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Inactive, frozen and thawed cytoplasmic extracts of 3T3 and SV-101 (3T3 transformed by SV-40 virus) cells contain an inhibitor which blocks the poly(U)-directed incorporation of [14C]phenylalanine into polypeptides, catalyzed by active extracts of these cells. This inhibition is not reversed by adding increased amounts of poly(U). Furthermore, little or no inhibitory activity is observed when poly(U) translation is assayed using precharged [14C]Phe-tRNA. These results suggest that the observed inhibition is not due to the degradation of poly(U) by a nuclease. The inhibitor appears to act primarily at the level of tRNA charging since the synthesis of both Phe-tRNA and Lys-tRNA is impaired in its presence. Evidence is presented which indicates that the inhibitory activity is not due to a high molecular weight protein or nucleic acid. However, the inhibitor appears to be adsorbed to a macromolecule. The inhibitory activity is completely destroyed by ashing.

RNA ligase, isolated from bacteriophage T4-infected Escherichia coli, catalyzes the conversion of synthetic [5'-32P]polyribonucleotides to a form resistant to alkaline phosphatase. The reaction requires ATP and leads to the formation of AMP and PPi. The purification procedure for T4-induced RNA ligase has been modified to yield stable enzyme preparations. The influence of secondary structure on the RNA ligase reaction has been investigated with these fractions. The conversion of synthetic polynucleotides to complex secondary structures was either without effect or inhibitory in the RNA ligase system. Purified RNA ligase was shown to catalyze an exchange reaction between ATP and PPi, but not between ATP and AMP. The ATP-PPi exchange reaction occurred in the absence of a polyribonucleotide substrate. The formation of an RNA ligase-adenylate complex was demonstrated by gel filtration, polyacrylamide gel electrophoresis, and ultracentrifugation in a glycerol gradient. The complex was dissociated by 5'-P-poly(A) to yield AMP while ATP was generated in the presence of PPi. These findings suggest that an enzyme-AMP intermediate occurs during the course of the RNA ligase reaction. Evidence is presented for the occurrence of an RNA ligase activity in eukaryotic cells.

Ternary complex formation between eukaryotic initiation factor 2 (eIF-2), initiator Met-tRNA and guanosine 5′-(β, γ-imino) triphosphate [GMP-P(NH)P] is strongly inhibited by mRNA in the Artemia salina system. Developing A. salina embryos contain a factor which displays a novel activity, namely the ability to counteract the mRNA-induced inhibition of ternary complex formation. This factor is heat-labile. It is proposed that the factor may play an important role in protein biosynthesis by preventing mRNA from inhibiting an early step of peptide chain initiation.

Cytosine arabinoside (ara-C) is metabolized via two enzymatic pathways: by phosphorylation to the nucleoside triphosphate arabinosyl-CTP or deamination to the inert metabolite uracil arabinoside. Resistance to cytosine arabinoside had been attributed to a decreased synthesis of arabinosyl-CTP, the active inhibitor of DNA synthesis. Steuart and Burke (Nature New Biol. 233, 109, 1971) suggested the increased deamination of cytosine arabinoside as an alternative mechanism of resistance in patients treated with this drug. In the present study the mechanism for the effect of cytosine arabinoside on nucleoside deaminase activity was investigated in cell culture. HeLa cells grown in medium with added cytosine arabinoside had a 4-fold increase in nucleoside deaminase activity. The increase is dependent on the concentration and time of exposure to cytosine arabinoside. The levels of four control enzymes were unaffected by this agent. No difference was found between the properties of nucleoside deaminase purified from cells grown in the presence of cytosine arabinoside and in controls. The early appearance of an increase in nucleoside deaminase in the absence of cell killing made selection of a mutant population improbable. Cycloheximide did not prevent the elevation in nucleoside deaminase indicating that the mechanism, unlike "classical induction," may not be dependent on the synthesis of a new protein. These studies show that the presence of cytosine arabinoside in tissue culture leads to an increased activity of nucleoside deaminase which could result in accelerated drug degradation. This process may provide a mechanism for resistance to the antimetabolite.

A 400-fold increase was observed in the specific activity of nucleoside deaminase (cytidine aminohydrolase, EC 3.5.4.5) in the spleen of mice infected with Friend murine leukemia virus. The enzyme was purified 100-fold from the spleen of leukemic mice. It was sensitive to SH reagents and stimulated by dithiothreitol and GSH. Different pH responses were observed in the presence or absence of dithiothreitol. Cytidine was deaminated at a greater rate than deoxycytidine; cytosine or its nucleotides were not deaminated. The molecular weight determined by gel filtration was 74 000.

Publisher Summary This chapter describes the purification procedure of cytidine deaminase enzyme from leukemic mouse spleen. Cytidine deaminase is widely distributed among mammalian tissues. This enzyme is responsible for the inactivation of cytosine arabinoside––one of the most useful chemotherapeutic agents available for the treatment of acute myeloblastic leukemia. In the mouse spleen, a marked increase in the specific activity of cytidine deaminase occurs following infection with Friend leukemia virus. Elevated levels of cytidine deaminase are also found in regenerating mouse liver, in human leukemic cells, following the treatment of the patient with cytosine arabinoside, and in HeLa cells cultured in the presence of cytosine arabinoside. The activity of cytidine deaminase is assayed by measuring the amount of labeled uridine formed by the deamination of 2-14C-labeled cytidine. High levels of cytidine deaminase are found in human polymorphonuclear leukocytes.

Summary Several ‘capped’ mRNAs (e.g., 9S globin mRNA) as well as ‘noncapped’ mengo viral mRNA greatly stimulate the activity of a novel ATPase isolated from Artemia salina embryos. Other RNAs tested [tRNAs, initiator Met-tRNA, poly r(U), poly r(A)] are much less stimulatory. The ATPase activity is also stimulated by mRNAs in the presence of 40S ribosomal subunits. However, the other RNAs listed above are inhibitory under the latter conditions. The ATPase activity is strongly stimulated by 40S ribosomal subunits in the absence of mRNAs. It is suggested that the ATPase, described here, might be involved in the binding of natural eukaryotic mRNAs to 40S peptide chain preinitiation complexes, a process known to depend on ATP hydrolysis.

An enzyme, purified 300-fold from Escherichia coli infected with bacteriophage T4, catalyzes the conversion of 5′-termini of polyribonucleotides to internal phosphodiester bonds. The reaction requires ATP and Mg ++ . For every 5′- 32 P terminus rendered resistant to alkaline phosphatase, an equal amount of AMP and PPi are formed. Various polyribonucleotides are substrates in the reaction; to date, the best substrate is [5′- 32 P]polyriboadenylate. With the latter substrate, no evidence of intermolecular reaction was obtained. However, the 5′- 32 P termini of poly(A) rendered resistant to alkaline phosphatase are also resistant to attack by RNase II, polynucleotide phosphorylase, and low concentrations of venom phosphodiesterase. Since the product formed with poly(A) lacks 3′-hydroxyl ends, as measured with these exonucleases, the enzyme appears to convert linear molecules of polyriboadenylate to a circular form by the intramolecular covalent linkage of the 5′-phosphate end to the 3′-hydroxyl terminus.

The 5′ terminus of the Rous Sarcoma Viral 30-40S RNA was characterized as follows: Unlabeled RNA was treated with polynucleotide kinase and (γ-32P) ATP. Degradation of the 5′-(32P) RNA with alkali yielded labeled pAp while degradation with venom phosphodiesterase yielded labeled 5′-AMP. Dephosphorylation with alkaline phosphatase was unnecessary for the RNA to accept32P indicating the presence of 5′-OH ends. This establishes that the base at the 5′ end of Rous Sarcoma Viral 30-40S RNA is adenine.

Increased activity of the enzyme nucleoside deaminase is found in the spleens of mice protected from lethal whole-body irradiation by transfusion of isogenic bone marrow cells. As few as 10,000 nucleated bone marrow cells produce a significant elevation in enzyme level. This increase precedes any detectable increase in hemoglobin synthesis and is temporarily associated with an increase in protein synthesis during the early stages of recovery. Hypertransfusion of the irradiated, protected mice eliminates most of the stimulus to erythroid differentiation and prevents the increase in nucleoside deaminase. The present studies indicate that this enzyme is present in a differentiated red cell precursor not found in the normal stem cell population.