Residual wall-loosening activity after removal of unbound Zea m 1 and after
protease treatment. A, After 30 min of extension of the heat-inactivated wall
in 50 mm sodium acetate (pH 4.5), the incubation solution was
changed for one containing 0.3 mg mL-1 Zea m 1d, and the wall was
extended for a further 30 min. The solution in the extensometer cuvette was
exchanged 5× with buffer without Zea m 1d (“Treatment”). The
negative control contained no Zea m 1d, whereas the positive control continued
extending in 0.3 mg mL-1 Zea m 1d. B, Destruction of wall extension
activity by Pronase. Heat-inactivated wheat coleoptiles were pre-incubated for
1 h with 0.1 mg mL-1 of Zea m 1d in 50 mm sodium
acetate, pH 4.5. After three washes with 50 mm MES, 1 mm EDTA, and 5 mm DTT (pH 6.0), they were then further pretreated for
1 h at room temperature with 2 mg mL-1 of Pronase in the washing
buffer. The walls were then assayed for residual Zea m 1 activity. At the time
indicated by the arrow, the buffer was switched from pH 7.5 buffer to pH 6.0
buffer (50 mm DMGA in both cases). All of the above experiments
were performed at least five times with similar results.