Incorporation of p-fluorophenylalanine into protein by a cell-free system.
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Bacterial Proteins↗
Biomedical subjects
F R Leach
Publications and source records attributed to F R Leach.
Effect of Bromouracil-containing Deoxyribonucleic Acid on Bacillus subtilis.
Gimlin, Dixie M. (Oklahoma State University, Stillwater), Sue D. Hardman, Betty N. Kelley, Grace C. Butler, and Franklin R. Leach. Effect of bromouracil-containing deoxyribonucleic acid on Bacillus subtilis. J. Bacteriol. 92:366-374. 1966.-Replacement of one-half of the thymine with bromouracil in Bacillus subtilis transforming deoxyribonucleic acid (DNA) resulted in a slight decrease in transforming activity, but, when used at high concentrations, this DNA preparation inhibited cell growth. Acid-hydrolyzed DNA, or addition of equivalent concentrations of the free base bromouracil in a transforming mixture, was without effect on cell growth. Treatment of the DNA preparation with deoxyribonuclease completely destroyed transforming activity and killing effect, whereas treatments with ribonuclease and trypsin were without effect on either transformation or killing activity. Growth of competent B. subtilis cells in test tubes was inhibited by high concentrations of both normal and bromouracil-containing DNA, with the bromouracil-containing DNA being significantly more inhibitory. This type of inhibition was also reflected in the time of division of the cells. The inhibitory effect was not due to viscosity, or to mutagenicity. The time course of killing paralleled transformation, and competency was required. These results can be interpreted as being due to uptake of homologous but imperfect DNA (containing bromouracil instead of thymine) by means of the systems involved in transformation, followed by either integration (resulting in lethal transformation, activation of a defective, nonlytic but lethal prophage) or interference with the recombination mechanism.
Journal Article↗
Use of firefly luciferase for ATP measurement: other nucleotides enhance turnover.
Firefly luciferase utilizes only ATP and a few closely related nucleotides as substrates for the formation of luciferyl adenylate which is an intermediate in the bioluminescent reaction sequence that oxidizes firefly luciferin. The enzyme shows two different time courses of light production depending on ATP concentration used: a flash with high concentrations of ATP (> 8 microM) or a fairly constant production of light with lower concentrations of ATP (< 1 microM). Many nucleotides, nucleotide-containing substances and other compounds, when added either prior to or 1 min after the addition of ATP, change the time course of light production. When added before ATP, these compounds yield a reaction mixture in which light production is fairly constant (at the level characteristic of the flash observed with that ATP concentration). When the compounds are added after ATP addition, light production is markedly stimulated and the higher rate of light production is maintained for several minutes. There is an increase in quanta of light produced per luciferase dimer from 1 to 5/min with the addition of any of several nucleotide analogues. These results are consistent with a stimulated release of the inhibitory product oxyluciferin, allowing turnover of the enzyme. This enzyme turnover permits more light output at high ATP concentrations, thus enhancing the sensitivity of enzyme determination.
Adenine Nucleotides↗
Stability of firefly luciferase in tricine buffer and in a commercial enzyme stabilizer.
A solution of firefly luciferase in AuthentiZyme Enzyme Stabilizer retains full activity when stored in an ice bath (0.5 degrees C) during one day. These solutions have the advantage that no additional protein (other than the luciferase) is present, which is desirable for proteolytic digestion and protein derivatization experiments. For longer-term experiments, firefly luciferase solutions in 0.05 mol/l Tricine buffer at pH 7.8, 10 mmol/l MgSO4, 1 mmol/l EDTA, and 1 mmol/l DTT which contain 100 micrograms/ml of bovine serum albumin are stable for 6 weeks if frozen and thawed only once.
Animals↗
Determination of sterilization effectiveness by measuring bacterial growth in a biological indicator through firefly luciferase determination of ATP.
A bioluminescence procedure for measurement of microbial ATP allows a rapid determination of the effectiveness of autoclave sterilization. This determination is achieved faster than detection of acid production in a biological indicator via a pH indicator. Bacterial outgrowth from spores on test strips of the biological indicator was detected by measurement of ATP using the firefly luciferase reaction. A measureable increase in ATP was found after 5 hours of incubation of a biological indicator that had been treated under sterilizing conditions that produced 75% sterility of the biological indicator as measured by acid production. This is a marked improvement over the 24-48 hours of incubation currently required.
Adenosine Triphosphate↗
In memoriam Dr Marlene DeLuca. 1987 O. M. Smith Lecture. Firefly luciferase: mechanism of action, cloning and expression of the active enzyme.
The untimely death of Marlene DeLuca in 1987 has deprived the scientific community of an outstanding expert on bioluminescence. Earlier in that year she was honoured as the thirty-ninth recipient of the Otto Mitchell Smith Lectureship Award at Oklahoma State University, Stillwater, Oklahoma. On 20 March 1987 Dr DeLuca presented a scientific lecture entitled 'Firefly Luciferase--Mechanism of Action, Cloning, and Expression of the Active Enzyme' and a popular lecture at the banquet that evening entitled 'Light and Life'. She was selected for her excellence in research, her oral presentation ability, and her personableness. Marlene was the first woman so honoured. To honour Dr Otto M. Smith the Alpha Delta Chapter of Phi Lambda Upsilon, a national chemistry honorary organization, inaugurated The Otto Mitchell Smith Lectureship in 1948 at Oklahoma State University. Former awardees include Nobel Laureates H. C. Brown, Stanford Moore, and Arthur Kornberg and the following prominent biochemists/molecular biologists: Robert A. Alberty, University of Wisconsin; Daniel E. Koshland, Brookhaven National Laboratory; Sol Spiegelman, University of Illinois; Carl Djerassi, Stanford University; and John T. Edsall, Harvard University. The lectureship honours Dr O. M. Smith, who was Director of the Research Foundation, professor, and Head of the Departments of Chemistry and Chemical Engineering. As a tribute to Dr DeLuca's outstanding contribution to bioluminescence we reproduce here the edited text of her Otto Mitchell Smith Lectureship and a selected bibliography of her work on firefly bioluminescence.
Awards and Prizes↗
Bioluminescent determination of 0.1 picomole amounts of guanine nucleotides.
A bioluminescence procedure for the determination of the guanylates has been optimized to allow measurement of 0.1 pmol amounts. Modifications of the Karl procedure include the use of purified firefly luciferase and nucleoside diphosphate kinase instead of a crude extract of firefly tails, the use of Tricine buffer instead of the inhibitory arsenate buffer, and optimization of the amounts of reagents and incubation times for each of the partial reactions. In the determination of GMP, background values varied widely with different lots of bovine guanylate kinase. Careful selection of a suitable lot of bovine brain guanylate kinase was essential for determination of lower amounts of guanylates. This establishes that selection of guanylate kinase must be based on experimental determination and not reported adenylate kinase activity. The wide variation in background was not eliminated by the inclusion of adenylate kinase inhibitors.
Adenylate Kinase↗