Abstract
The cycle of protein-carboxyl methylation and demethylation was studied in intact blood platelets. Platelets rapidly incorporated L-[methyl-3H]methionine and after a delay of about 20 min, they evolved [3H]methanol. This evolution, and the amount of [3H] methanol liberated by treatment with base, was inhibited in a dose-dependent fashion by the cyclic nucleotide phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine, papaverine, dipyridamole, and RA233 (2,6-bis(diethanolamino)-4-piperidinopyrimido[5,4-d] pyrimidine). Each of these compounds increased the incorporation of [3H]methionine into platelets. The effects of RA233 were studied in more detail. Inhibition of [3H]methanol production was not potentiated by stimulators of the adenylate cyclase or the guanylate cyclase. The majority of the base-labile radioactivity was trichloroacetic acid precipitable. Thin layer chromatography of extracts of platelets incubated with L-[35S]methionine showed that RA233 did not induce a cellular accumulation of [35S]S-adenosylhomocysteine, and that it actually increased the amount of cellular [35S]S-adenosylmethionine. Discontinuous polyacrylamide gel electrophoresis at acid pH using the cationic detergent benzyldimethyl-n-hexadecylammonium chloride of platelets incubated with [3H]methionine showed incorporation of radioactivity into more than 30 protein bands, including one which co-migrates with calmodulin. The incorporation into the majority of these bands was inhibited by RA233 in a dose-dependent fashion. It is suggested that caution should be used in ascribing the pharmacological effects of known phosphodiesterase inhibitors to increases in cyclic nucleotides, because some of these effects could be due to inhibition of protein carboxyl methylation.
Highlights
The cycle of protein-carboxyl methylation and de- a mechanism not involving the accumulation of cyclic nucleomethylation was studied in intact blood platelets. tides
Inpreliminaryexperiments, we confirmed that washed platelets rapidly concentrate radioactivity when they are incubated with [methyl-’HH]methionine,and this radioactivity subsequently appears in S-adenosylmethionine, protein, and a volatileproduct witha distillation profile identical withthat
DMTA)/SAH was 0.39 inthecontrolplatelets,and platelets treated with RA233
Summary
Therunning gel was diluted with 20 ml of 1:l (v/v) methanol/water This was subcontained acrylamide (7.5%, w/v), N,N’-methylenebisacrylamide jected to fractional distillation, and the’H and 14C of each fraction, (0.26%, w/v), urea (15% w/v), ferrous sulfate (7.7 p ~ )a,nd ascorbic and of the residue left in the boiling flask (about 1 ml) were deteracid (0.43 mM) inpotassiumphosphate buffer(150 mM), pH 2.0. The gel was fixed in two changes of methanol/acetic acid/water (4:1:5) stained withCoomassie blue, destained, hydrated withtwo changes of distilled water, and impregnated with M sodiumsalicylate containing 100 mM acetic acid and 1%(v/v) glycerol It was dried onto filter paper without applied heating using a rotary vacuum pump protected with a trap and exposed to Kodak x-ray film a t -80 “C for 1 week. Tlme of lncubatlon (rnln) prostacyclin was a gift from the samecompany
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