Isolation of IbHypSys. A, alkalinization of the
medium of suspension cultured cells of tomato (Sl), petunia
(Ph), tobacco (Nt), Arabidopsis (At), and
sweet potato (Ib). Ten μl of a crude extract from sweet potato
(I. batatas) leaves were added to 1 ml of cells. The change in pH was
recorded after 30 min. The data represent the average of three separate
experiments. B, lower panel, the crude extract was dissolved
in 0.1% trifluoroacetic acid, H2O and chromatographed through a
preparative reversed-phase C18-HPLC column with a 90-min gradient from 0 to
40% acetonitrile at a flow rate of 4 ml/min (Stage III of purification).
Upper panel, alkalinization assays of 10 μl from each fraction
when added to 1 ml of tomato suspension culture as described under
“Experimental Procedures.” C, further purification of the
peptide eluting in the fraction of the peak in the upper panel of
B, marked (*) as described in Stages IV and V of the
purification. Lower panel, the peptide mixture was separated on a
narrow bore reversed-phase C18 HPLC column with 10 mm potassium
phosphate, pH 6, at a flow rate of 0.25 ml/min. A 90-min gradient was
performed from 0 to 40% with an elution buffer consisting of 10 mm
potassium phosphate in 50% acetonitrile, pH 6. One-minute fractions were
monitored at 220 nm (Stage V of purification). D, the concentration
dependence of the purified isopeptides from the final purification in Stage V
in the alkalinization assay. The change in pH of the suspension cell medium in
response to the increasing concentration of peptides was recorded 30 min after
the addition of peptide. The change in pH was compared with distilled
H2O controls of equal volume (10 μl). The data represent the
average of three separate experiments.