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S J McNall

Publications and source records attributed to S J McNall.

7 recordsLinked to original sources
PubMed1988

Phosphorylase phosphatase. Comparison of active forms using peptide substrates.

Phosphorylase phosphatase isolated from rabbit skeletal muscle can be activated in several ways. Trypsin-Mn2+ treatment of the purified Mr = 70,000 complex or addition of Mn2+ alone to the isolated inactive catalytic subunit gives enzyme species that readily dephosphorylate phosphorylase a and the type 2 regulatory subunit of cAMP-dependent protein kinase as well as synthetic phosphopeptides corresponding to the phosphorylation sites of these proteins. In contrast, enzyme activated by phosphorylation of the regulatory subunit using factor FA (glycogen synthase kinase 3) and Mg2+-ATP and thought to be of physiological significance dephosphorylates the protein substrates but not the phosphopeptides. Likewise, the active catalytic subunit isolated following FA treatment could not act on the peptides unless Mn2+ ions (maximal effect at 250 microM) were added. Mg2+ and Ca2+ could not substitute for Mn2+. Such differences in substrate specificity are not seen with p-nitrophenyl phosphate which is dephosphorylated by all forms of the phosphatase. The results suggest that the primary sequence surrounding the phosphorylation site of the substrate is not all that is necessary for recognition by the FA-activated form of the enzyme. They are interpreted in terms of constraints within the enzyme that are relaxed following exposure to Mn2+ or by the additional determinants present in larger protein substrates.

Animals↗
PubMed1988

Stimulation of protein phosphatase activity by insulin and growth factors in 3T3 cells.

Incubation of Swiss mouse 3T3-D1 cells with physiological concentrations of insulin resulted in a rapid and transient activation of protein phosphatase activity as measured by using [32P]phosphorylase a as substrate. Activation reached a maximum level (140% of control value) within 5 min of addition and returned to control levels within 20 min. The effect of insulin was dose-dependent with half-maximal activation occurring at approximately 5 nM insulin. This activity could be completely inhibited by addition of the heat-stable protein inhibitor 2, which suggests the presence of an activated type-1 phosphatase. Similar effects on phosphatase activity were seen when epidermal growth factor and platelet-derived growth factor were tested. These results suggest that some of the intracellular effects caused by insulin and growth factors are mediated through the activation of a protein phosphatase.

Animals↗
PubMed1985

Forskolin activation of serotonin-stimulated adenylate cyclase in the liver fluke Fasciola hepatica.

Properties of forskolin activation of adenylate cyclase in the liver fluke Fasciola hepatica are described. Forskolin stimulated adenylate cyclase activity in cell-free fluke particles to levels more than 30-fold above the basal rate. This activation was not dependent on guanine nucleotides and, upon washing of the particles, was rapidly reversed. Forskolin potentiated the activation of adenylate cyclase by serotonin (5-HT) and lysergic acid diethylamide (LSD), resulting in both an increase in the maximal level of enzyme activity and a decrease in the apparent activation constant (KA). The 5-HT antagonist 2-bromo-LSD did not inhibit enzyme activation by forskolin. Furthermore, forskolin had no effect on specific [3H]LSD binding to fluke particles. Activation of adenylate cyclase by sodium fluoride or guanine nucleotides was modified in a complex manner by forskolin with both stimulatory and inhibitory effects present. The results suggest that forskolin does not interact directly with the 5-HT receptor coupled to adenylate cyclase. Instead, it appears that forskolin effects are, at least in part, due to its ability to alter the interaction between the regulatory and catalytic components of adenylate cyclase. Incubation of intact flukes with forskolin increased their cAMP levels 2- to 3-fold. The concentration dependence of this response was similar to that for forskolin activation of adenylate cyclase in fluke particles, with 300 microM forskolin giving the maximum response. Forskolin and other agents that increased fluke cAMP levels also stimulated fluke motility.

Adenylyl Cyclases↗
PubMed1984

Novel serotonin receptors in Fasciola. Characterization by studies on adenylate cyclase activation and [3H]LSD binding.

Serotonin (5-HT) receptors coupled to adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in the liver fluke Fasciola hepatica have been characterized by adenylate cyclase activation studies and by direct binding studies using [3H]-d-lysergic acid diethylamide ([3H]LSD) as a radioligand. Inhibition of 5-HT stimulation of adenylate cyclase by a series of 5-HT antagonists revealed a potency order of LSD = 2-bromo-LSD greater than methiothepin greater than metergoline = cyproheptadine greater than methysergide greater than spiroperidol. [3H]LSD binding to a cell-free fluke particle preparation was rapid, stereospecific, and proportional to protein concentration. Scatchard analysis indicated multiple binding sites which, when resolved into two components, gave for the high affinity site an apparent dissociation constant of 25 nM and a receptor concentration of 160 fmoles/mg protein. The ability of a series of compounds to compete for [3H]LSD binding sites correlated closely with their ability to inhibit 5-HT stimulation of adenylate cyclase. [3H]LSD binding sites were most concentrated in the anterior region of the fluke which was consistent with the higher levels of 5-HT activated adenylate cyclase found in this region. GTP and 5'-guanylyl imidophosphate, a poorly hydrolyzable GTP analog, decreased the affinity of the agonist 5-HT for the binding sites but had little effect on the affinity of the antagonist 2-bromo-LSD. Calcium at concentrations above 300 microM significantly reduced both [3H]LSD binding and 5-HT activation of adenylate cyclase. The results indicate that [3H]LSD can be used to label the 5-HT receptors coupled to adenylate cyclase activity. The pharmacological specificity and other characteristics of the fluke receptors appear to differ from the properties of reported mammalian 5-HT receptors. As a result, serotonin receptors in the fluke represent sites that may be amenable to selective manipulation by new chemotherapeutic agents useful in the treatment of these parasite infections.

Adenylyl Cyclases↗
PubMed1984

Desensitization of serotonin-stimulated adenylate cyclase in the liver fluke Fasciola hepatica.

Incubation of the intact liver fluke Fasciola hepatica with serotonin (5-HT) resulted in a time-dependent decrease in both 5-HT-stimulated adenylated cyclase activity and specific [3H]LSD binding in the subsequently prepared cell-free fluke particles. In control fluke particles, the activation of adenylate cyclase by 5-HT was biphasic, indicating a high and low affinity form of the 5-HT receptor with half-maximal activation constants (KA) of 0.35 and 2.8 microM respectively. In contrast, 5-HT activation of desensitized particles occurred through a single set of low affinity sites having a KA value of 6.3 microM. The maximal level of 5-HT activation of adenylate cyclase was also reduced in the desensitized particles. Lysergic acid diethylamide (LSD)-stimulated adenylate cyclase activity was also less in the desensitized particles. However, unlike with 5-HT, activation by LSD occurred through a single set of sites for both control and desensitized particles. [3H]LSD binding studies showed that the affinity of LSD for the 5-HT receptors in the control and desensitized particles was similar. Thus, the decrease in [3H]LSD binding and serotonin-stimulated adenylate cyclase activity in the desensitized particles appeared to be due to a preferential loss or inactivation of the high affinity form of the 5-HT receptor. A similar time-dependent loss in 5-HT-stimulated adenylate cyclase and in [3H]LSD binding occurred in cell-free fluke particles upon the addition of 5-HT or LSD. These effects were not due to protein denaturation or metabolism of the ligand during the incubation procedure. This cell-free desensitization was reversible and temperature dependent, and was not affected by ATP or other nucleotides.

Adenosine Triphosphate↗
PubMed1983

Photoreactivity of lysergic acid diethylamide and its possible utility as a photoaffinity labeling reagent.

Aqueous solutions of lysergic acid diethylamide (LSD) are extremely sensitive to light in the near-ultraviolet region of the spectrum. This rather efficient photoreaction yields a variety of products which have very low affinity for LSD-binding sites on plasma membranes from Fasciola hepatica. Since this photoreaction may be elicited by normal white fluorescent lighting in the laboratory, it represents a potential source of error in determining the binding affinity of LSD. Utilizing this photoreactivity advantageously, [3H]LSD was used to photolabel membrane proteins. Covalent binding of [3H]LSD was shown to be a function of the duration of illumination and was inhibited by 5-hydroxytryptamine and nonradioactive LSD. Sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis of [3H]LSD labeled membranes from F. hepatica showed two proteins which were selectively labeled by the photoreactive [3H]LSD. This method of direct photolabeling with non-derivatized [3H]LSD may allow identification of LSD-binding proteins in a variety of systems.

Affinity Labels↗