Confirmation on the Catalytic Centers of Endopeptidases
As shown in Table 2, the dominant endopeptidases were aspartic
endopeptidases, which had obviously higher relative abundance than
metalloendopeptidase and subtilisin-like proteases. Cysteine
endopeptidases showed extremely low relative abundance. Therefore,
pepstatin A, AEBSF, and EDTA-2Na were selected for the protease
inhibitor assay. Without inhibitors (Fig. 2a), the Tricine–SDS–PAGE
profiles of pH 7-OB incubated at pH 3–9 were similar to the
corresponding results in Fig. 1a. In the presence of pepstatin A (Fig.
2b), the hydrolysis of oleosin-H was obviously inhibited at pH 3–6,
while the hydrolysis of 11S globulins was obviously inhibited at pH
3–4. However, the hydrolysis of oleosin-H was still obvious at pH 4–6,
especially at pH 5. In the presence of AEBSF (Fig. 2c), the
Tricine–SDS–PAGE profiles were almost the same as those in Fig. 2a,
which supported the results that aspartic endopeptidases were the
dominant endopeptidases. Generally, aspartic endopeptidases are
sensitive to pepstatin A (Simoes & Faro, 2004), but some ones are
insensitive to it (Toogood, Prescott, & Daniel, 1995; Kocabıyık &
Özel, 2007). Therefore, it was suggested that there were at least two
types of aspartic endopeptidases in pH 7-OB. One type (optimal at pH 3)
could be inhibited by pepstatin A, but the other (optimal at pH 5) could
not. This could be well supported by the three kinds of aspartic
endopeptidases (aspartic proteinase-like, 2.938‰; aspartyl protease
2-like, 0.205‰; aspartic proteinase A1-like, 0.177‰) with high relative
abundances in Table 2.
In one previous study, it was clarified that one aspartic endopeptidase,
which was bound to peanut OBs, could be greatly washed from OBs by
alkaline pH (Chen et al., 2018). Therefore, pH 9.5-OB was prepared to
examine whether AEBSF could exert obvious inhibition activity. In the
absence of inhibitors (Fig. 2d), it was found that the hydrolysis of
oleosin-L at pH 4 and 6 and 11S globulins at pH 3 was obviously weaker
than that in pH 7-OB (Fig. 2a), indicating that aspartic endopeptidases
were greatly removed from OBs by pH 9.5 washing. However, the hydrolysis
of oleosin-H at pH 5 was still obvious. In the presence of pepstatin A
(Fig. 2e), the hydrolysis of oleosin-H at pH 5 was inhibited to some
extent, which could be attributed to the residual aspartic
endopeptidases in pH 9.5-OB. In the presence of AEBSF (Fig. 2f), the
hydrolysis of oleosin-H at pH 5 was greatly inhibited. These results
revealed that subtilisin-like proteases showed higher resistance to pH
9.5 washing than aspartic endopeptidases, and they exhibited sharp
optimum at pH 5. One subtilisin-like protease from soybean also showed
sharp optimum at pH 5 (Boyd, Barnaby, Tan-Wilson, & Wilson, 2002).
Until now, all examined plant metalloendopeptidases had optimal activity
at neutral and alkaline pH (Marino & Funk, 2012). Therefore, the
effects of EDTA-2Na on proteolytic activity in isolated sesame OBs was
conducted at pH 8 and 50 °C. It was found that hydrolysis of oleosin-H
could be inhibited by EDTA-2Na (data not shown). Thus, it was suggested
that metalloendopeptidase contributed to the low proteolytic activity at
neutral and alkaline pH, while the proteolytic activity in the pH range
of 3–6 was dominantly attributed to aspartic endopeptidases. In
addition, it should be noted that subtilisin-like proteases also
contributed to the proteolytic activity at pH 5.