H. J. KOCH 



The facts seemed at first to be consistent with the idea that these substances act as 

 inhibitors because of their oxidation-reduction properties. Working further on this 

 assumption we tested different dyes in order to determine the limits of their activity 

 in relation to their position on the rH scale. Discrepancies appeared rapidly, sub- 

 stances as far apart as Bindschedler's green (E' = +0-224) an d safranine T 

 (E' = —0-289) both being active. 



A list of the dyes so far tested is given in Table I ; they belong to very different 

 chemical groups. When we looked for characteristics common to these widely differ- 

 ent substances, it appeared that all the inhibitors of salt transport were basic dyes, 

 whereas the inactive ones were acidic dyes (Koch, Evans and Schicks, 1953.) 



However the mere fact that a substance of nearly the same molecular weight as 

 these dyes was a basic compound was not enough to confer on it an inhibiting action : 

 substances like quinine proved to be inactive. 



The only further salient feature of the dye inhibitors was the presence of a quatern- 

 ary NH 4 group. When tetramethyl ammonium chloride was tested it also proved to 

 be an inhibitor of salt transport. 



Now some quaternary ammonium compounds at least are known as inhibitors of 

 cholinesterases, and the cholinenergic properties of methylene blue on the heart of 

 vertebrates has been described by Heymans (1923) and R. P. Cook (1926). From the 

 purely biochemical point of view the anti-cholinesterase activity of basic dyes has 

 been investigated by Rentz (1940) and especially by Massart and Dufait (1941) on 

 horse-serum cholinesterase. The latter authors have shown that the anti-cholin- 

 esterase activity of basic dyes is dependent on the quaternary NH 4 , because it dis- 

 appears in the leuco-form where N is no longer present as a quaternary compound. 



All this suggested that the inhibitory action of basic dyes on salt transport might 

 be due to their influence as anti-cholinesterases. However such an interpretation of 

 the inhibition of salt transport by means of basic dyes required further evidence, 

 especially because basic dyes have been also described as inhibitors of dehydrogenases 

 (Quastel and Wheatley, 1931). 



In order to elucidate the effect of basic dyes on the respiratory activity of the gills, 

 experiments were conducted with Warburg manometers in which the dyes could be 

 tipped in from a side arm at a certain moment. The oxygen consumption was mea- 

 sured with pure 2 and with an 8 mM solution bathing the gills. The gills were pre- 

 pared exactly as for an active transport experiment : they were ligatured at the base. 



While acidic dyes had no effect on respiration, basic dyes increased the respiratory 

 intensity (Safranine T from 30 to 126 cu. mm. per hour). Therefore the inhibition of 

 active transport was not caused by a depression of respiration. 



THE PRESENCE OF CHOLINESTERASE AND ITS LOCATION IN THE 



GILL EPITHELIUM 



A suspension of the gills of Eriocheir sinensis (obtained by means of three minutes' 

 treatment in 25 cc. of bicarbonate Ringer in a mixing blender) clearly exhibits 

 cholinesterase activity when tested in the presence of acetylcholine according to the 

 method of Ammon. 



Measurements of the activity showed that 1 g. of fresh gill tissue is able to hydrolyse 



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