Labeling of α‐P of Nucleoside Triphosphates by in vivo Incorporation of ³²P in Rat Liver

Abstract

R a t liver RNAs labeled in vivo by 32P and hydrolyzed by snake venom phosphodiesterase into 5'-nucleotides show a specific radioactivity always higher for AMP than that found for GMP, UMP and CMP. The difference of labeling is found on ribosomal and nuclear RNA as well as on the internal chain of tRNA.The high labding of AMP of (32P]RNA compared to the other 5'-phosphates is not due to contaminants but to the very high radioactivity of the cc-P of the ATP precursor in comparison to that of other nucleoside triphosphates, especially after a short incorporation time. The inequality of labeling decreases with time.It has been previously reported that rat liver RNA labeled in wivo by 32P and hydrolyzed by snake venom phosphodiesterase into 5'-nucleotides, shows a specific radioactivity always higher for AMP than that found for GMP, UMP and CMP [1-31. The difference of labeling is found on ribosoinal and nuclear RNR as well as on the internal chain of tRNA [2]. The meaning of these facts is not clear.I n this paper, we demonstrate that the high labeling of AMP of [32P]RNA is not due t o contaminants such as adsorbed free nucleotides or poly A, but t o the very high radioactivity of the cc-P of the ATP precursor in comparison to that of other iiucleoside triphosphates, especially after a short incorporation time. The inequality of labeling decreases with time. The consequences of these facts are discussed. MATERIAL AND METHODS AnimalsMale Wistar rats (Commentry strain) received an intraperitoneal injection of I00 pC/100 g of Na,H3'P0, (CEA, Saclay, France). They were killed by clecapitation and the liver rapidly removed and chilled at 0". All subsequent operations were performed a t 4". Cell Fractionation and R N A ExtractionA 2001, homogenate (w/v) in 0.25 M sucrose was centrifuged 2 hours at 40000 rev./min (Spinco, rotor 40); the upper three quarters of the supernatant were withdrawn by automatic aspiration.Enzyme. Phosphodiesterase or orthophosphoric diester phosphohydrolase (EC 3.1.4.1 ).They correspond t o the soluble phase of liver and will be used for extraction of soluble RNA.A loo/, homogenate was centrifuged 10 min a t 8500 x g. The postmitochondrial supernatant was carefully removed. One tenth of its volume of loo/, sodium deoxycholate wm added and the suspension was mixed in a loose-fitted homogenizer (teflon-glass) and centrifuged 130 min at 50000 rev./min (Spinco, rotor 50). The supernatant was removed and the tubes were drained and cut just above the pellet of ribosomes.The nuclei were isolated in 2.2 M sucrose containing 0.1 mM MgC1, according the method of Chauveau, Mouk and Rouiller [4].Extraction of soluble RNA from the liver supernatant has been described previously [2]. Ribosomal RNA was extracted from pelleted ribosomes with sodium dodecyl sulfate and phenol in presence of 1 01, hydroxyquinoline and 0.1 01, naphthalene disulfonate [ S ] . The RNAs were purified by four successive precipitations. Degradation by Snake Venom Phosphodiesteraseand Isolation of 5'-Nucleotides The degradation was carr...

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