Martin Lubin

On the role of intracellular potassium in protein synthesis

Abstract 1. 1. Mutants of Escherichia coli B that can be readily depleted of K+ were used to study the role of K+ in cell growth. When cells were depleted of K+, cell division and protein synthesis stopped, but ribonucleic acid synthesis continued. 2. 2. The effect of K+ and Na+ on protein synthesis was measured over a wide range of concentrations both in intact mutant cells and in the cell-free polyuridylic acid system. Na+ was found to antagonize the action of K+. NH4+ produced 2- to 4-fold more incorporation in the cell-free system than did K+. 3. 3. In the cell-free system, the rate of protein synthesis at low K+ concentration was found to be limited by the transfer of amino acid from aminoacyl soluble ribonucleic acid to polypeptide. This result was consistent with the finding of unbalanced ribonucleic acid synthesis in K+-depleted mutant cells. 4. 4. The intracellular NH4+ concentrations was found to be low, and appeared to be controlled at levels of 5–10 mM. 5. 5. The results suggest that decreased cell K+ limits the rate of cell growth by a specific effect on protein synthesis.

Pre-ribosomal particles formed in potassium-depleted cells: Studies on degradation and stabilization

Abstract 1. 1. When cells of mutant strain B207 of Escherichia coli were incubated in a sodium salts medium, they failed to grow, but they accumulated RNA (K + -depletion RNA) without any increase in protein content. About half of the K + -depletion RNA was contained in particles (K + -depletion particles) which appear to be similar to chloramphenicol particles. The conditions that promote degradation or stabilization of K + -depletion particles were studied. 2. 2. K + -depletion particles appeared in the analytical ultracentrifuge as twin peaks, with sedimentation coefficients of about 14 S and 18 S. When cells containing labeled K + -depletion particles were transferred to a medium supporting growth, the particles were rapidly converted to 30-S and 50-S ribosomes, without evidence of prior degradation. 3. 3. K + -depletion particles were more sensitive to degradation than were the other components of K + -depletion RNA. Degradation of K + -depletion RNA was rapid if intracellular K + was high, but when intracellular K + was largely replaced by Na + , the rate of degradation decreased to one-sixth. In contrast, pulse-labeled RNA was degraded rapidly in the presence of high intracellular levels of either K + or Na + . Hence the RNA portion of K + -depletion particles appears to be degraded by an enzyme that is strongly activated by K + .

On the distinction between peptidase activity and peptide transport

Abstract 1. 1. Mutants of a glycine-requiring strain of Escherichia coli have been isolated and used to show that active transport of peptides, and peptidase activity, are separable functions of the bacterial cell. 2. 2. One mutant, lacking the transport system for glycylglycine but processing the peptidase, grew well in media containing high levels of glycylglycine but failed to grow at low levels. 3. 3. Another mutant, lacking the peptidase but possessing the transport system, was unable to grow on glycylglycine supplied at high or low levels. In this mutant, intracellular glycylglycine reached as much as one hundred times the extracellular level. 4. 4. Studies of competition between peptides show that glycylglycine is carried by a transport system with broad specificity.

Transport of proline inEscherichia coli

Abstract Mutants of Escherichia coli , blocked in proline biosynthesis, and requiring very high external proline supplements for growth, have been studied. The mutants show normal permeability for inward and outward proline diffusion, and have no apparent defect in biosynthetic systems involved in protein biosynthesis. The high proline requirement has been related to the lack of the specific process responsible for concentrative uptake of proline at 37°. The mutant strain also lacks the ability to carry out rapid exchange at 0° between an intracellular pool and a low level of extracellular [ 14 C]proline. This suggests that concentrative uptake at 37° and exchange at 0° are mediated by closely related processes.

Selective synthesis of ribosomal protein during recovery from unbalanced growth

Abstract 1. 1. Cells of Escherichia coli were permitted to accumulate RNA under conditions of unbalanced growth. A study was then made of the readjustment of rates of RNA and protein synthesis during the recovery period. Two methods were primarily used to produce accumulation of RNA without simultaneous synthesis of protein: incubation of mutant cells in a sodium salts medium and methionine starvation of a relaxed-control amino acid auxotroph. 2. 2. During the recovery period, cells displayed the following properties: (a) protein synthesis was rapid, but RNA synthesis was slowed down until the RNA/protein ratio reached the level characteristic of steady-state growth; (b) in the initial burst of protein synthesis, the specific activity of the protein that sedimented with ribosomes was about 3 times greater than the specific activity of the supernatant protein; and (c) the protein of high specific activity remained as a stable constituent of ribosomes. 3. 3. The results indicate that E. coli cells have the capacity to regulate synthesis of ribosomal protein, perhaps by a mechanism involving messenger activity of the unbalanced RNA in pre-ribosomal particles.

Capacity for synthesis of a pyrimidine biosynthetic enzyme in mammalian cells

Abstract 1. 1. The control of formation of the first enzyme unique to the pyrimidine pathway was studied in Sarcoma-180 cells growing in culture. Pyrimidine starvation, induced by the addition of 6-azauridine, produced a three- to four-fold increase in the level of aspartate transcarbamylase relative to total cell protein. 2. 2. The level of this enzyme was not changed either by the addition of preformed pyrimidines to growing cultures, or by the simultaneous addition of uridine and 6-azauridine. 3. 3. The results show that the level of aspartate transcarbamylase is under the control of end-product pyrimidines, and that the cells have a potential forincreased enzyme synthesis ordinarly not expressed.

Measurement of Mechanical Properties of Muscle under Servo Control

Accurate measurement and analysis of the mechanical events in active muscle requires the use of high-speed equipment. A hydraulic servo-valve, controlled by analog units (integrators, adders, inverters), can be used to control the speed of shortening of muscle at rates as high as 1 mm per millisecond. The apparatus can be used for isometric, isotonic, and controlled release experiments. Both release and stretch, at high or low speeds, can be produced during a single contraction cycle. Force is measured by an unbonded strain gauge of high natural frequency and low compliance. To maintain constant force on the muscle, a signal proportional to measured force is fed into an error detector, whose output controls the servovalve piston. The instrumentation described can provide the necessary and sufficient information to specify completely both transient and steady-state mechanical properties of muscle.