product is formed. The absorbance o

product is formed. The absorbance of the samples at 405 nm can
be measured with a microtiter plate (ELISA) reader and is directly
correlated to the level of RT activity. A fixed amount (4–6 ng) of
recombinant HIV-1 RT was used. The inhibitory activity of the
lectin was calculated as percent inhibition as compared to a
control without the protein (Wang and Ng, 2001). The mushroom
ribosome-inactivating protein hypsin was used as a positive
control (Lam and Ng, 2001).
Assay for ribonuclease activity
The assay was performed because some lectins display this
activity (Liu et al., 2007). Ribonuclease activity of RLL toward
yeast tRNA (Sigma) was assayed by determining the generation of
acid-soluble, UV absorbing species with the method of Mocket al.
(Mock et al., 1996). The lectin was incubated with 200mg of tRNA
in 150mg 100 mM morpholino-ethanesulfonic acid buffer (pH
6.0) at 371C for 1 h. The reaction was terminated by adding 350ml
of ice-cold 3.4% perchloric acid. After leaving on ice for 15 min, the
sample was centrifuged (15,000g, 15 min) at 41C. The OD 260
of the supernatant was read after appropriate dilution. One unit of
enzymatic activity was defined as the amount of enzyme that
brings about an increase of one per minute in OD 260 nm in the
acid-soluble supernatant (Mock et al., 1996). Bovine pancreatic
RNase A (Sigma) was used as a positive control.
Assay for antifungal activity
The assay was carried out since some lectins demonstrate this
activity (Ciopraga et al., 1999). The assay for antifungal activity
toward Fusarium oxysporum, Rhizoctonia cerealis, Rhizoctonia
solani, and Sclerotinia sclerotiorum was carried out in
10015 mm petri dishes containing 10 ml of potato dextrose
agar (PDA). After development of the mycelial colony, sterile
blank paper disks (0.625 cm in diameter) were laid at a distance of
0.5 cm away from the rim of the mycelial colony. An aliquot
(15ml) of the lectin was added to a disk. The dishes were
incubated at 231C for 72 h until mycelial growth had enveloped
the disks containing the control and had produced crescents of
inhibition around disks containing samples with antifungal
activity (Wang and Ng, 2006; Ye et al., 2001). The mushroom
ribosome-inactivating protein hypsin was used as a positive
control (Lam and Ng, 2001).
Results and discussion
Purification of RLL
The fraction of the fruiting body extract unadsorbed on DEAEcellulose (D1) but not the adsorbed fractions (D2,3,4) contained the
hemagglutinating activity (Table 1). Fraction D1 was divided into