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Degradation of gastrin by isolated pig hepatocytes
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DEGRADATION OF GASTRIN BY ISOLATED PIG HEPATOCYTES L.A. Christiansen & S. Gammeltoft Dept. of Surgical Gastroenterology, Rigshospitalet and Dept. of Clinical Chemistry, Bispebjerg Hospital, Copenhagen, Denmark Hepatocytes were isolated from pig liver by the collagenase method and incubated at 37°C in Krebs-Ringer-bicarbonate buffer, pH=7.4 containing l0 mg/ml albumin. Gastrin concentrations were measured by radioimmunoassay using antibody 2604, which measures the four main components with almost equimolar potency. Hepatocytes (2 x 106 ceZls/ml) were incubated with synthetic human gastrin-14, -17 and -34. After 60 minutes 65% of G-14, 30% of G-17 and 0-15% of G-34 immunoactivity had disappeared. The disappearance rate was exponential, concentration- and temperature dependent, suggesting an enzymatic process. The degrading activity was associated with the cells and was not released into the medium during incubation. Incubation of hepatocytes with 125-I G-17 resulted in decreasing immunoreactivity in the supernatant and gelchromatography (Sephadex G-25 superfine) showed conversion to bigger and smaller gastrin components as well as to 125-I labelled iodide and tyrosine. 125-I labelled G-17 and degradation products were not accumulated in the cells and no specific receptor binding could be demonstrated. The presence of gastrin-degrading activity in isolated hepatocytes supports the hypothesis that the liver participates in the degradation of gastrin in pigs.
ACTION OF CCK-8 ON KINETIC PARAMETERSOF PANCREATICADENYLATE CYCLASE J Christophe and [q Svoboda, Depart.~lent of 3iochemistry, Universit6 l i b r e de Druxelles, Drussels, Selgium. Pancreatic adenylate cyclase is a multicomponent enzyme in dynamic e q u i l i b r i u m between i n a c t i v e and active states. The a c t i v e state is induced by the occupancy o f a s p e c i f i c regulatory s i t e by a guanyl nucleeside triphosphate such as GTP or p[I~H]ppG. Deactivation is observed a f t e r hydrolysis of GTP by a GTPase or a f t e r the release o f p[IIH]ppG (,which is GTPase-resistant). lie tested the bearing on k i n e t i c parameters of the 100 kJ/,mol reduction, provoked by CCK-8, of the a c t i v a t i o n energy of the nucleotide a c t i v a t i o n process. The kinet i c constants of p[IJH]ppG and GTP a c t i v a t i o n and d e a c t i v a t i o n rates were estimated with a two-step incubation n~thod based on the use of GDPBS as i n h i b i t o r of nucleotide a c t i v a t i o n and of d i b u t y r y l c y c l i c GFP (3t2-cGMP) as antagonist of CCK-8 action. CCK-8 ~as shown to increase the p[HH]ppG activat i o n rate (kon) of pancreatic adenylate cyclase dose-dependently : the value of kon was 0.1-0.2 min- ;Jithout CCK-8, i t increased two-fold with I nM CCK-8, and became superior to I min-l with CCK-8 concentrations above 10 nM. The d e a c t i v a t i o n rate ( k o f f ) of GTP + CCK-3-activated adenylate cyclase was the same (6 rain-l) in the presence of GDPBS alone or of ODPBS + the CCK-8 antagonist 3t2-cGMP, suggesting t h a t CCK-3 exerted no e f f e c t on t h i s r a t e . The k o f f with Bt2-cG~ alone was lower (3 rain-l) and might r e f l e c t the rate of d i s s o c i a t i o n of CCK-8 provoked by 3t2-cGMP,when the l a t t e r was added to the adenylate cyclase assay medium. In conclusion, the g a s t r o i n t e s t i n a l hormone CCK-8, by lowering the a c t i v a t i o n energy of the a c t i v a t i o n process, increased the a c t i v a t i o n rate of the enzyme without decreasing i t s d e a c t i v a t i o n r a t e . There resulted an increased proportion of GTP-activated enzyme.
DEGRADATION OF GASTRIN BY ISOLATED PIG HEPATOCYTES L.A. Christiansen & S. Gammeltoft Dept. of Surgical Gastroenterology, Rigshospitalet and Dept. of Clinical Chemistry, Bispebjerg Hospital, Copenhagen, Denmark Hepatocytes were isolated from pig liver by the collagenase method and incubated at 37°C in Krebs-Ringer-bicarbonate buffer, pH=7.4 containing l0 mg/ml albumin. Gastrin concentrations were measured by radioimmunoassay using antibody 2604, which measures the four main components with almost equimolar potency. Hepatocytes (2 x 106 ceZls/ml) were incubated with synthetic human gastrin-14, -17 and -34. After 60 minutes 65% of G-14, 30% of G-17 and 0-15% of G-34 immunoactivity had disappeared. The disappearance rate was exponential, concentration- and temperature dependent, suggesting an enzymatic process. The degrading activity was associated with the cells and was not released into the medium during incubation. Incubation of hepatocytes with 125-I G-17 resulted in decreasing immunoreactivity in the supernatant and gelchromatography (Sephadex G-25 superfine) showed conversion to bigger and smaller gastrin components as well as to 125-I labelled iodide and tyrosine. 125-I labelled G-17 and degradation products were not accumulated in the cells and no specific receptor binding could be demonstrated. The presence of gastrin-degrading activity in isolated hepatocytes supports the hypothesis that the liver participates in the degradation of gastrin in pigs.
ACTION OF CCK-8 ON KINETIC PARAMETERSOF PANCREATICADENYLATE CYCLASE J Christophe and [q Svoboda, Depart.~lent of 3iochemistry, Universit6 l i b r e de Druxelles, Drussels, Selgium. Pancreatic adenylate cyclase is a multicomponent enzyme in dynamic e q u i l i b r i u m between i n a c t i v e and active states. The a c t i v e state is induced by the occupancy o f a s p e c i f i c regulatory s i t e by a guanyl nucleeside triphosphate such as GTP or p[I~H]ppG. Deactivation is observed a f t e r hydrolysis of GTP by a GTPase or a f t e r the release o f p[IIH]ppG (,which is GTPase-resistant). lie tested the bearing on k i n e t i c parameters of the 100 kJ/,mol reduction, provoked by CCK-8, of the a c t i v a t i o n energy of the nucleotide a c t i v a t i o n process. The kinet i c constants of p[IJH]ppG and GTP a c t i v a t i o n and d e a c t i v a t i o n rates were estimated with a two-step incubation n~thod based on the use of GDPBS as i n h i b i t o r of nucleotide a c t i v a t i o n and of d i b u t y r y l c y c l i c GFP (3t2-cGMP) as antagonist of CCK-8 action. CCK-8 ~as shown to increase the p[HH]ppG activat i o n rate (kon) of pancreatic adenylate cyclase dose-dependently : the value of kon was 0.1-0.2 min- ;Jithout CCK-8, i t increased two-fold with I nM CCK-8, and became superior to I min-l with CCK-8 concentrations above 10 nM. The d e a c t i v a t i o n rate ( k o f f ) of GTP + CCK-3-activated adenylate cyclase was the same (6 rain-l) in the presence of GDPBS alone or of ODPBS + the CCK-8 antagonist 3t2-cGMP, suggesting t h a t CCK-3 exerted no e f f e c t on t h i s r a t e . The k o f f with Bt2-cG~ alone was lower (3 rain-l) and might r e f l e c t the rate of d i s s o c i a t i o n of CCK-8 provoked by 3t2-cGMP,when the l a t t e r was added to the adenylate cyclase assay medium. In conclusion, the g a s t r o i n t e s t i n a l hormone CCK-8, by lowering the a c t i v a t i o n energy of the a c t i v a t i o n process, increased the a c t i v a t i o n rate of the enzyme without decreasing i t s d e a c t i v a t i o n r a t e . There resulted an increased proportion of GTP-activated enzyme.