Jon R. Katze

Administration of queuine to mice relieves modified nucleoside queuosine deficiency in Ehrlich ascites tumor tRNA

Abstract The tRNA from Ehrlich ascites tumor cells is deficient in the modified nucleoside Q (queuosine). Continuous infusion of Q base (queuine) to tumor-bearing mice reverses the deficiency of Q in Ehrlich ascites tRNA, and coincidently, causes an inhibition of tumor growth.

Substrate and inhibitor specificity of tRNA-guanine ribosyltransferase

We have tested as inhibitors or substrates of tRNA-guanine ribosyltransferase (EC 2.4.2.29) a number of compounds, including derivatives of 7-deazaguanine, pteridines, purines, pyrimidines and antimalarials. Virtually all purines and pteridines that are inhibitors or substrates of the rabbit reticulocyte enzyme have an amino nitrogen at the 2 position. In addition the 9 position and the oxygen at the 6 position may be important for recognition by the enzyme. Saturation of the double bond in the cyclopentenediol moiety of queuine reduces substrate activity and queuine analogs that lack the cyclopentenediol moiety, such as 7-deazaguanine and 7-aminomethyl-7-deazaguanine, are relatively poor substrates for the enzyme. While adenosine is not an inhibitor, neplanocin A (an adenosine analog in which a cyclopentenediol replaces the ribose moiety) is a poor inhibitor. The incorporation of 7-aminomethyl-7-deazaguanine into the tRNA of L-M cells results in a novel chromatographic form of tRNAAsp, indicating that L-M cells cannot modify this Q precursor (in Escherichia coli) to queuosine. The specific incorporation of 7-deazaguanine and 8-azaguanine into tRNA by L-M cells also results in novel chromatographic forms of tRNAAsp. With intact L-M cells, the enzyme-catalyzed insertion into tRNA of queuine, dihydroqueuine, 7-aminomethyl-7-deazaguanine, or 7-deazaguanine is irreversible, while guanine or 8-azaguanine incorporation is reversible; suggesting that it is the substitution of C-7 for N-7 which prevents the reversible incorporation of queuine into tRNA.

Salvage of the nucleic acid base queuine from queuine-containing tRNA by animal cells

Abstract The incorporation of queuine into tRNA and its fate upon tRNA turnover has been studied in the Vero and L-M cell lines. An assay was developed using [ 3 H]dihydroqueuine to detect the queuine acceptance and, thus, the queuine content of tRNA in intact cells. While L-M cells can use only queuine, Vero cells can use either queuine or its nucleoside, queuosine, to form queunine-containing tRNA. Since queuosine is not a substrate for the enzyme which incorporates queuine into tRNA, Vero cells must generate queuine from its nucleoside. When Vero cells are labelled with [ 3 H]dihydroqueuine, the half life of acid insoluble radioactivity is 52 days in queuine-free medium and 3.1 days in queuine-containing medium, indicating that [ 3 H]dihydroqueuine is salvaged from tRNA and reused by Vero cells, but that exogenous queuine can compete with the salvaged [ 3 H]dihydroqueuine. When L-M cells are labelled with [ 3 H]dihydroqueuine, the half life of the acid insoluble radioactivity is 1.2 days in the presence or absence of queuine, indicating the absence of queuine salvage in L-M cells.

Q-factor: a serum component required for the appearance of nucleoside Q in tRNA in tissue culture.

Abstract Animal serum contains an activity, designated Q-factor, which effects an increase in nucleoside Q-containing tRNA in tissue culture. The appearance of Q-positive tRNA Asp in the L-M cell line cultivated serum-free has been used as an assay to partially characterize Q-factor from fetal bovine serum and to determine that bovine amniotic fluid contains 100 fold more Q-factor than does fetal bovine serum. Q-factor is dialyzable, 500 molecular weight or less, and binds tightly to activated charcoal and dextran. Using Q-factor, evidence is presented that the Q-negative tRNA Asp species are precursors of the Q-positive species.

Regulation of glutamate synthase from Bacillussubtilis by Glutamine

Abstract Glutamate synthase, an important enzyme in the assimilation of ammonia, was measured in cultures of Bacillus subtilis grown with different nitrogen sources. An attempt was made to correlate the specific activity to the intracellular levels of five metabolites of glutamate metabolism: aspartate, glutamate, glutamine, alanine and NH 4 + . An inverse relationship was found between the activity of glutamate synthase and the pool level of glutamine. We propose that the intracellular concentration of glutamine is an important element in controlling the level of glutamate synthase.

Identifying inhibitors of queuine modification of tRNA in cultured cells

Altered queuine modification of tRNA has been associated with cellular development, differentiation, and neoplastic transformation. Present methods of evaluating agents for their ability to induce queuine hypomodification of tRNA are tedious, time-consuming, and not readily amenable to examining cell-type or tissue specificity. Therefore, a rapid, small-scale assay was developed to identify agents that alter queuine modification of tRNA in cultured cells. Monolayer cultures (2cm2) of Chinese hamster embryo cells depleted of queuine for 24 h were evaluated for their ability to incorporate [3H]dihydroqueuine into acid precipitable material (tRNA) in the presence and absence of potential inhibitors. Known inhibitors of the queuine modification enzyme tRNA-guanine ribosyltransferase (e.g., 7-methylguanine, 6-thio-guanine, and 8-azaguanine) were very effective in blocking incorporation of the radiolabel, and the dose-dependent results exhibited small standard deviations in independent experiments. The data indicate that the method is rapid, reliable, and potentially useful with a variety of cell types.

Why is tumor tRNA hypomodified with respect to Q nucleoside

Nucleoside Q is found exclusively in the first position of the anticodon in tRNATyr, tRNAHis, tRNAAsn, and tRNAAsp (Kasai et al. 1975b, 1976). Its function is unknown, and long-term Q-deficient, germ-free mice appear normal (Reyniers et al. 1981). However, queuirie metabolism is altered in neoplasia. Many tumors exhibit Q-deficient tRNA (Katze 1975; Okada et al. 1978; Roe et al. 1979) and queuine addition to tumor-bearing mice can reverse tumor tRNA Q deficiency (Katze and Beck 1980; Nishimura et al., this volume pp. 401–412) and may inhibit tumor growth (Katze and Beck 1980). It is unlikely that Q-deficient tRNA is necessary for the neoplastic state since the careful study of Marini and Mushinski (1979; Mushinski and Marini, this volume pp. 121–132) shows widely varying, repeatable isoaccepting patterns (largely due to differences in Q content) for the Q family of tRNAs from several Balb/c mouse plasmacytomas with similar growth rates and size at harvest. These large differences among such a similar group of tumors suggest that the alteration in Q metabolism is secondary to the neoplastic process.

Queuosine Metabolism: Possible Relation to B-Cell Activation by C8 Derivatives of Guanosine

Abstract The observed enhancement of B-lymphocyte activation by 8-bromoguanosine and 8-mercaptoguanosine is hypothesized to occur via a “binding protein” which requires a guanine nucleoside as the syn conformer for productive interaction. In addition, because of the 7-substituent, Q nucleoside also is hypothesized to bind as the syn conformer and, therefore, to be a potential B-lymphocyte activator.

Mass spectrometry of the nucleic acid base queuine

Abstract Mass spectrometry has played a major role in the structure elucidation of members of the Q family of bases and nucleosides, which are widely distributed in nature. The volatile N -(aminomethylene)-2,2,2-trifluoroacetamide (AMT) - trimethylsilyl (TMS) derivative of the nucleic acid base queuine has been prepared and its electron ionization mass spectrum studied. A deuterated analog of queuine has been prepared and tested for use as an internal standard for quantitative measurements of queuine in plant and animal materials by selected ion monitoring.