VOL. 12 (1953) 



FNZYMES OF FATTY ACID METABOLISM 



311 



constant of the reaction has not yet been determined but it appears to favor the S-/3- 

 hydroxyacyl derivatives. Nothing can as yet be said as to the chain-length specificity of 



crotonase and consequently the occurrence of several 

 enzymes of this type is not excluded. 



Crotonase would also appear to convert S-vinyl- 

 acetyl CoA to the j8-hydroxybutyryl derivatives^*. If 

 so, an equilibrium would be established between the 

 S-acyl CoA derivatives of crotonic, vinylacetic, and 

 ^-hydroxybutyric acid. This might explain the obser- 

 vation that vinylacetate can be either oxidized or re- 

 duced by extracts of C. kluyveri^'^^. The failure of 

 crotonate to replace vinylacetate in this system^^ may 

 have been due to failure of the bacterial extracts to 

 activate crotonate. 



Ethylene reductase. Ethylene reductase was de- 

 tected in liver extracts^^ by a method similar to that 

 employed by Fischer and Eysenbach to study fuma- 

 rate reductase^'^. Leucosafranine is oxidized by S- 

 crotonyl-N-acetyl thioethanolamine, but not by free 

 crotonate, in the presence of an enzyme from liver. 

 The reaction is shown in Fig. 13. Here again a natural compound, in this case crotonyl- 

 S-CoA, could be replaced by its readily synthesized thioethanolamine analogue. The 



2 3 4 

 MINUTES 



Fig. 12. Optical crotonase test. Tris 

 (hydroxy methyl) arainomethane- 

 HCl-buffer pH 7.5, 100 /iiM; egg 

 albumin, o.i mg; ethylenediamine 

 tetraacetate, 1.5 //M; S-crotonyl 

 CoA, /^ 0.5 fiM. 2.0 juM of AMP 

 in blank cell. Volume, 1.5 ml; 

 d = 0.5 cm; temp., 25°. 



H 





+ CH„ — CH = CH— CO — S— CH, — CH„— NH— CO — CH, 



^^^^NHj-HCl 



H,N/^'^\N/"^/^^NH,Cl 



+ CH,— CH, — CH.— CO — S— CH, — CH, — NH— CO— CH3 



■-*%: 



Fig. 13. Reaction of the ethylene reductase assay. 



enzyme assay, in which the appearance of colour 

 from the leucodye is followed, is illustrated in 

 Fig. 14. By the use of this assay ethylene re- 

 ductase was 'purified about 80-fold from sheep 

 liver extracts through steps involving acetone 

 fractionation, adsorption and elution from calci- 

 um phosphate gel, and ammonium sulfate frac- 

 tionation. Solutions of the purified enzyme are 

 yellow in colour. A colourless, almost inactive 



Fig. 14. Optical ethylene reductase test. Phosphate 



buffer pH 7.1, 140 /j.M; leucosafranine T, 0.5 /iM; 



S-crotonyl-N-acetyl thioethanolamine, 2.6 /<.!/. Volume, 



2.1 ml; d = 0.5 cm; temp. 17°. 



2 3 



HfllNUTES 



References p. 313 1 31 4- 



