Asynchronous and synchronous HeLa S-3 suspension cultures treated with colcemid and vinblastine (VLB) resulted, in the former case, in suppression of 3H-leucine incorporation into protein which became increasingly apparent as the percentage of M-phase-arrested cells increased. In the latter case, where no cells were allowed to enter M-phase, no significant inhibition of incorporation occurred. M-phase cells were collected by shake-off from asynchronous monolayers, or alternatively monolayers presynchronised 10 h before shake-off, either in the presence or absence of alkaloids. In both cases, alkaloid-treated cultures showed marked inhibition of protein synthesis, while controls were largely unaffected. The results indicate that marked suppression of protein synthesis in alkaloid-arrested M-phase population is primarily due to the inability of cells to complete mitosis. Arrested cells showed no increase in size or protein content with time, but began disintegrating approximately 6-7 h after alkaloid treatment. The necessity for cells to traverse M-phase quickly is emphasised, otherwise any transient decline of protein synthesis during M-phase becomes grossly exaggerated.

Most mitotic HeLa cells divided into two daughter cells with half the volume of the parent; no additional reduction in volume was detectable during late telophase or the early part of G1. Synchronous growth throughout the next generation cycle was exponential, without an additional and sudden rise in volume being detectable as cells entered M-phase. Since the wet weight/dry weight ratio and the protein per unit mass of cells remained constant throughout the cycle, measurements based on volume changes accurately reflected growth parameters and cell water content. Nuclear magnetic resonance (n.m.r.) t1 (spin-lattice) proton relaxation times for intracellular water for M-phase cells measured at both 32 MHz (25 degrees C) and 80 MHz (37 degrees C) in a Bruker spectrometer had the same values as normal interphase cells. Furthermore, cells arrested in metaphase by nitrous oxide or alkaloids gave the same t1 values. Artificial manipulation of cell hydration by adjustment of the tonicity of the external medium led to t1 values that correlated well with the level of intracellular water. On this basis, a 40% increase in hydration raised t1 values by a factor of 1.29 at 32 MHz and 1.5 at 80 MHz. This is considerably less than the increase by a factor of 1.9 in t1 time reported with the 40% rise in cell water accompanying mitosis, measured at 30 MHz and 25 degrees C under isotonic conditions by others. The protein-synthesizing ability of hydrated cells was reduced to only half the normal level after a doubling of intracellular water. The data from these several different analyses, taken together, strongly indicate that the water content of mitotic cells is very similar to that of interphase cells, and that certain features unique to the mitotic phase of the cell cycle cannot be ascribed to an elevated (free) water content.

Linear kinetics of incorporation of labelled amino acids into protein have been reported in many cell types from bacteria to mammals. Using HeLa S-3 cells as a paradigm in carefully controlled kinetic studies of this growing mammalian cell system, detailed analysis of many of the factors which are involved under steady growth state conditions indicates that linearity is more apparent than real. Quasi-linear kinetics are not primarily due to the time-scales of experiments being too short for adequate distinction between linearity and shallow exponential kinetics, nor to the fact that conventional assay procedures of biological material are too variable (although both have been taken into account), but to the highly complex pattern of protein turnover following synthesis. Synthesis is a zero-order reaction which would inevitably give exponential kinetics if it were not offset by a series of first-order degradation reactions operating at different intensities during the subsequent history of each cohort of protein molecules produced by the cell. These interactions, however, give a net result for incorporation of label into protein which closely approximates linearity, and hides the true complexity of the underlying processes. These findings on labelling kinetics are discussed throughout in relation to the growth characteristics of the cells.

Publisher Summary Since life is based on protein chemistry, it follows that there is probably some “primitive” or basic mechanism, common to all organisms, by which amino acids have been obtained since the origin of life on earth. Different life forms may subsequently have made modifications in the manner by which they accumulate or store amino acids, but similarities in dealing with amino acids should still be more striking than differences in the diversity of organisms which have been studied. As protocols for the preparation and extraction of pools vary significantly from laboratory to laboratory, it is difficult to compare and draw conclusions from the artificial end points that they provide. Furthermore, pools of amino acids are often referred to as if they are distinct and individual entities in their own right. It has been accepted for a long time that amino acids experience difficulty in entering cells because they are virtually insoluble in lipid, and membrane theory predicts, (but does not demonstrate, that they will be excluded. We shall not cease from exploration And the end of all our exploring Will be to arrive where we started And know the place for the first time. T. S. ELIOT (“Four Quartets”)

The turnover of nascent proteins in HeLa S-3 cells was estimated simultaneously with synthesis by the difference between the expected accumulation of radioactivity, based on rate determinations from very short pulse treatments, from that observed by continuous exposure for 100 min. The results were similar to those from conventional pulse-chase analysis, but not identical for reasons which are discussed. They confirm that extensive protein turnover accompanies synthesis in the growing cell, and therefore net synthesis is much lower than gross synthesis. Cells retained all proteins synthesised during heat treatment at 45 degrees C, indicating the complete inhibition of protein degradation, cells retaining all proteins synthesised. This particular perturbation experimentally validated the deficit method, and its potential usefulness in future turnover studies.

Etude sur cellules Hela de l'absorption d'aminoacides selon les conditions operatoires et la structure chimique ou le pouvoir rotatoire des aminoacides

Intracellular acid-extractable pools preloaded with amino acids before the cells were placed in lower concentrations of labelling amino acid showed a massive infiltration of the label at resuspension. In some the overshoot was rapid, in others it was slower developing, but in all cases the final level returned to the control value. Some amino acids can produce an overshoot in preloaded pools of others, whereas in certain combinations, e.g. alanine preloaded cells challenged with leucine, no discernible change in uptake kinetics was seen. Discharge of the preloaded pool is a necessary prerequisite for overshoot since cells placed in challenges of equal concentration to the preloading conditions do not exhibit this phenomenon. There is no energy requirement for the rapid phase of overshoot to occur since it follows similar kinetics at 2 degrees and 37 degrees C. The results are discussed in terms of exchange reactions occurring within the acid-extractable pool which is composed of amino acids in a complexed form, and a hypothesis is offered to explain the phenomenon. This hypothesis is consistent with the cyclical perfusion model of Wheatley and Inglis (1980), and does not contravene Fick's law ans the second law of thermodynamics.

The acid-extractable pool in mammalian cells is composed of amino acids bound to or complexed with other molecules, possible proteins. It does not appear to consist of free amino acid molecules restrained within the cell by a supposedly impermeable plasma membrane. Attempts to characterize the pool have involved permeabilizing cells to macromolecules by standard procedures, and extraction of amino acids by a variety of chemical and physical methods. The molecules seem to be held by covalent bonds of a partially ionic nature, or in the form of a complex which may involve more than one other moiety. Simple ionic and hydrogen bonding have been ruled out, and disulphide bonds are not involved. Pools can be as effectively extracted with the lower alcohols as with organic acids. Further characterization will require the exceedingly difficult task of releasing amino acids from cells without disrupting their links with other moieties. The findings are consistent with the behaviour of pools in relations to protein synthesis (Wheatley and Inglis, 1980), and may also explain differential discharge kinetics.

Amino acid displacement is a separate and easily distinguishable phenomenon from discharge of the acid-extractable pool. It is characterized by its extreme rapidity for certain amino acids, with a resetting of the concentrations of different species in the pool at a new level, from which normal discharge kinetics then continue. It is possible to insert a labelled amino acid into the acid-extractable pool by displacement by the same mechanism as labelled amino acid can be ejected from the pool. The amount of displacement which occurs is dependent on the total number of potential displacing molecules available and not simply on their concentration in the medium. Differences occur between amino acids in their ability to displace the pool or to be displaced from the pool. The major determining factor is the strength with which a resident amino acid in the pool is complexed. Displacement for the more weakly complexed amino acids occurs at 0--2 degrees C, and requires no energy supply. The results are discussed in relation to the nature of the pool itself, and the implications of displacement on the uptake of amino acids into the cell.

HeLa cells exposed to 10mM leucine in medium for 30 mins at 37 °C showed greatly increased uptake of tritiated leucine when returned to medium containing 0.1mM levels of labelled amino acid. Further analysis of the phenomenon demonstrates that the steepness of the gradient is essential for rapid uptake to be observed, that no stereo-specificity is involved, and that the peak of uptake is followed by discharge with first order kinetics back to equilibrium. The effect of 14 different amino acids examined individually as 10mM supplements to the labelling medium ranged from slight reduction (lysine, proline) to complete suppression of the ‘stimulated uptake’ (phenylalanine, isoleucine).

The three major routes by which amino acids are released from the acid-extractable pool in mammalian cells are described. The kinetics with which different amino acids are displaced and the rates at which they are discharged show considerable variation, suggesting that each has a different affinity for some complexing agent, the chemical nature of which remains unknown. The rate of efflux from cells has a direct bearing on the kinetics of uptake into the pool, and this review draws attention to the need to re-evaluate uptake and its regulatory controls in terms of what is happening on the efflux side of the movement of amino acids through the cell.

A working model is proposed accounting for the relationship between pools and protein synthesis in heterotrophic eukaryotic cells. A constant intracellular perfusion system moves amino acids past the selection mechanism for protein synthesis but operates quite independently of the latter. By maintaining as high a flux as possible, the cell maximizes its chances of retrieving all the required amino acids for protein synthesis even under adverse conditions such as gross imbalances or restriction of the availability of amino acids. The intracellular acid soluble pool is largely, probably completely for most amino acids, on the efferent limb of the proposed cyclical perfusion system, i.e. amino acids in this pool are no longer directly retrievable for protein synthesis.