Fractionation of the mycobacillin-synthesizing enzyme system

Abstract

The mycobacillin-synthesizing enzyme system was highly purified by fractionation at 30-55% (NH4)2SO4 saturation. The enzyme concentrate on Sephadex G-200 gel chromatography was resolved into three distinct fragments. Each of the fragments on further purification by DEAE-cellulose ion-exchange chromatography behaved as a single-component system, as clearly indicated by the sharpness of the peaks in the elution diagram. None of the fragments alone nor any two of them in all possible combinations possessed mycobacillin-synthesizing activity, which was restored only when the three fragments were used together in the test system.

Functional characterization of constituent enzyme fractions of mycobacillin synthetase

“…The incubation mixture and the method of isolation of radioactive mycobacillin were the same as described by Ghosh et al (1983), and that the radioactive material was mycobacillin was further confirmed by silica-gel t.l.c (Kieselgel F 1500;Schleicher und Schiill, Dassel, Germany) with the solvent system propanol/conc. NH3 (2:1, v/v), the radioactivity in the scraped material being found in the range of the RF value of authentic mycobacillin.…”

“…The producer organism B. subtilis B3 (Majumder & Bose, 1958) The exchange was studied by using the method of Stulberg & Novelli (1960) as described by Ghosh et al (1983).…”

“…The biosynthesis occurs through sequential activation, by ATP-Pi exchange reaction, of the mycobacillin amino acids starting from L-proline (Sengupta & Bose, 1972, 1974, and the enzyme complex involved in the synthesis is a threefraction enzyme (A, B and C) called mycobacillin synthetase (Ghosh et al, 1983). All the fractions are localized in cytosol in the early exponential phase (Sengupta & Bose, 1971), but are subsequently translocated to plasma membrane during the stationary phase (Mukhopadhyay et al, 1985).…”

Accumulation of Peptides by Mycobacillin-negative Mutants of Bacillus subtilis B3

“…Cell-free extracts of the strains were prepared and treated with (NH,),SO,. The precipitate collected between 30% and 55% saturation (Ghosh et al, 1983) was dissolved in buffer (50 mM-Tris/HCl pH 7.2, containing 0.25 mM-EDTA, 10 mM-MgCl, and 1 mM-DTT) and dialysed for 10 h against 2 litres of the same buffer, and used as a source of the enzyme. The incubation mixture (2 ml) used for the synthesis of peptides contained 5 mg enzyme, 100 pmol Tris/HCl buffer pH 7.2.…”

“…The present communication describes the isolation of some mycobacillin-negative: mutants from an auxotrophically tagged mutant of the producer B. subtilis B3 and the use of thew blocked mutants in whole-cell fermentation and cell-free in vitro synthesis, both to isolate and characterize some peptides that might be involved in mycobacillin biosynthesis, and to locate the enzyme defect in the three fractions (Ghosh et al, 1983) of the mycobacillin synthetase complex.…”