Evidence against a general role of NADP-glycohydrolase in differentiation of Streptomyces griseus.

and production of streptomycin by Actinomyces streptomycini is postulated to involve NADPglycohydrolase (E. C. 3. 2. 2. 6)4>. A-factor was reported to stimulate the formation of NADPglycohydrolase in Str-Amymutants and the product, phospho-adenosinediphosphoribose, was found to act as a competitive inhibitor of the enzymes of carbohydrate catabolism4> and the citrate cycles' during development of aerial mycelium. It was the aim of the present work to determine whether this mechanism of control by NADP-glycohydrolase exists in other streptomycetes. As a model vie used Streptoryces griseus JA 5142 forming both aerial mycelium and leukaemomycin, a daunomycin-type anthracycline antibiotic'). This strain (Amy' Lkm+) was compared with two antibiotic-blocked and non-aerial-mycelium-forming mutants, JA 5142/39 and JA 5142/86 (AmyLkmstrains)'). Strain JA 5142/86 has been shown to produce both aerial mycelium and antibiotic in surface cultures upon addition of a novel bioregulator'>. The bioregulator, distinguished from A-factor by chromatographic properties and preliminary results of physicochemical analysis, was recently isolated from the culture medium of different strains of Streptomyces griseus (GRAFE et al., to be published). When it was added at zero time to agar cultures of strain JA 5142/86, and the cultures incubated at 28°C, development of aerial mycelium (Fig. 1) and production of leukaemomycin became evident by 48 hours. Cultures of strain JA 5142/86 with and without addition of purified bioregulator of strain JA 5142 and of strain JA 5142/39 were grown separately under the same conditions and on the same medium'). After appropriate intervals, the surface mycelia were scraped from the cellophane-coated agar and homogenized by 90-second and sonic disruption at 0°C in 0.05 M tris buffer, pH 7.2 (Labsonic 1510, Braun Melsungen, F.R.G.). After 15 minutes centrifugation (23,000 xg, 0°C), the supernatant was used for the spectrophotometric assay of NADP-glycohydrolase according to ZATMAN et al.8) with the modifications reported'). The test mixture contained in a volume of 1.125 ml; 0.1 M tris buffer pII 7.4, 1 mm NADP and mycelium extract (approximately 0.02 mg of protein). Incubation time was 10 minutes (35°C). Formation of the cyanide complex of intact NADP was measured at 340 nm. Fig. 2 shows the specific activities of the enzyme in the mycelium. Both the original strain, Streptomyces griseus JA 5142 (Amy+ Lkm+), and its AmyLkmmutant, JA 5142/39, possessed high levels