Louise S. Prestidge

The initial kinetics of enzyme induction

Abstract Kinetics of induction of several enzymes of Escherichia coli have been investigated under conditions where non-specific nutrient effects and permeability mechanisms are not important. Measurements made over time intervals of a few minutes permitted detection of initial events brought about by addition or removal of inducers or inhibitors. With each enzyme a lag of about 3 min between addition of inducer and appearance of enzyme at 37° was noted. This lag was dependent on temperature and independent of inducer concentration, in contrast to an inhibitor which showed the opposite behavior. The induction lag, therefore, does not represent the time required for penetration of the inducer. Studies with inhibitors suggest that some metabolism involving the synthesis of a ribonucleic acid is required before enzyme synthesis can proceed. The formation of enzyme ceased about 5 min after inducer was removed or after glucose was supplied to the culture, suggesting that the enzyme-forming system is unstable. A model for the enzyme forming apparatus of the cell is suggested which is consistent with the kinetic data and with other information regarding induction. A repressor prevents function of the genetic material of the bacteria. The inducer is thought to prevent the repressor from acting. This freed genetic material forms a special, unstable ribonucleic acid which interacts with the ribosomes to provide an active template for enzyme synthesis. The time required for enzyme formation to commence is attributed principally to the interval during which the repressor is lost and the unstable ribonucleic acid is formed. A possible similarity between repressor and unstable ribonucleic acid is suggested. Instability appears to be spontaneous at temperatures where metabolism goes on, rather than being caused by use of the system for enzyme production.

A study of the ribonucleic acid of normal and chloromycetin-inhibited bacteria by zone electrophoresis☆

Abstract 1. 1. Bacterial RNA migrates in only two bands on starch electrophoresis. The major band consists of the two kinds of nucleoprotein particles previously found by ultracentrifugation. RNA in the two bands is bound with different affinities to protein; these two forms of RNA have different base ratios and molecular sizes, and do not appear to be metabolically related. 2. 2. Most of the RNA of rapidly growing E. coli or B. megatherium is in the form of nucleoproteins. Less than 15%, if any, is present as protein nucleates. 3. 3. RNA made in the presence of chloromycetin is bound to protein but has a different mobility than does most of the RNA of normal E. coli. In contrast to normal RNA it is readily freed from protein by sonic treatment. During exposure to chloromycetin one of the two kinds of normal nucleoprotein particles disappears. 4. 4. The mobility of DNA in the starch electrophoresis apparatus depends on molecular size. Conclusions regarding the attachment of DNA to proteins could not be made in these experiments. 5. 5. The effects of variables such as methods of preparation of extracts, pH, buffers, and age of cells upon the starch electrophoresis patterns of bacterial extracts have been investigated.

Effects of azatryptophan on bacterial enzymes and bacteriophage

The influence of a number of amino acid analogs on the ability of Escherichia coli to form proteins was investigated. The analog of tryptophan, 7-azatryptophan, is incorporated into proteins and permits synthesis of proteins and nucleic acids, but the majority of enzymes, and also bacteriophages T1 and T2, did not appear in active forms. Two enzymes, asparate carbamyl transferase and D-serine deaminase, were formed in about half the maximal amounts in the presence of azatryptophan, but not when the tryptophan supply was limited by various other means. Kinetics of inhibition showed that under the experimental conditions 1 and 1.8 minutes were required to establish and reverse the inhibition of β-galactosidase formation, respectively. Phage formation was much more strongly inhibited by the analog than was bacterial growth, thereby providing a kind of chemotherapy. Inhibition of phage development was observed in both the early and late periods after infection. When added a few minutes after the time of infection, azatryptophan inhibited the formation of phage DNA much more strongly than the development of resistance to ultraviolet light; this suggests that the former is not necessary for the latter phenomenon. Protein made in the presence of azatryptophan and isolated in inactive, centrifugable particles had none of the properties of phage protein, but was associated with DNA of the phage type. The analog acts so as to cause major imperfections in the phage protein rather than by simply being incorporated in place of tryptophan.

Inactivation of enzyme formation by ultraviolet light. I. Action spectra and size of the sensitive unit.

Abstract The action spectra for loss of ability to form the inducible enzymes β-galactosidase and tryptophanase by Escherichia coli , and also for colony formation, resemble the absorption spectra of nucleic acids. A minimum size of the sensitive unit for enzyme formation of 300,000 molecular weight units is computed. This suggests that the sensitive unit is either high molecular weight RNA or DNA and not soluble RNA. On the basis of previously reported quantum yields, the size of the target is estimated to be somewhat larger than the minimum, perhaps of mass 700,000. This result is in fair agreement with present estimates of the size of a functional gene or of the RNA of a ribonucleoprotein particle. These data do not permit a choice between RNA and DNA as target materials.