Determination of aldolase in animal tissues.

Aldolase catalyzes the reversible cleavage of 1 mole of fructose-l ,6diphosphate (HDP) into 1 mole of glyceraldehyde-3-phosphate and 1 mole of dihydroxyacetone phosphate (1, 2). Although the enzyme has been obtained in crystalline form by Warburg and Christian (3) and by Taylor, Green, and Cori (4) only fragmentary data concerning the content of aldolase in different mammalian tissues have been reported (1,5,6). Meyerhof and $ohmann measured the activity of the enzyme by following the format.ion of the alkali-labile phosphate of the triose phosphates (1). This method was also used by Herbert et al. (7) in purification of the enzyme. Although this method, when employed under proper conditions, is perfectly reliable, it is inconvenient, owing to t.he necessity of performing many blank det.erminations of inorganic and alkali-labile P. Warburg and Christian employed a spectrophotometric test based on the reduction of diphosphopyridine nucleotide (DPN) which occurs when glyceraldehyde phosphate, formed from HDP by aldolase, is allowed to react with DPN in the presence of crystalline triose phosphate dehydrogenase and arsenate (3). This method does not lend itself to routine use, owing to its technical requirements. Furthermore, as will be shown, it has been found to be subject to large negative errors when used in assaying certain crude tissue extracts, owing to the presence of enzymes capable of destroying DPN. More recently, Dounce and Beyer have published details of a method (6) of assaying aldolase based on calorimetric determination of trioses by the Barker and Summerson procedure (8) for determination of lactic acid. Although the method appears to be quite reliable with crystalline aldolase, its application to crude tissue extracts or homogenat.es yields much less certain results, as the authors have pointed out. In the course of a reexamination of the findings of Warburg and Christian concerning serum aldolase levels in tumor-bearing rats (5) a method for quantitative determination of aldolase was devised which is free from some of the difficulties of the several methods outlined above and which allowed a quantitative survey of the enzyme in rat tissues to be made. Principle of Method-HDP is incubated with the buffered test sample