VOL. 4 (1950) 



TEST FOR FUMARASE AND ACONITASE 



213 



■0.20 



bl- 



under these experimental conditions the Michaelis constant for fumarase as 

 determined by the method of Lineweaver and Burk* was 4.1-10-=^ (moles x Hter-i) 

 with sodium L-malate as substrate. 



The enzymatic activity of fumarase can also be followed with sodium fumarate as 

 the substrate. Due to the high specific absorption of fumaric acid, only limited amounts 

 of this substrate, which are not sufficient to saturate the enzyme, can be used in the 

 spectrophotometric test. The rates, therefore, are slower and fall off more rapidly than 

 with L-malic acid as the substrate. However, with an active enzyme preparation the 

 equilibrium is quite rapidly established from 

 either direction. 



b) Aconitase. For the measurement of 

 aconitase activity the test system was the 

 same as that for fumarase except that the 

 substrate used was either 0.03 M sodium cit- 

 rate or o.oi M sodium D,L-isocitrate (kindly 

 supplied by Dr S. Ocho.\). Since the enzyme 

 is unstable in dilute solutions, all estimations 

 were carried out immediately following di- 

 lution in 0.1 M phosphate buffer. The en- 

 zymatic acti\'ity followed a zero order course 

 for several minutes and was proportional to 

 the amount of enzyme added (Fig. 2). 



The specific activity (units/mg protein) 

 of aconitase preparations when tested with 

 isocitrate was always found to be greater 

 than with citrate. Considerable variation in 

 the relative activities was found in different 

 fractions during purification. Although no 

 evidence was obtained of a separation of the 

 enzyme activity for the two substrates, the 

 respective activities, for the sake of conve- 

 nience, are referred to as citrase and isoci- 

 trase. Thus, in a crude heart extract, a ratio 

 isocitrase/citrase activity of 2.1 was found, 



while the purified preparation^ had a ratio of 7.5. Similarly, the fractions obtained 

 from yeast by acetone and ammonium sulphate precipitation, showed considerable 

 variation in the relative citrase and isocitrase activities. The ammonium sulphate 

 precipitate obtained at 50% saturation showed an isocitrase/citrase ratio of 2.0, while 

 the fractions obtained between 60 and 80% saturation showed a ratio of about 7.0. 



The Michaelis constant of aconitase measured with sodium citrate as substrate 

 was found to be i.i- lO"^ and for D-isocitrate 4-10"* M. 



c) Aspartase. This enzyme was measured in the same manner as the other hydrases 

 with 0.15 M sodium aspartase as the substrate. A high concentration of substrate i.s 

 required for maximal activity of this enzyme. With substrate concentration sufficient 

 for enzyme saturation, proportionaUty between enzyme concentration and increments 

 in optical density was found (Fig. 2). 



The Michaelis constant of aspartase was found to be in the neighbourhood of 



References p. 214. 



0.16 



0.12 



0.08 



0.04 



Fig. 2. Quantitative determination of fumarase, 



aconitase and aspartase. Relation of enzyme 



concentration to activity per minute. 



