Search

1. Guanine deaminase activities in homogenates and supernatant fractions of liver and brain of rat and mouse were elevated by administration of guanine to the animals. The maximum induction in mouse tissues occurred within 24h and in rat tissues within 48h. 2. Mitochondria of rat (but not mouse) liver and brain contain an inhibitor of supernatant guanine deaminase, and this was also increased by guanine treatment. 3. Administration of ethionine, cycloheximide or actinomycin D prevented the guanine-dependent increase in deaminase activity and also the increase in mitochondrial inhibitory activity; chloramphenicol suppressed only the latter.

The nucleotide composition of 4s RNA from livers of rats fed with a diet containing 0.3% D-ethionine was found to be identical with that from untreated animals. In contrast, one single modified nucleotide was absent in 4s RNA from livers of rats fed with a 0.3% L-ethionine diet. The minor nucleo=tide was also absent in liver 4s RNA from rats fed with a 0.3% L-ethionine diet followed by ten days of normal food. It was identified after dephosphorylation by ultraviolet absorption spectra, cochromatography with authentic material and mass spectra as N(6)-dimethyladenosine. It is concluded that S-adenosylethionine, the primary product of L-ethionine in the liver, causes strong and selective inhibition of the specific RNA-methylase responsible for adenosine to N(6)-dimethyl=adenosine methylation in rat liver 4s RNA. Compared to the strong inhibition of N(6)-dimethyladenosine formation described here, L-ethionine-dependent ethylation of liver 4s RNA is far less efficient. The quantitation of l-methyladenosine, ribothymidine and 3'-terminal adenosine in this 4s RNA as well as its aminoacid acceptor activity is typical for tRNA; hence it may be concluded that N(6)-dimethyladenosine is a component of rat liver tRNA. This may demonstrate the first evidence for the existence of specifically methyl-deficient mammalian tRNA. A possible correlation between the activity of L-ethionine as a liver carcinogen and its ability to induce the formation of methyl-deficient tRNA by selectively inhibiting the synthesis of N(6)-dimethyladenosine on the tRNA level in the same organ is discussed.

The purpose of the present study was to elucidate the metabolic requirements of autophagocytosis. Two model systems were used for this purpose: a) glucagon-induced autophagocytosis in the rat liver, and b) the wave of autophagocytosis which occurs when isolated flounder kidney tubules are incubated in vitro. In the rat liver, protein synthesis was inhibited by the administration of cycloheximide (1.5 mg/kg) to rats 2 hours prior to glucagon injection. In flounder kidney tubules, protein synthesis was inhibited at least 90% by adding cycloheximide, actinomycin D, pactamycin and puromycin to the medium. In both systems the inhibition of protein synthesis failed to inhibit the formation of autophagic vacuoles or their subsequent transformation into autolysosomes, as depicted from electron microscopic histochemical preparations. In flounder kidney tubules no differences were found in the levels of p-nitrophenylphosphates, beta-DL-glycerophosphatase, N-acetyl-beta-D-glucosaminidase, arylsulphatase, beta-D-galactosidase or acid proteinase when tubules were incubated up to 5 hours in the presence or absence of protein synthesis inhibitors. When ethionine was administered to rats 2 hours before glucagon injection, a decrease of approximately 75% in the ATP levels was observed. After ethionine administration, glucagon failed to induce the formation of autophagic vacuoles. The incubation of flounder kidney tubules in the presence of cyanide or in a nitrogen atmosphere decreased the ATP levels to less than 10% of controls and blocked autophagy. On the other hand, cyanide had little effect on acid hydrolase levels at 1 hour of incubation. A wide variety of other inhibitors were also shown to block autophagy. These results further support the hypothesis that, in the formation of antophagic vacuoles, preexisting enzyme and membrane pools are utilized. On the other hand, the esotropy-exotropy membrane conformational changes occurring in the formation of autophagic vacuoles seem to be energy dependent and can therefore be blocked by lowering intracellular ATP levels.

Mutants resistant to selenomethionine were isolated, and their properties studied. Mapping studies indicate that the mutation sites are located near the eth-1(r) locus in linkage group I, about ten map units away from the mating type locus. The sites of new mutation are either allelic to or very close to eth-1(r). They are resistant not only to selenomethionine but also to ethionine, while the ethionine-resistant mutant, eth-1(r), is sensitive to selenomethionine. The selenomethionine-resistant mutants are also temperature-sensitive mutants. However, they can grow at higher temperatures in medium containing 1 M glycerol.-It is very unlikely that the resistance is due to a change in the permeability of the membrane. Aryl sulfatase of se-met(r) mutants is not repressed by a high concentration of methionine (5 mM), although inorganic sulfate (2 mM) still can cause total repression. The gamma-cystathionase levels of the mutants are normal, but the S-adenosylmethionine synthetase levels are only one-tenth of that observed in the wild-type strain. The heat-stability of this enzyme in the mutant is also different from that of the wild-type enzyme suggesting that the mutation might affect the structural gene of S-adenosylmethionine synthetase.