM for ABAL and acetaldehyde to 2–3 μM for capronaldehyde. The highest relative V-values have been observed with ABAL (100) and isobutyraldehyde (64), and the lowest with acetaldehyde (14). Affinity for NAD⁺ is affected by the aldehyde present at the active site: K_m for NAD⁺ is 70 μM with ABAL, 200 μM with isobutyraldehyde and capronaldehyde, and>800 μM with acetaldehyde. The kinetic behaviour at 37°C is quite complex; according to enzymatic models, NAD⁺ activates the enzyme (K_act 500 μM) while NADH competes for the regulatory site (K_in 70 μM). In the presence of high NAD⁺ concentrations (4 mM), ABAL promotes further activation by binding to a low-affinity regulatory site (K_act 10 mM). The data show that the enzyme is probably an E₃ aldehyde dehydrogenase, and suggest that it can effectively metabolize aldehydes arising from biogenic amines.     

α-cyano-substituted analogoues of decarboxylated S-adenosylmethionine as enzyme activated, irreversible inhibitors of S-adenosylmethionine decarboxylase

A pair of α-cyano analogues of decarboxylated S-adenosylmethionine (2a and 2b) were synthesized as potential enzyme activated, irreversible inhibitors of the[pyruvoyl enzyme S-adenosylmethionine decarboxylase (AdoMet-DC). Each of these analogues acts as an irreversible inactivator for ADoMet-DC from Escherichia coli (IC₅₀ values of 9 and 50 μM, respectively). These analogues also inactivate human AdoMet-DC, with K_I values of 246.6 and 7.2 μM, and k_inact values of 0.29 and 0.03 min⁻¹, respectively.     

Formation of adenosine 3′,5′-monophosphate from adenosine in mouse thymocytes

The effect of adenosine on the mouse thymocyte adenylate cyclase-adenosine 3′:5′-monophosphate (cyclic AMP) system was examined. Adenosine, like prostaglandin E₁, can cause 5-fold or greater increases in thymocyte cyclic AMP content in the presence but not in the absence of certain cyclic phosphodiesterase inhibitors. Two non-methylxanthine inhibitors potentiated the prostaglandin E₁ and adenosine responses, while methylxanthines selectively inhibited the adenosine response. Adenosine increased cyclic AMP content significantly wihtin 1 min and was maximal by 10 to 20 min with approx. 2 and 10 μM adenosine being minimal and half-maximal effective doses, respectively. Combinations of prostaglandin E₁, isoproterenol and adenosine were near additive and not synergistic. Of the adenosine analogues tested, only 2-chloro- and 2-fluoroadenosine significantly increased cyclic AMP. Thymocytes prelabeled with [¹⁴C] adenine exhibited dramatic increases in cyclic [¹⁴C]AMP 10 min after addition of adenosine or prostaglandin E₁ which corresponded to simultaneously determined increases in total cyclic AMP. Using [¹⁴C]adenosine, the percent of total cyclic AMP increase due to adenosine was only 16%. Adenosine was also shown to elicit a 40% increase in particulate thymocyte adenylate cyclase activity. Therefore, the increased content of cyclic AMP seen in mouse thymocytes after incubation with adenosine was due primarily to stimulation of adenylate cyclase and only partially to conversion of adenosine to cyclic AMP. The increased cellular content of cyclic AMP may be, in part, responsible for various immunosuppressive effects of adenosine.