GPDase

The previously discussed enzymes may all be used for solid-phase AM assays. For homogeneous AM-type EIA, lysozyme, malate dehy-rogenase (MDase), glucose-6-phosphate dehydrogenase (GPDase), and ribonuclease A (RNase A), are used in addition to BGase. 

GPDase (D-glucose-6-phosphate NADP+ oxidoreductase EC 1.1.1.49) is frequently used for AM-type El A, particularly for substances in sera (Table 10.19). This enzyme is widely distributed (Keller, 1971 Lohr and Waller, 1974). However, unlike its mammalian... 

Catalytic properties and assay of glucose-6-phosphate dehydrogenase The reaction catalyzed by the bacterial GPDase... 

Mg + stimulates the enzyme up to 10 mM but inhibits its activity at higher concentrations. Other bivalent cations are often inhibitory. Another important inhibitor is phosphate, though NADP+ may reverse to a certain extent this inhibition (Glaser and Brown, 1955). There is a rather pronounced pH dependence of the L. mesenteroides GPDase activity (Ldhr and Waller, 1974), namely 7.8. 

GPDase activity may be determined by several methods (Lohr and Waller, 1974), among which the spectrophotometrical determination of NADH at 340 nm is the most convenient. 

Chemiluminescent substrates have also been developed for lucifer-ase (Neufeld et al., 1965), GPDase (Haggerty et al., 1978), catalase (Neufeld et al., 1965) and BGase (Puget et al., 1977). 

In this method an appropriate enzyme (e.g., GPDase) and the antigen are co-immobilized on a solid phase. Hexokinase-labeled antibody produces an accelerated conversion of glucose, ATP and NAD+ since the enzymes are in each other s vicinity. 

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