Amino Acid Activation in Subcellular Fractions of Tetrahymena pyriformis*

Abstract

SYNOPSIS. The presence of amino acid activating enzymes was demonstrated in the ciliated protozoan Tetrahymena pyriformis. By employing a sensitive hydroxamate assay procedure, the activation of L‐valine was assayed in various subcellular fractions of the ciliate, and some characteristics of the enzyme activity in the most active fraction were determined. Most of the activity resided in pH 5 fractions isolated from high speed supernatants of ciliates disrupted by various physical and chemical methods. No activity could be demonstrated in isolated cilia, in pellicles with attached kinetosomes, in microsomes or in macronuclei, providing these organelles were thoroughly washed. A washed mitochondrial preparation isolated by the Mager and Lipmann procedure activated L‐valine; mitochondria isolated by the procedure of Hogg and Kornberg did not. The pH 5 fraction isolated from the 102,000 X g supernatant of digitonin‐lysed ciliates was stable for several weeks when stored in 0.1 M Tris buffer, pH 7.6 at – 25 C. The activity of this fraction with respect to L‐valine activation was dependent on the presence of ATP1 and magnesium in the reaction mixture. The optimal concentrations of these components and of L‐valine and hydroxylamine were determined, and the linearity of activity with time and enzyme concentration was demonstrated. Valine activation was not modified by dialysis of the pH 5 fraction, or treatment with RNase, or the addition of boiled pH 5 fraction.

STUDIES ON NUCLEAR STRUCTURE AND FUNCTION IN TETRAHYMENA PYRIFORMIS

“…CULTURE' CONDITIONS : Nuclei can be routinely isolated by this method only from growing populations in well aerated cultures . Recently, we have been able to isolate nuclei successfully from stationary phase cells by including 0 .01% of spermidine (Rosenbaum and Holz, 1966) in the homogenization medium and extending homogenization times, or by doubling the octanol concentration in Medium B .…”

Flagellar Regeneration in Protozoan Flagellates

“…At the present time there is not suffi ient evidence to discount either possibility. Although a self-contained flagellar protein synthetic system may seem improbable on the basis of the available biochemical evidence showing that cilia have little RNA (4,39) and activating enzyme activity (27) and, ultrastructurally, an absence of ribosomes, these investigations have all been carried out on fullgrown cilia and flagella.…”

Amino Acid Selection in Protein Biosynthesis

“…Aminoacyl-s-RNA synthetases have been reported in extracts of the ciliate Tetrahymena pyrijormis (Rosenbaum & Holz, 1963) and from extracts of the trypanosomid flagellate Crithidia oncopelti (Chesters, 1966). Many isolations from bacterial sources have been described, e.g.…”

“…Nucleoli may also contain these enzymes (Brandt & Finamore,I 963). Macronuclei and cilia preparations from Tetrahymena pyriformis were, however, found to be inactive (Rosenbaum & Holz, 1963). More than a slight cytoplasmic contamination of isolated particles has in most cases been excluded by careful particle isolation and thorough washing in aqueous and non-aqueous media.…”