Lividomycin inactivated

Partial purification of the lividomycin-inactivating enzyme was attempted by Mitsuhashi and coworkers, who briefly described their discovery that the enzyme obtained by fractionation with ammonium sulfate and column chromatography on Sephadex G-lOO inactivates lividomycins A and B, but not kanamycin A, indicating involvement of two different enzymes in the phosphorylation of lividomycin and kanamycin. However, it was definitely proved by H. Umezawa and coworkers that kanamycin-neomycin phosphate transferase I phosphorylates the 5"-hydroxyl group of lividomycins. The observation by Mitsuhashi and coworkers was probably occasioned by instability of the enzyme and the higher activity of the enzyme in phosphorylating lividomycins than in phosphorylating kanamycin A. 

The extrachromosomal nature of lividomycin resistance has not been proved conclusively. However, on storage loss of resistance to lividomycin along with inactivating enzyme from strains of Ps. aeruginosa occurs [198]. 

Neomycins, Paromomycins, Lividomycins. Although the tetra- and pen-tacyclic aminoglycosides have been known for some time (neomycin 1949, paromomycin 1959, lividomycin 1967), only a few chemical modifications have been carried out. Not until after the discovery of the enzymatic inactivation mechanisms were several deoxy compounds and acylated derivatives (HABA, etc.) prepared. 

Escherichia coli K12 ML1629, . coU K12 ML1410 R81, and E. coli K12 J5 Rll-2 showed similar resistant patterns to aminoglycosidic antibiotics, and the S-100 fraction contained kanamycin-neomycin phosphate transferase I. This enzyme solution also inactivated lividomycins A and B (6 and 7) in the presence of adenosine 5 -triphosphate. A crude powder of the inactivated lividomydn A extracted by column chroma-... 

Inactivated lividomycin A does not melt up to 210 , and has [a]n 4-52.2° (c 1.5, water). The empirical formula of lividomycin A monophosphate was shown by elementary analysis. It gives positive ninhydrin, Rydon-Smith, and Hanes reactions. On high-voltage paper-electrophoresis at 3.0 kV for 20 minutes, with 1 3 36 (v/v) formic acid-acetic acid-water, the inactivated lividomycin A moved 10.8 cm towards the cathode, while lividomycin A moved 12.3 cm. It showed no u.v. maximum (except end absorption). A band at 970 cm (phosphoric ester) was observed in the i.r. spectrum. The inactivated lividomycin A consumes 6.2 moles of periodate per mole in 24 horrrs, as does lividomycin A (5.9 moles per... 

The lividomycin A-Sepharose 4B was packed in a column (1.0 X 11.7 cm 9 ml) and washed with 20 mM Tris hydrochloride buffer containing 10 mM magnesium acetate, 60 mM potassium chloride, and 10 mM 1,4-dithiothreitol, and 5 ml of the supernatant liquor from the disrupted cells was passed through the column at the rate of 40 ml/hour. The enzyme adsorbed was successively eluted with a gradient of sodium chloride (0 to 0.8 M) in 20 mM Tris-hydrochloric add buffer, pH 7.2. The enzyme that phosphorylates and inactivates both lividomycin A and kanamycin appeared in the eluate made with 0.4 to 0.6 M sodium chloride. By this procedure, the enzyme was purified 25- to 40-fold in phosphorylating activity for both kanamycin A and lividomycin A. The purified fraction obtained showed two bands in disc-gel eledrophoresis, and one of the bands had the activity as regards phosphorylating kanamycin A and lividomycin A. 

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