ANTI-NUTRITIONAL CONSTITUENT OF COLOCASIA ESCULENTA ...

180 = molecular weight ofthe glucose;
60 = incubation time in minutes;
N = number ofmolecules ofglucose hberated by hydrolysis in each sugar (n = 1 for sucrose
and lactose, n = 2 for maltose) [Rodriguez-Castilla et al., 1996].
B.C.2.3 ATPase [Vaslirhelyi et aL (1986)]
The Na+!K'"-ATPase activity of freeze-thawed samples was determined by measuring the
release of inorganic phosphate (Pi) associated with the hydrolysis of ATP. The samples
(250 )l1) were added to 4750 )l1 of Reagent 1 [final concentration per litre: 100 mM of
NaCl., 20 mM of KCl., 2.5 mM of MgCh, 0.5 mM of ethylene glycol tetraacetic acid
(EGTA), 50 mM oftris-HCI (pH 7.4),1 mM ofATP, 1 mM of phosphoenolpyruvate, 0.16
mM of nicotineamide adenine dinucleotide (NADH), 5 kU of pyruvate kinase, 12 kU of
lactate dehydrogenase]. After 300 s, 65 J.L1 of 10 mM ouabain was added to inhIbit ouabain­
sensitive ATPase activity. The Na+JK+-ATPase is composed of the stoichiometric of two
obligatory major polypeptides, the a-subunit (- 112 kDa) and the f3-subunit (- 45 kDa).
The binding sites for ATP, cations and ouabain are localized in the a-subunit, which is
responsible for the atalytic activity ofthe enzyme. The change in absorbance was monitored
at 340 llID. The Na+JK+-ATPase activity was calculated from the difference in A 340 .
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