Assay of Two Activities Forming Different Products

In one type of study, the HPLC assay method has been applied to the question of assaying the activity of two enzymes that can use the same substrate but form different products. In another, the HPLC method was applied to the case of related two different enzymes that can use the same substrate and form the same product. It was also applied to the case a single enzyme that can use either of two substrates to form two different products. In a separate application, the so-called reconstitution approach, a multienzyme pathway is reconstructed by the addition of enzymes one after the other to form a multienzyme complex and to thereby reconstitute a naturally occurring multienzyme complex. And finally, this approach allows us to use the HPLC method to study an intact, naturally occurring multienzyme system. 

2 ASSAY OF TWO ACTIVITIES FORMING DIFFERENT PRODUCTS FROM THE SAME SUBSTRATE (Pahuja et al., 1981) 

Glutamic acid is used by two different activities glutamine synthetase and glutamic acid decarboxylase. 

Glutamine synthetase and glutamic acid decarboxylase catalyze the conversion of glutamic acid to glutamine and y-aminobutyric acid (GABA), respectively. Since both activities are involved in the metabolism of this amino acid, it would be useful to be able to study both simultaneously. And indeed, an HPLC method was developed to measure both activities in crude extracts. 

In contrast, when ATP is omitted and pyridoxal phosphate is added for the decarboxylase assay, the formation of GABA can be detected (Fig. 10.3). The activities, were prepared by homogenizing bovine brain. After centrifugation at lOOOg to remove debris, the supernatant solution was used directly for experiments. Bovine retinas were also prepared in the same manner. 

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