Nejvíce citovaný článek - PubMed ID 7084821
Effect of inorganic phosphate and benzyl thiocyanate on the activity of anhydrotetracyline oxygenase in Streptomyces aureofaciens
Cell protein profiles of submerged cultures of Streptomyces aureofaciens cultivated in the absence or presence of 12 microM benzyl thiocyanate (BT) were analyzed by one-dimensional SDS polyacrylamide gel electrophoresis. Substantial increase in the intensity of the 13, 35, 37, 60, and 100 kDa protein bands was observed in cultures treated with BT. Similar increase in the 35, 37, and 60 kDa bands was found in a mutant blocked in the last chlortetracycline biosynthesis step. Effect of BT on the solid medium-grown cultures was also observed, with a more intensive substrate mycelium pigmentation and alteration in the spore size and shape as the most characteristic features. Earlier studies of BT effect involving those on the stimulation of chlortetracycline biosynthesis are summarized and a possible signal-transducing mechanism is discussed from the point of view of adaptation of S. aureofaciens to the uncoupling of oxidative phosphorylation.
- MeSH
- bakteriální proteiny metabolismus MeSH
- elektronová mikroskopie MeSH
- Streptomyces aureofaciens účinky léků metabolismus ultrastruktura MeSH
- tetracykliny biosyntéza MeSH
- thiokyanatany farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bakteriální proteiny MeSH
- benzyl thiocyanate MeSH Prohlížeč
- tetracykliny MeSH
- thiokyanatany MeSH
Anhydrotetracycline oxygenase was purified to homogeneity from Streptomyces aureofaciens, a producer of tetracycline. The enzyme was purified 60-fold in a 40% yield by a two-step procedure using a combination of hydrophobic chromatography and ion-exchange h.p.l.c. Purified anhydrotetracycline oxygenase was homogeneous according to SDS/polyacrylamide-gel electrophoresis, isoelectric focusing, ion-exchange h.p.l.c. on a Mono Q HR 5/5 column and size-exclusion h.p.l.c. on a TSK G 3000 SW column. The enzyme consists of two subunits of Mr 57,500, as determined by SDS/polyacrylamide-gel electrophoresis.
The localization of anhydrotetracycline oxygenase and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was studied by determining the enzyme activities in subcellular fractions obtained by differential centrifugation of the mycelia of Streptomyces aureofaciens after lysozyme treatment. Glucose-6-phosphate dehydrogenase was a typical cytoplasmic enzyme both in the low- and high-production strain. Anhydrotetracycline oxygenase was found in the membrane fraction of the low-production strain. In the high-production strain, it was detected in several fractions, the highest activity being found in cytoplasm. The presence of 10 microM benzyl thiocyanate in the culture medium significantly changed the distribution of the latter enzyme in both strains. The redistribution of the enzymes is discussed with respect to tetracycline over-production.
- MeSH
- frakcionace buněk metody MeSH
- glukosa-6-fosfátdehydrogenasa metabolismus MeSH
- hořčík farmakologie MeSH
- kinetika MeSH
- oxygenasy metabolismus MeSH
- Streptomyces aureofaciens účinky léků enzymologie MeSH
- thiokyanatany farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- anhydrotetracycline oxygenase MeSH Prohlížeč
- benzyl thiocyanate MeSH Prohlížeč
- glukosa-6-fosfátdehydrogenasa MeSH
- hořčík MeSH
- oxygenasy MeSH
- thiokyanatany MeSH
Mycelia of a low- and a high-production strain of Streptomyces aureofaciens were converted into protoplasts and divided into five subcellular fractions in order to localize exopolyphosphatases (EC 3.6.1.11), triphosphatase (EC 3.6.1.25), inorganic diphosphatase (EC 3.6.1.1), apyrase (EC 3.6.1.5) and glucokinase (EC 2.7.1.2). The highest specific activity of enzymes hydrolyzing polyphosphates was found in cytoplasmic vesicles and membranes. Triphosphatase was detected in the periplasmic fraction. Periplasmic vesicles and cytoplasm exhibited a high activity of diphosphatase. Apyrase was found mainly in the fractions of membranes and cytoplasmic vesicles. Glucokinase was a cytoplasmic enzyme. The enzymes were released from membrane structures into cytoplasm or periplasmic space if benzyl thiocyanate (10 microM) was present in the growth medium.
- MeSH
- draslík farmakologie MeSH
- fosfatasy metabolismus MeSH
- glukokinasa metabolismus MeSH
- hořčík farmakologie MeSH
- kationty MeSH
- kinetika MeSH
- sodík farmakologie MeSH
- Streptomyces aureofaciens účinky léků enzymologie MeSH
- subcelulární frakce enzymologie MeSH
- thiokyanatany farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- benzyl thiocyanate MeSH Prohlížeč
- draslík MeSH
- fosfatasy MeSH
- glukokinasa MeSH
- hořčík MeSH
- kationty MeSH
- sodík MeSH
- thiokyanatany MeSH
ATP diphosphohydrolase activity and inorganic pyrophosphatase reached two maxima during cultivation of the low- and high-producing variant of Streptomyces aureofaciens under conditions of phosphate limitation, i.e. after 30 and 70 h of cultivation. Increased levels of inorganic phosphate in a medium inhibitory to biosynthesis of chlortetracycline markedly decreased the levels of both enzymes. The ATP diphosphohydrolase activity was detected both in the supernatant and membrane fractions of the cell-free preparation of the mycelium.
- MeSH
- apyrasa metabolismus MeSH
- chlortetracyklin biosyntéza MeSH
- fosfatasy metabolismus MeSH
- fosfáty farmakologie MeSH
- kultivační média MeSH
- membrány enzymologie MeSH
- pyrofosfatasy metabolismus MeSH
- Streptomyces enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- apyrasa MeSH
- chlortetracyklin MeSH
- fosfatasy MeSH
- fosfáty MeSH
- kultivační média MeSH
- pyrofosfatasy MeSH
