コハク酸脱水素酵素の組織化学的検出
1953 年 66 巻 785-786 号 p. 277-285
詳細
1953 年 66 巻 785-786 号 p. 277-285
The reduction of 2, 3, 5-triphenyltetrazolium chloride (TTC) by embryo slices of Phaseolus vulgaris was studied.
Excised radicle and hypocotyl were sliced longitudinally with a razor blade and incubated in reaction mixtures containing 10-2% TTC, 10-2M sodium succinate and 0, 088M phosphate buffer (pH6, 8) at 28°C in dark. Then the appearence of red colouring was observed in various time intervals.
Under these conditions, the colouring, indicating the reduction of TTC to triphenyl formazan, was appeared at the procambial region, and was facilitated by addition of sodium succinate (Table 1). Effects of temperature and some inhibitors were studied under the existence of succinate.
(1)The reducing activity of the tissues was completely damaged by pretreatment at 52°C for 30min. (Table 2, 3), and influenced by the incubation temperature (Table 4).
(2) The reduction was also completely inactivated by monoiodoacetic acid at the concentration of 10-3M (Table 5, 6).
(3) Malonate, known as a specific and competitive inhibitor of succinic dehydrogenase, inhibited the reduction only slightly when added in the reaction mixture (Table 7, 8). When the slices, however, were pretreated with a 10-2M malonate solution for 37min, the inhibition increased intensively (Table 9).
(4) The reduction was prevented completely by pretreatment with 10-1M lead nitrate solution (Table 10). When these slices were washed with a dilute solution of ammonium sulphide after the treatment, their procambial regions became stained brown just at the corresponding places formerly stained red. This brown precipitate was proved as lead sulphide, and these facts suggest that the inactivation by lead salt may be due to the combination between lead ions and enzyme protein (probably succinic dehydrogenase).
(5) When the phosphate buffer was replaced with acetate buffer, the reaction was completely inhibited (Table 11).
(6) Potassium cyanide inhibited the reduction at the concentration of 10-3M (Table 12, 13). This inhibition may be due to the inactivation of succinic dehydrogenase as was pointed out by Tsou.
Standing on the basis of these results it was concluded that the site of staining, i.e. the procambial region, was that of succinic dehydrogenase, although it was not confirmed with certainty yet what was the direct carrier of hydrogen atom to TTC.
