Siccanin at 3 mug/ml completely inhibited the growth of Trichophyton mentagrophytes. The primary site of action of siccanin on T. mentagrophytes is succinate dehydrogenase in the terminal electron transport system. At a concentration of siccanin giving 50% inhibition of growth (0.3 mug/ml), respiration of intact cells was inhibited more strongly than any other cellular functions tested, including the syntheses of cellular ribonucleic acid, deoxyribonucleic acid, phospholipid, protein, and cell wall fractions. In addition, at the same concentration siccanin did not cause any detectable damage in the permeability of the cells. Furthermore, the oxidation of succinate in mitochondrial preparation is more sensitive to the antibiotic than respiration in intact cells. Oxidation of other substrates tested was less sensitive to siccanin than that of succinate. The antibiotic inhibited both phosphorylation and oxidation, without causing changes in the P:O ratio. Siccanin at 0.03 mug/ml, which caused 50% inhibition of succinate oxidation in mitochondria, had effect neither on the exchange reaction between inorganic phosphate (P(i)) and adenosine triphosphate (ATP) nor on that between adenosine diphosphate and ATP. An ATP phosphohydrolase activity was also insensitive to the antibiotic. At very high concentrations, however, the antibiotic slightly inhibited the P(i)-ATP exchange reaction. From those results, it was concluded that siccanin inhibits fungal growth by inhibiting the respiratory electron transport system.

Adenosine Triphosphatases↗

PubMed1970

A simple procedure for the preparation of UDP-N-acetylglucosamine-14C.

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Carbon Isotopes↗

PubMed1970

Feedback inhibition of L-glutamine D-fructose 6-phosphate amidotransferase by uridine diphosphate N-acetylglucosamine in Neurospora crassa.

The enzyme, l-glutamine d-fructose 6-phosphate amidotransferase (EC 2.6.1.16) of Neurospora crassa, which catalyzes the formation of glucosamine 6-phosphate was shown to be subject to feedback inhibition by uridine diphosphate N-acetyl-d-glucosamine (UDP-GlcNAc). The conclusion is based on the following observations. UDP-GlcNAc, the direct precursor of chitin, did not accumulate in the cell even when its utilization for the synthesis of cell wall chitin was interrupted by the antibiotic polyoxin D, a competitive inhibitor of the chitin synthetase (EC 2.4.1.16). Furthermore, the cellular level of UDP-GlcNAc rose in a short period of time when the amidotransferase was bypassed in vivo by the addition of glucosamine to the growing medium of the fungus. The amidotransferase was purified from N. crassa approximately 85-fold. Kinetic studies showed that UDP-GlcNAc was a potent and specific inhibitor of the amidotransferase, and that it did not alter the Michaelis constant for either l-glutamine or d-fructose 6-phosphate, suggesting that the inhibitor binds at a site on the enzyme distinct from the active site.

Anti-Bacterial Agents↗

PubMed1970

Mechanism of action of the antifugal agent polyoxin D.

The antibiotic polyoxin D was shown to inhibit the incorporation of (14)C-glucosamine into cell wall chitin in Neurospora crassa at levels which were comparable with those required for inhibition of fungal growth. At the same time, the antibiotic increased the accumulation of a nucleotide, which was identified as uridine diphosphate (UDP)-N-acetylglucosamine, indicating inhibition of chitin synthesis. Chitin synthetase (UDP-N-acetylglucosamine: chitin N-acetylglucosaminyl transferase, EC 2.4.1.16) of N. crassa was found to be strongly inhibited by polyoxin D, as determined by the transfer of (14)C-N-acetylglucosamine from (14)C-UDP-N-acetylglucosamine to the particulate fraction. The inhibition was competitive with respect to UDP-N-acetylglucosamine and specific for chitin synthetase. The K(i) for polyoxin D in the reaction was 1.40 x 10(-6)m, and the K(m) for UDP-N-acetylglucosamine was 1.43 x 10(-3)m. The formation of osmotically sensitive, protoplast-like structures, when the fungus Cochliobolus miyabeanus was grown in the presence of polyoxin D, also suggested that the primary site of action of polyoxin D was in the formation of cell wall structures.

Antifungal Agents↗

PubMed1970