Biomedical subjects
Publications and source records attributed to M Svoboda.
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(1) The kinetic parameters of rat pancreatic adenylate cyclase were evaluated, using GTP, p[NH]ppG or GTP gamma S as nucleotide activator, cholecystokinin as peptide hormone, and GDP beta S and dibutyryl cyclic GMP as inhibitors of guanosine triphosphate and CCK-8, respectively. The time courses of activation and the degree of activation at steady state (EA/ETOT) were compatible with a simple two-state model of activation-deactivation based on a pseudo-monomolecular activation process (rate constant kappa+1), and a deactivation process (rate constant kappa off) that included, depending on the activating nucleotide, the hydrolysis of GTP (rate constant kappa 2) and/or the dissociation of the intact nucleotide (rate constant kappa-1), so that EA/ETOT = kappa+1/(kappa+1 + kappa 2 + kappa-1). (2) The hormone CCK-8 increased the value of kappa+1 with GTP dose-dependently, from 0.2 to 10.9 min-1. The value of kappa-1 increased 0.01 to 0.3 min-1 but the value of kappa 2 was unaltered at 7 min-1, so that EA/ETOT increased 15-fold, from 4% to 61%. (3) A cholera toxin pretreatment at 30 micrograms/ml allowed also a large increase in EA/ETOT with GTP (up to 51%) but the underlying mechanism was different. It consisted of a 14-fold decrease in the kappa off value of the GTP-activated enzyme (from 7 min-1 to 0.5 min-1) that corresponded to a reduction in GTPase activity. When testing the system with p[NH]ppG, two added effects of the cholera toxin pretreatment were observed: a 4-fold increase in the value of kappa+1 (from 0.2 to 0.8 min-1) and the occurrence of a significant 0.3 min-1 value for kappa-1.
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The toxicity of the rodenticidal product Pyriminyl (Vacor) was tested in 4 dogs, 9 cats, 11 coypus and 16 ducks. In the tested dogs the 1000mg dose per kg l. w. was lethal only in 17% of cases. The results demonstrate that the LD50 for the dog is higher than stated in the literature (1000 mg/kg l. w.). Dogs show a high variability in their sensitivity to the product, since some of them died at lower doses. It can be assumed that 1 to 2% field bait will not induce any lethal intoxication in dogs. Doses of 300 mg/kg l. w. and higher were lethal for cats. The dose of 200 mg/kg killed 66% of the cats tested. There is a high possibility of poisoning cats in field deratization campaigns with Vacor. Coypus show clinical symptoms of intoxication after ingesting a Vacor bait at a 100mg dose per kg l. w.; however, they remain alive. Doses of 300 mg/kg l. w. and higher kill all the animals tested. The high sensitivity of coypu to Vacor suggests that coypu can be poisoned during Vacor deratization of coypu farms. Ducks were highly resistant to Vacor. Doses from 710 to 3000 mg per kg l. w. were endured without clinical changes, and the same was the case when the birds were given a diet with a 2% concentration of Vacor for six days. The toxicological examination demonstrated the different amounts of Vacor in liver for different periods of time from the ingestion of the toxic agent, including the dependence on the animal species.
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The activation energy of adenylate cyclase by p[NH]ppG in rat pancreatic plasma membranes was estimated to be 141-189 kj/mol. When a high concentration of secretin or CCK-8 (C-terminal octopeptide of cholecystokinin-pancreozimin) was added to the assay medium, the activation energy was reduced to 73 kj/mol. This hormone effect was exerted on the activation energy of the activation process of adenylate cyclase by p[NH]ppG. Indeed, when plasma membranes were preactivated with p[NH]ppG alone or with p[NH]ppG and CCK-8 and then washed, there resulted a persistent activation with low activation energy (65 and 48 kj/mol, respectively). A similar low activation energy was observed in membranes preincubated with GMP and CCK-8. The latter treatment could not induce persistent activation but facilitated the activation by p[NH]ppG, suggesting that the step of p[NP]ppG activation requiring a high activation energy in the absence of hormone had developed during preincubation with GMP and CCK-8, and had not been reversed by membrane washing. By contrast, EDTA pretreatment did not influence p[NH]ppG activation while provoking a reversible deactivation of persistently activated adenylate cyclase.
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1. The activation of rat pancreatic adenylate cyclase by guanosine 5'-(beta-gamma-imido)triphosphate (p[NH]ppG) and GTP, and by the two gastrointestinal hormones pancreozymin (as C-terminal octapeptide) and secretin was correlated with the binding of [8-3H]guanosine 5'-(beta-gamma-imido)triphosphate to rat pancreatic plasma membranes. 2. The low basal adenylate cyclase activity was stimulated 17-fold by p[NH]ppG (after a 2 min lag period), 3,5-fold only by GTP, 21-fold by C-terminal octapeptide of pancreozymin, and 8-fold by secretin. GTP inhibited competitively the activation of adenylate cyclase by p[NH]ppG with a Ki,app almost identical with the Ka,app (0.3 micron). p[NH]ppG and GTP enhanced the stimulation by secretin more markedly than that by the C-terminal octapeptide of pancreozymin, leading to the same maximal activity. Both hormones suppressed the lag period of activation by p[NH]ppG. 3. The binding of [8-3H]p[NH]ppG was dependent on time, temperature and Mg2+ and it was also a saturable and reversible process. Scatchard plots with a concavity upward were linearized after co-addition of ATP, Mg2+ and an ATP-regenerating system that abolished low-affinity sites for p[NH]ppG without saturating higher affinity sites, GTP, ITP and UTP inhibited [8-3H]p[NH]ppG binding to the high-affinity sites in concentration ranges identical with those found for adenylate cyclase activation. Considerable binding of [8-3H]p[NH]ppG was still evident at 20 degrees C, but enzyme activation was not observed any more, except in the presence of hormones.
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1. The subcellular distribution of adenylate cyclase activity in rat pancreatic homogenates was examined after differential centrifugation. Divalent cations exerted significant effects on this distribution. In addition, the ratio of adenylate cyclase activities in the presence of the C-terminal octapeptide of cholecystokinin-pancreozymin and secretin was lower in the crude 'mitochondrial' fraction than in 'microsomal' fractions. This difference was due to the lability of cholecystokinin-pancreozymin receptors compared to secretin receptors. The Km,app of activation was affected more than the V by this lability. Such a degradation of cholecystokinin-pancreozymin receptors was markedly delayed by isolation and storage in the presence of a phospholipid mixture. 2. A simple, reasonably rapid (6 h), and easily reproducible method was developed to prepare a stable semi-purified plasma membrane fraction, characterized by a 10-fold increase in the specific activity of adenylate cyclase with respect to the whole homogenate. At variance with data obtained on crude subcellular fractions, the V of adenylate cyclase activity observed in this preparation, under maximal concentration of the C-terminal octapeptide of cholecystokinin-pancreozymin, was higher than that obtained with secretin or the vasoactive intestinal polypeptide.
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