Immunospecific binding of tRNA precursors containing N6-(delta 2-isopentenyl) adenosine

Antibodies specific for N6-(delta 2-isopentenyl) adenosine (i6A) were immobilized on Sepharose and this adsorbent (Sepharose-anti-i6A) was used to selectively isolate bacteriophage T4 tRNA precursors containing i6A/ms2i6A from an unfractionated population of 32P-labeled T4 RNAs. The results showed that antibodies to i6A selectively bound only those tRNA precursors containing i6A/ms2i6A. Binding of tRNA precursors by antibody and specificity of the binding was assessed by membrane binding using 32P-labeled tRNA precursor. Binding was highly specific for i6A/ms2i6A residues in the tRNA precursors. This binding can be used to separate modified from unmodified precursor RNAs and to study the biosynthetic pathways of tRNA precursors.     

Effect of ribosylzeatin isomers on the enzymatic degradation of N6-(delta2-isopentenyl) adenosine.

Cytokinin oxidase has been partially purified from cultured tobacco tissue. This enzyme converts N6-(delta2-isopentenyl)-adenosine to adenosine. The reaction is inhibited by the two isomers of ribosylzeatin [n6-4-hydroxy-3-methylbut-2-enyl)adenosine]. Trans-ribosylzeatin inhibits the reaction more than the cis-isomer.     

Purification of antibodies for the cytokinin N6-(delta 2-isopentenyl) adenosine by affinity chromatography.

Isopentenyl adenosine antibodies useful in the investigations of the "cytokinin" functions of isopentenyl adenosine were purified by affinity chromatography. Using different affinity columns, the antibodies were purified to near complete purity. Analyses of the purified proteins revealed the presence of isopentenyl adenosine binding proteins in normal rabbit serum, which presence supports a suggested role for isopentenyl adenosine and its related compounds in animal cell division in vivo.     

Conformational changes in yeast tRNATyr revealed by EPR spectra of spin-labelled N6-(delta2-isopentenyl)-adenosine residue.

Temperature-induced conformational changes in the anticodon region of yeast tRNATyr were studied by EPR spectroscopy. The spin label 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl was attached to the N6-(delta2-isopentenyl)-adenosine residue in tRNATyr, previously made reactive by iodination. The labelled tRNATyr gave an asymmetrical triplet spectrum typical of rapidly tumbling nitroxide, with a rotational correlation time (tauc) of 0.65 ns. Spin-labelled tRNATyr was exposed to heating and cooling in three different buffers each with or without MgCl2. In each case the Arrhenius plot of --log tauc vs. inverse absolute temperature gave two straight lines, intersecting at a critical temperature (tcr). Above tcr, the anisotropy of the spectrum was not reduced and the activation energy of motion increased, indicating that the transition is associated with a conformational change of the macromolecule. Transitions in 0.05 M potassium phosphate (pH 8.0) and 0.02 M Tris - HC1 (pH 7.0) were observed at potassium phosphate (pH 8.0) and 0.02 M Tris - Hc1 (pH 7.0) were observed at approx. 37 degrees C. When 0.01 M mgCl2 was present in these buffers, transitions were shifted to 46 degrees and 53 degrees C, respectively. Transitions in 0.01 M sodium cacodylate were observed at temperatures which are significantly lower. Since all these transitions occur at temperatures considerably below those required to melt the helical regions of tRNA, and at least approximately 10 degrees C below those reported to break tertiary interactions, it is supposed that they reflect some reorientation of the anticodon region, e.g. a change in tilt of the bases.     

Investigation of immunoadsorbent efficiency and capacity for the isolation of tRNAs containing N6-(delta 2-isopentenyl)adenosine derivatives

Antibodies directed to modified nucleosides recognize the nucleoside (antigen) when it is present in an intact tRNA molecule. The general application of anti-nucleoside immunoadsorbent chromatography, however, has been greatly impeded by the apparent inefficiency and low capacity of conventional immunoadsorbents for transfer RNA. Antibodies specific for isopentenyladenosine (i6A) were employed to investigate the efficacy of various immunoadsorbents with respect to immobilization of antibody protein and with respect to their ability to bind i6A-containing tRNAs. Biologically active anti-i6A was recovered in high yield (80-88%) by affinity chromatography on i6A-adipate-Sepharose 4B or i6A-butane diglycidyl ether-Sepharose 4B using either 15% pyridine in phosphate-buffered saline or 0.2 M acetic acid as eluents. The binding capacity of various anti-i6A antibody immunoadsorbents was evaluated. While both anti-i6A antibody-protein A-agarose-iminothiolane (ITL) and anti-i6A antibody-protein A-agarose-dimethyl suberimidate showed a high capacity for i6A-tRNA, the latter column is much less efficient with respect to antibody immobilization. Under optimal conditions, the ITL immunoadsorbent binds 5-6 nmol of i6A/mg of antibody protein. With respect to bulk tRNA, 1 mg of antibody protein (ITL immunoadsorbent) binds all of the i6A-tRNA in a 1-mg sample.     

Amino acid transport in a water-mould: the possible regulatory roles of calcium and N6-(delta2-isopentenyl)adenine.

Transport of amino acids in the water-mould Achlya is an energy-dependent process. Based on competition kinetics and studies involving the influence of pH and temperature on the initial transport rates, it was concluded that the 20 amino acids (L-isomers) commonly found in proteins were transported by more than one, possibly nine, uptake systems. This is similar to the pattern elucidated for some bacteria but unlike those uncovered for all fungi studied to date. The nine different systems elucidated are: (i) methionine, (ii) cysteine. (iii) proline, (iv) serine-threonine, (v) aspartic and glutamic acids, (vi) glutamine and asparagine, (vii) glycine and alanine, (viii) histidine, lysine, and arginine, and (ix) phenylalanine-tyrosine-tryptophan and leucine-isoleucine-valine as two overlapping groups. Transport of all of these amino acids was inhibited by azide, cyanide, and its derivatives and 2,4-dinitrophenol. These agents normally interfere with metabolism at the level of the electron transport chain and oxidative phosphorylation. Osmotic shock treatment of the cells released, into the shock fluid, a glycopeptide that binds calcium as well as tryptophan but no other amino acid. The shocked cells are incapable of concentrating amino acids, but remain viable and reacquire this capacity when the glycopeptide is resynthesized.