Abstract
Abstract A prototrophic revertant SP22 has been isolated from a double auxotroph of Rhodopseudomonas spheroides M29.5 requiring methionine and leucine for growth. As compared to M29.5, the specific activity of threonine deaminase in cell-free extract of SP22 is about 4-fold higher; the specific activity of aspartokinase, on the other hand, is about one-fifth to one-tenth of that found in M29.5. DEAE-cellulose chromatography of crude extracts of SP22 reveals two threonine deaminase activities, designated as PR-TDI and PR-TDII. The ratio of activities of PR-TDI and PR-TDII in extracts is about 0.67. The deaminase of the double auxotrophic strain M29.5 is indistinguishable from the enzyme obtained from the wild type R. spheroides and no such enzyme activity is detected in extracts prepared from SP22. The activity of PR-TDII is much more stable to storage at 4° and during heat treatment at 55°. PR-TDI activity, on the other hand, like the M29.5 enzyme, is susceptible to rapid inactivation under various experimental conditions. l-Isoleucine protects the deaminase of M29.5 against heat inactivation, whereas this amino acid increases the rate of heat inactivation of the PR-TDI activity. In contrast to the M29.5 threonine deaminase, the enzymes of the mutant strain do not require pyridoxal phosphate for activity during successive purification steps, indicating tightly bound coenzyme on the enzyme molecules. Studies with the feedback inhibitor l-isoleucine reveal that both wild type and PR-TDI activities are strongly inhibited by this amino acid, whereas the activity of PR-TDII is much less sensitive to isoleucine inhibition. The molecular weights of all three deaminases, as judged by gel filtration on Sephadex G-200, are not significantly different from one another. These results, and the data of the kinetic studies presented in the following paper, indicate that the two enzymes obtained from the prototrophic revertant are different from each other as well as from that of the auxotrophic strain M29.5.
Highlights
MethodsGrowth of Bacteria-The synthetic medium S of Lascelles, [8] as modified by Datta [6] but without added yeast extract, was used for all liquid cultures
A prototrophic revertant SP22 has been isolated from a double auxotroph of Rhodopseudomonas spheroides M29.5 requiring methionine and leucine for growth
L-Isoleucine protects the deaminase of M29.5 against heat inactivation, whereas this amino acid increases the rate of heat inactivation of the PR-TDI activity
Summary
Growth of Bacteria-The synthetic medium S of Lascelles, [8] as modified by Datta [6] but without added yeast extract, was used for all liquid cultures. Isolation of Mutant-An exponentially growing culture of AJ29.5 in minimal medium containing 30 pg per ml each of Lmethionine and n-leucine was centrifuged and the cells were washed twice with minimal medium and resuspended in a small volume of minimal medium. Aliquots of 0.1 ml were plated on several minimal agar plates not supplemented with amino acids and the plates were irradiated for 60 set with ultraviolet light provided by a General Electric germicidal lamp at a distance of 30 cm. The irradiated plates were incubated at 30”, and after a few days several colonies appeared (at an approximate frequency of one colony for every million viable cells plated). Twenty colonies were selected randomly from three plates and were purified by repeated (three times) single colony isolation on minimal medium
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