Studies on tricarboxylic acid cycle of Trichophyton

Abstract

There exists a considerable literature on the tricarboxylic acid cycle (TCA cycle) in bacteria and Candida, but little work has been done on that in Trichophyton. On the other hand, sufficient knowledge concerning the acting mechanism of antifungal agents on fungi is not yet obtained.This report deals with the biochemical assay of cell free extract of Trichophytons on some dehydrogenase and fumarase activities which concern with TCA cycle, and the effects of several antifungal agents on these enzyme activities.The strains of Trichophyton used in this study were Tr. mentagrophytes strain 840 obtained from the Department of Dermatology, Juntendo University, and Tr. rubrum isolated from patient in Tonan Hospital. The material which was scraped from two-months' Sabouraud culture was removed to 200ml Sabouraud glucose broth and then shake culture was made at 25°C for one week. The pellets of organism were washed and dried, then ground in mortar with sea sand. The supernatant of centrifuged material was dialysed, then used as cell free extract. The enzyme activities such as iso-citric dehydrogenase (ICDH), alpha-ketoglutaric dehydrogenase (α-KG), succinic dehydrogenase (SDH) and malic dehydrogenase (MDH) were measured colorimetrically with TTC reduction system. The fumarase activity alone was measured qualitatively with paper chromatography. Several antifungal agents such as bisethylmercuri phosphate (BEMP), salicylic acid, trichomycin, griseofulvin, naphthiomate N and vitamin K3 were added respectively to cell free extract in order to observe the effect of these agents on enzyme activities.The endogenous respiration of each sample was makedly inhibited though dialysis procedure, while enzyme activities were not influenced. Therefore, every cell free extract were dialysed in this study. Activities of all four dehydrogenases tested were found in both Tr. rubum and mentagrophytes. The values of each enzyme were as follows: 1) Tr. rubrum: The average±standand error of 7 samples. Endogenous respiration (ER) 0.4±0.05 milimicromoles/mg protein/hour, SDH 0.9±0.07, MDH 1.6±0.21, α-KG 1.2±0.20, ICDH 2.9±0.42. 2) Tr. metagrophytes: The average±standard error or 6 samples. ER 0.4±0.08, SDH 1.0±0.18, MDH 2.2±0.41, α-KG 1.0±0.16, ICDH 4.2±0.59.As to Tr. mentagrophytes, the spot of malic acid (Rf 41) was identified on paper chromatography as the proof of presence of fumarase activity. The changes of enzyme activities of two species induced by six different antifungal agents were as follows: 1) 30, 000×solution of BEMP: No effect was observed on ER in both species, while the activities of SDH, MDH, α-KG and ICDH were markedly inhibited in both species. 2) 600×solution of salicylic acid: The MDH activity in both species was increased while activities of SDH, α-KG, ICDH and ER were inhibited. 3) 9, 000×solution of trichomycin: No effect of the solution was observed in both species, while ER of Tr. mentagrophytes was slightly inhibited and MDH activity of that species was slightly increased. 4) 9, 000×solution of griseofulvin: No effect of the solution was observed in both species, while ER of Tr. rubrum was slightly increased. 5) 9, 000×solution of napthiomate N: No effect was observed in both species, while ER of Tr. mentagrophytes was inhibited and MDH of that species was slightly increased. 6) 15, 000×solution of vitamin K3: α-KG activity of Tr. rubrum was inhibited, while SDH, MDH and ICDH activities of two species were markedly increased.