Search

Chang liver cells from exponentially growing suspension cultures have been separated by sedimentation at unit gravity. Determinations of the protein content per cell showed that the fractionation procedure resulted in good separation of cells of different size. On the other hand, the DNA content of individual cells from the fractions, as determined cytofluorimetrically, indicated considerable heterogeneity in the size of cells from the same stage of the division cycle. On the basis of earlier results on intermitotic growth and the variation in the length of the cell cycle in homogeneous cell populations, a mathematical model has been constructed and tested using a computer program. The present results on the size distribution of cells from the different stages of the mitotic cycle are consistent with a regeneration of size heterogeneity in each cell generation, as a result of the dispersion of intermitotic times. The variation in cell cycle times may be related to a probabilistic event in the G1 period. In the mathematical model it was necessary to include a mechanism by which the regeneration of abnormally large cells is prevented. The experimental data are compatible with a gradually increasing inhibition of growth in cells larger than a certain size (circa 400 pg protein per cell).

During the normal 4-day growth cycle of Chang's liver cells in tissue culture, ornithine δ-transaminase increased and returned to the initial level in a fairly regular and reproducible manner. These changes in activity appeared to be the net result of synthesis and degradation of enzyme. Synthesis of enzyme was prevented or decreased by omitting as essential amino acid from the medium or by the addition of puromycin. Inhibition of synthesis allowed a minimum degradation rate to be determined. Synthesis of enzyme began again when the missing amino acid was restored. If the missing amino acid was glutamine or phenylalanine, the subsequent rise of ornithine δ-transaminase took place as a single continuous stage after transferring the cells to complete medium. In contrast, when arginine was the amino acid omitted from the preceding incubation medium, the subsequent increase of enzymatic activity was biphasic. The addition of actinomycin either initially or after prior incubation of cells in medium lacking an essential amino acid stimulated the first phase rise; the second phase increase then was not observed. Leucine, valine, isoleucine, or ornithine, each of which is inhibitory to ornithine δ-transaminase, increased enzymatic activity when added at a 6 mm concentration and extended the period of normal increase of the enzyme. Other amino acids were without effect. It is proposed that the biosynthesis of ornithine δ-transaminase in Chang's liver cells is repressed by an as yet unidentified metabolic product. In addition, it is suggested that the enzyme is synthesized in response to messenger ribonucleic acid of an appreciable lifetime.

Experiments have been carried out to study the fine structure of the development of arginase in the young chick embryo. Considerable periodic fluctuations were found in the speed of accumulation of the enzyme. The results suggest that enzyme synthesis may be regulated by a feedback control. It is tempting to investigate whether enzyme repression, the feedback mechanism which seems to play an important part in the regulation of enzyme synthesis in microorganisms, may not also be of importance in the changes in the enzyme pattern of the embryonic cells that are characteristic of early embryonic development. Studies along this line of thinking are in progress.

An increase of arginase activity has been produced in Chang's liver cells in suspension cultures by increasing the concentration in the growth medium of lysine, leucine, valine, or ornithine. These amino acids are known to inhibit enzymes in the arginase-initiated reaction sequence from arginine to proline. An increase in arginase also occurred with o-aminobenzaldehyde, a compound which reacts with a metabolite (Δ1-pyrroline 5-carboxylate) of the same reaction chain. A stimulation of arginase has been also shown following substitution of citrulline for arginine in the growth medium. A decrease in arginase was found after addition of proline, a product of the same reaction sequence. Experiments in which protein synthesis was inhibited by puromycin indicate that the rate of accumulation of enzyme protein was involved rather than an activation or inactivation of preformed enzyme. When arginase was stabilized by the addition of manganese to the growth medium, the compounds mentioned above produced the same results as before, although greater in magnitude, indicating an effect on the rate of synthesis rather than on the rate of degradation of the enzyme during general protein turnover. These results suggest that synthesis of arginase in Chang's liver cells during a normal 4-day growth cycle is regulated, in part at least, by product repression.

The effects of temporary glutamine deficiency on the protein and nucleic acid metabolism of Chang's liver cells in suspension cultures have been studied. It was observed that cells maintained in a glutamine-free medium showed a reduced incorporation of labeled precursors into protein and RNA. At the same time, the activity of the ribosomes and the proportion of polyribosomal aggregates in cell extracts diminished. These effects were reversed when the glutamine content of the medium was restored. The restoration of a normal rate of amino acid incorporation by intact cells as well as by cell-free systems was time dependent, and took place within a few hours after glutamine addition without preceding increase in the prevailing low rate of RNA synthesis. The addition of actinomycin D at concentrations that strongly inhibited the RNA metabolism of the cells did not prevent the increase in protein synthesis or the reappearance of polyribosomal aggregates. These facts suggest that the restoration of protein synthesis in the cells after glutamine starvation was not dependent on a production of new messenger RNA. The experimental data are consistent with the hypothesis that previously synthesized messenger RNA, preserved in the cells in a stable form, was brought into action in response to the reestablishment of an adequate cellular environment.

Publisher Summary Studies concerned with the kinetics of metabolic processes having a prolonged duration place certain demands on the sampling procedure, which may be difficult to handle practically. For example, investigating the pattern of synthesis of metabolites during the cell cycle necessitates the collection of samples from synchronized cell cultures at regular, frequent intervals over periods of one or more days in order to yield coherent results. The apparatus described here is assembled to facilitate the collection of samples at frequent, regular intervals, from cell cultures during experiments having duration of several days. For this technique to be of use in monitoring the progress of cellular processes, it is necessary to establish that the cell samples stored in the cold, and analyzed later, give the same results as samples freshly collected at these time points. Obviously, such control experiments have to be carried out for each individual type of experiment to ascertain whether the metabolites to be assayed are stable under the storage conditions used.

A rapid increase in specific arginase activity occurred in Chang liver cells immediately after addition of the missing nutrient to cells preincubated in a medium from which one essential amino acid had been omitted. The rapid change in enzymic activity offered an opportunity for studying specific enzyme regulation during short time experiments. 1. 1. The experimental results indicate that the increase in specific arginase activity, which could be immediately stopped by the addition of puromycin, was due to a synthesis of the enzyme more rapid than in normally growing cells. 2. 2. Actinomycin D at concentrations sufficient to inhibit DNA directed RNA synthesis did not prevent the increase in arginase activity, suggesting that the synthesis of the enzyme took place in response to a preformed stable messenger RNA. 3. 3. A few hours after the addition of the missing nutrient to a prestarved culture, arginase synthesis was again retarded. 3.1. (a) This decrease in the rate of enzyme formation could be prevented by addition of actinomycin D. The decrease in the rate of enzyme synthesis in absence of actinomycin, therefore, might not be due to a destruction of the specific messenger RNA, but rather to a depression of the rate at which the stable arginase messenger was translated. 3.2. (b) When proline was added to the culture medium the decrease in the rate of enzyme formation was enhanced. The experimental results suggest a regulation of arginase synthesis on the level of translation by means of an actinomycin sensitive repressor. The activity of this repressor seems to depend on the presence of a low molecular co-repressor, which accumulates in the cells in presence of proline. 4. 4. When proline was added to the deficient medium during the starvation period, the increase in arginase activity immediately after the addition of the missing nutrient was partially prevented. 4.1. (a) An increased rate of arginase formation was produced when these cells, prestarved in presence of proline, were transferred to a “normal” growth medium without added proline. 4.2. (b) This stimulation of arginase synthesis, brought about by the removal of proline, was not prevented by the addition of actinomycin D and therefore seemed not to be dependent of a formation of new messenger RNA. These results again suggest a proline dependent repression of arginase synthesis at the level of translation of a preformed, stable messenger RNA.