Activation of Protein Synthesis upon Dilution of an Arachis Cell Culture from the Stationary Phase

Abstract

When a stationary phase cell culture of Arachis hypogaea L. is diluted into fresh media, there occurs a 10-fold increase in the rate of protein synthesis. The kinetics of the activation of amino acid-incorporating capacity show a lag of 10 to 15 minutes with maximal activity reached at 2 hours after dilu- previously described (23). Both for maintaining the culture and for experimental analyses, cells were diluted by inoculating 25 ml of a 14-day suspension into 225 ml of fresh medium.Protein Synthesis. Rates of protein synthesis were measured by incubating 10-ml aliquots of the cell suspension with 0.1 ,uc "4C-leucine (300 mc/mmole) for 5 min at 25 C. The reaction was stopped by adding 2.5 ml of cold 16% trichloroacetic acid, 1 mM leucine, and the cells were pelleted by centrifugation. The pellet was homogenized in 5 ml of 5% trichloroacetic acid, 1 mM leucine, and analyzed for hot trichloroacetic acid-insoluble radioactivity (15). In experiments where uptake of 14C-leucine was measured, the cells were harvested on Miracloth and washed with water prior to homogenization in 5% trichloroacetic acid, 1 mm leucine. An aliquot of the trichloroacetic acid-soluble radioactivity was then taken for determination of 14C leucine uptake into the soluble pool.Polyribosome Analysis. Total ribosomes were isolated by a procedure utilizing high pH for the maintenance of polyribosomal stability (1, 4). Forty-milliliter aliquots of the diluted cell suspension were harvested by filtering through Miracloth, and the cells were immediately immersed in liquid nitrogen. The frozen cells were homogenized in a mortar and pestle kept in Dry Ice and the "Dry Ice powder" of the cells was transferred to a Dounce homogenizer containing 6 ml of 20 mm KCl, 5 mm magnesium acetate, 5 mm mercaptoethanol, 150 mM tris acetate, pH 8.5, 400 mm sucrose, and 0.5% Nonidet P-40 detergent (Shell Chemical Company) (6). The cells were homogenized gently, and the slurry was passed through Miracloth to remove the whole cells and crude cell wall fragments. The homogenate was centrifuged for 10 min at 20,000g to remove nuclei and mitochondria, and the post mitochondrial supernatant was layered on 2.5 ml of 1.5 M sucrose in TKM buffer (50 mm tris, pH 8.5, 20 mm KCl, 10 mm magnesium acetate) and centrifuged for 90 min at 105,000g in a Spinco Ti 50 rotor. The pellet was dissolved in 0.5 ml of TKM buffer and any undissolved material was removed by low speed centrifugation. For analysis of polyribosome content, ribosomes equivalent to 100 jug of RNA were layered on 5 ml of 15 to 38% linear sucrose gradient in TKM buffer. The gradients were centrifuged at 50,000 rpm for 40 min in SW 50.1 rotor and fractionated as described earlier (23). For RNAase treatment, an equivalent aliquot of ribosomes was treated with 10 ,ug/ml RNAase for 10 min at 30 C and layered on the gradient as above. This unusually severe RNAase treatment was required because of the effectiveness of the TKM suspension buffer in inhibiting RNAase activity (ref. 4).Analysis of mRNA Synthesis. Twent...

Response of Cytosolic-Isozyme and Plastid-Isozyme Levels of 3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthase to Physiological State of Nicotiana silvestris in Suspension Culture

“…Suspension-cultured cells were grown as previously described (2) The soluble protein content was determined on each day after stationary-phase cells were diluted into fresh medium. As has been found elsewhere (10), a marked change in the protein content occurred late in the lag phase ( Fig. 2A), increasing from 88 mg protein/g dry weight at the time of dilution to 176 mg/g on d 2.…”

“…A rationale and supporting evidence that part or all of the aromatic amino acid pathway is present in both subcellular locations of higher plants have been presented (9,10).…”

Protein Synthesis in Embryos of Dormant and Germinating Agrostemma githago L. seeds

“…In a number of other experimental systems, e.g. in activated cell cultures (29), the rate of protein synthesis was also found to increase during the lag phase between the induction and the onset of growth. The discrepancies with other reports on Agrostemma (18) and on seeds with the same type of dormancy (9,19,20) can be traced back to the pitfalls listed in the Introduction.…”

Caution in the interpretation of plant ribosome studies (Short Communication)

“…For example, in studying polyribosome metabolism during normal and hormonally induced growth of pea stems, Davies & Larkins (1972) sedimented ribosomes through a 4ml cushion of 1.5M-sucrose in a Spinco type 40 rotor for 90min at lOOOOOg (0.9x 107g-min). Verma & Marcus (1974) in a study of polyribosome formation in plant tissue culture sedimented ribosomes through 2.5ml of 1 Msucrose for 90min at 105000g (0.95x 107g-min).…”

“…Such changes are interpreted as a change in the translational capacity of the tissue, which may be part of the molecular mechanism utilized by plant cells to modulate their rate of growth (see, e.g., Beevers & Poulson, 1972;Davies & Larkins, 1972;Fountain & Bewley, 1973;Verma & Marcus, 1974;Travis et al, 1973).…”