Biomedical subjects
Publications and source records attributed to H N Torres.
Electrofusion of rat adrenal and Leydig cells generated hybrids capable of synthesizing simultaneously both testosterone and corticosterone, under stimulation of lutropin or adrenocorticotropin. Evidence was obtained indicating that under such circumstances, heterologous lutropin receptor--adrenal adenylate cyclase complexes were formed.
The soluble form of adenylate cyclase was extracted and purified from wild-type Neurospora crassa mycelia. Brain or N. crassa calmodulin significantly enhanced this enzyme activity in assay mixtures containing Mg2+-ATP as substrate. EGTA reverses this calmodulin activation.
Adenylate cyclase activity associated to wild type Neurospora membranes is highly dependent on Mn2+ and insensitive to fluoride, guanyl nucleotides, and cholera toxin. These membranes are able to interact with components of detergent extracts from turkey erythrocyte ghosts. The reconstituted cyclase system is catalytically active in the presence of Mg2+ and it is activated by guanyl-5'-yl imidodiphosphate plus isoproterenol and fluoride. When detergent extracts were prepared from avian erythrocyte membranes treated with cholera toxin, the reconstituted system was stimulated by guanyl-5'-yl imidodiphosphate in the absence of isoproterenol and cyclase activities were higher than those observed with extracts from membranes not treated with the toxin. Dose-response curves for isoproterenol and fluoride in the reconstituted system were similar to those reported for avian erythrocyte and liver membranes, respectively.
A monoclonal antibody against Neurospora soluble adenylate cyclase was obtained. The antibody inhibits cyclase activities from several lower eucaryotic organisms but not activities associated to testicular cytosol or turkey erythrocyte membranes.
A soluble form of adenylate cyclase was extracted from mycelia of Neurospora crassa wild-type strains. This enzyme activity was purified by chromatography on hexyl-amino-Sepharose, agarose and Blue Sepharose and preparative polyacrylamide-gel electrophoresis. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, peak fractions from the later purification steps showed a main polypeptide band with an apparent molecular weight of about 66 000. The following hydrodynamic and molecular parameters were established for the Neurospora soluble adenylate cyclase activity: sedimentation coefficient, 6.25 S; Stokes radius, 7.3 nm; partial specific volume, 0.74 ml/g; molecular weight, 202 000; frictional ratio, 1.65. The isoelectric point of this enzyme activity was 4.65. The enzyme was not activated by GTP, [beta gamma-imido]GTP, fluoride or cholera toxin.
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Cyclic nucleotide phosphodiesterase activities in soluble Neurospora crassa mycelial extracts were resolved into two peaks, phosphodiesterase I and II, by chromatography on DEAE-cellulose columns. Phosphodiesterase I hydrolysed cyclic AMP and cyclic GMP equally well. Phosphodiesterase II was active on cyclic GMP but scarcely active on cyclic AMP. Phosphodiesterase I was resolved by gel filtration and sucrose-density-gradient centrifugation into three peaks having molecular weights of about 57 000, 125 000 and 225 000. This suggests that this enzyme activity has at least three aggregation forms, tentatively defined as monomeric, dimeric and tetrameric. Similarly, phosphodiesterase II was resolved into two forms, having molecular weights of about 170 000 and 320 000. Evidence on the interconversion between phosphodiesterase I forms was obtained.
Erythrocyte soluble phosphodiesterase activities show at least two interconvertible states: "aggregated' and "non-aggregated'. In the former state the existence of a high affinity component for cyclic AMP is favoured and the system is more active with cyclic AMP than with cyclic GMP as substrate. When the system is in the "non-aggregated state', no activity with cyclic GMP is detected. Conversion to the "non-aggregated state' is enhanced by dilution or by addition of magnesium ions. Upon isoelectric focusing of erythrocyte soluble extracts. Phosphodiesterase activities can be resolved in two peaks: one specific for cyclic AMP located at pH 4.66 and the other specific for cyclic GMP focused at pH 4.95. Evidence would indicate that aggregation affects the enzyme specific for cyclic AMP.
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Cytosolic adenylate cyclase activity from rat seminiferous tubules was purified by chromtography in DEAE-cellulose, hydroxylapatite and Bio-Gel A-0.5 m as well as by centrifugation in sucrose gradients. In all these purification steps, fractions with adenylate cyclase activity also contained binding activity for L-T3. Binding studies indicate the existence of two L-T3 receptor components associated to adenylate cyclase activity. The component exhibiting the highest hormone affinity has the lowest binding capacity.
Testicular, soluble adenylate cyclase has been purified by anion-exchange chromatography, gel filtration, and isoelectric focusing. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peak fractions from the latter purification step showed only one polypeptide band with an apparent molecular weight of about 69 000. The following hydrodynamic and molecular parameters have been established for this enzyme: sedimentation constant, 4.3; Stokes radius, 3.95 nm; partial specific volume, 0.74 mL.g(-1); molecular weight, 74 000; fractional ratio, 1.4.
Cytosolic adenylate cyclase activity from rat seminiferous tubules is inhibited by L-triiodothyronine (L-T3). In a typical dose-response curve, using Mn-ATP as substrate, no effect is observed at 10(-10) M L-T3; about 15 to 25% inhibition is found in the range between 10(-9) and 10(-6) M L-T3 and finally a sharp enzyme inhibition is evident at increasing hormone concentrations from 10(-6) to 10(-4) M. Incubation of decapsulated testes with L-T3 leads to a decrease of intracellular cyclic AMP levels. Dose-response relationships for such effect are similar to those found for adenylate cyclase activity. In this case a clear response is observed at 10(-8) M L-T3.
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Insulin decreased markedly the adenylyl cyclase activity associated with fat cell membranes purified by centrifugation in sucrose gradients. The hormone effect was not readily evident in crude membrane preparations. The kinetics of this effect indicate that some time was required for the onset of the insulin-induced inactivation. This lag period decreased when the insulin concentration was increased. The hormone dose dependence for adenylyl cyclase inactivation measured at a fixed time (3 min) showed a 10 to 15% decrease in activity at 1 to 30 muU per ml insulin; 30 to 40% at 100 to 1000 muU per ml; and 75% at 0.1 U per ml. The insulin effect was completely abolished by 0.1 mM GMP-P(NH)P, 10mM fluoride, or 50 ng per ml glucagon, or by increasing the Mn++ concentration to 4 mM. In addition, it was partially reversed by the addition of a fraction from the sucrose gradient, which contained soluble factors. The kinetics of the adenyl cyclase-catalyzed reaction were studied using ATP or AMP-P(NH)P as adenylyl donor, and Mn++ or Mg++ as divalent cation, in the absence or presence of insulin. With ATP and Mg++ there was a striking reduction of the transient reaction rates after 1.5 min of incubation. Under these conditions the insulin effect was not evident. On the contrary, with ATP and Mn++ this spontaneous reduction of activity was less evident; however, in the presence of insulin there was a clear and marked reduction of the transient reaction rate measured after 1.5 min of incubation. With AMP-P(NH)P the kinetic data were qualitatively similar to those observed with ATP. It is concluded that under certain assay conditions adenylyl cyclase may be converted to an inactive enzyme form, and that such a conversion is more evident in the presence of Mg++ than with Mn++. In the latter case, insulin appears to enhance the rate of this conversion.
The kinetic behavior of the adenylyl cyclase activity associated with fat cell membranes purified by centrifugation on sucrose gradients was studied. Under most of the conditions explored, with either Mn++ or Mg++ as the divalent cation in the assay mixtures, the time courses of the reaction were not linear. In the absence of modifiers (i.e., basal activity) or in the presence of insulin, the rate tended to decrease with time; on the other hand, with fluoride or GMP-P(NH)P the curves were concave upwards. To simplify analysis of the results, two kinetic components were defined: an "initial component" corresponding to the transient rate measured between zero time and 1.5 min of assay and a "final component" corresponding to the transient rate determined between 3 and 5 min. Over the entire range of Mn++ concentration explored (0.5 to 6.0 mM), the basal initial rates were slightly higher than the final ones. With Mg++ in the range between 1.5 and 2.5 mM, the final rates were fourfold lower than the initial ones. Higher or lower Mg++ concentrations gave velocity ratios equivalent to those observed with Mn++. Insulin clearly decreased the final rates at Mn++ concentrations up to 2.5 mM. With higher concentrations the effects were completely reversed. The effects of insulin on initial rates measured with Mn++, or the initial or final rates measured with Mg++, were less evident. Stimulation of adenylyl cyclase activity by fluoride was most pronounced on the final rates. In addition, this stimulation was higher with Mg++ than with Mn++. Isoproterenol stimulation of adenylyl cyclase was negligible in the presence of Mn++ (0.5 to 6.0 mM). With Mg++ (0.5 to 6.0 mM), stimulation was more evident on the final rates. GMP-P(NH)P inhibited the initial but activated the final components of the reaction. The extent of this inhibition or activation was more pronounced with Mg++ than with Mn++. Under conditions which lead to maximal inactivation of the final component, adenylyl cyclase activity was tenfold or more higher with Mn++ than with Mg++. Similar effects were observed with GMP-P(NH)P on the initial component. However, insulin, isoproterenol and fluoride decreased the Mn++ dependence of the final component. With fluoride, the final rates measured with Mg++ were almost equivalent to those found in assays containing Mn++. Under the conditions used for measurements of adenylyl cyclase activity, the enzyme system slowly interconverts between active and inactive forms.