VOL. 4 (1950) 



ENZYMES FROM PIG HEART 



207 



Malic Dehydrogenase. — The optical test for malic dehydrogenase activity is based on Reaction 4. 



(4) Oxalacetate + DPNred ^ /-malate + DPNqx 



The test is carried out in the Beckman spectrophotometer, at wave-length 340 m/z, using cells of 

 i.o cm light path. It is based on the fact that the early rate of oxidation of reduced diphosphopyridine 

 nucleotide (DPNred) by oxalacetate is proportional to the enzyme concentration within certain limits. 

 One enzyme unit was defined as the amount of enzyme causing a decrease in optical density of o.oi 

 per minute calculated for the third 15 second period after the start of the reaction. The reaction 

 mixture, in a final volume of 3.0 ml, contained 0.025 M glycylglycine buffer pfj 7.4, 0.4-10"* M 

 DPNj-ed. enzyme, and 0.25-10-^ M oxalacetate. The volume was made up with water adjusted to a 

 temperature of 22-23°. The blank cell contained no DPN. The reaction was started, after taking a 

 zero time reading of the optical density, by addition of either oxalacetate or enzyme. 



ir> 0.20 



0.15 



0.10 



0-05 



0.01 0.02 0.03 



CC Pig heart extract 



Fig. I. Optical test for oxalosuccinic carboxylase (Reaction 3). Proportionality of rate to enzyme 



concentration. 



II 



y^ j 



II ^^ 



PREPARATION OF ENZYME 



Extraction. — Acetone-dried pig heart was prepared by the method described by 

 Straub^°. The dry material was ground to a fine powder in a mechanical mortar. The 

 powder was extracted with o.i M phosphate buffer pn 7.4 at room temperature following 

 the method of Straub^". 



Ammonium Sulphate Fractionation. ■ — - The clear extract was cooled to 0°, brought 

 to 50% saturation with solid ammonium sulphate, and the mixture was filtered with 

 suction in the cold room using filter-aid (Hyflo-Supercel) to facilitate filtration. The 

 precipitate was discarded and the supernatant was brought to 60% saturation with 

 solid ammonium sulphate. The mixture was filtered as before. The supernatant was 

 discarded and the precipitate was dissolved in cold 0.04 M phosphate buffer pfj 7.4 to 

 give a concentration of about 3% protein. The solution was clarified by filtration and 

 dialysed against 0.04 M phosphate buffer pjj 7.4 at 2-^" for 4-5 hours. 



Ethanol Fractionation. — The dialysed solution was fractionated with ethanol at 

 low temperature. Details of the procedure have been described elsewhere^^. The most 

 active fraction was usually obtained between 20 and 30% ethanol by volume at -5°. 

 The precipitate was collected by centrifugation at -5°, dissolved in cold o.oi M phosphate 

 buffer Ph 7.4, and dialysed for a few hours at 2-3° against the same buffer. 

 References p. 210. 



