Polyclonal Antiboby Preparation And Inhibitor Screening Of The Polygalacturonase Of Winter-Jujube(Ziziphus Jujuba Mill.) Fruits

Polygalacturonase (E.C.3.2.1.15) have been found in ripening fruits, abscission zone, pollen and other parts of plant, which are well known to catalyze aspects of pectin modification and disassemble as one of cell wall degrading enzymes. PG may play originally important role in Jujube fruits softening. In this paper, PG from ripening winter-jujube fruits was routinely purified by the following methods. The crude enzyme extract was concentrated with polyethylene glycol 20000(PEG-20000) and applied to a column of Carboxymethyl-Sepharose CL6B(l.6cm by 4Ocm),the column was eluted with a linear salt gradient (0.O4mol/L-0.2mol/L sodium acetate pH5.5), active fractions were pooled and concentrated with PEG-20000, then fractioned on a column of Sephadex G-75 superfine (1.0cm by 80 cm) in 0.O4mol/L sodium acetate and l.Ommol/L Dithiothreitol (DTT), active fractions were pooled concentrated and stored at -70C until required. Two white male rabbits were injected intramusculary and subcutaneously with purified PG protein as antigen, the antiserum was also purified by using saturated ammonium sulfate. The titer of antiserum was 1:500. For western bloting experiment, The result indicated the antiserum could specilly reacted with the purified PG, but the antiserum reacted with two polypeptides in the total protein isolated from mature fruit, one of them had the same size with the PG, the other protein may be isoenzymes of PG or functionally unrelated protein that share sufficient homology at the epitope to reacted with the antibody. The PG showed different response towards the inhibitors, divalent metal ions Ca2. Mg2 Zn2 Hg2 partly inhibited PG activity, Mn2 Cu2 stimulated the PG activity. EDTA can also increased the activity. Trivalent metal ions Fe3~ activated the enzyme activity, but Al3 strongly inhibited the activity. Low concentrations of monovalent salt were slightly stimulated the enzyme, while the inhibition were occurred with the higher concentration. The activity wasstimulated by 150% on 1.0% of SDS. The non-ionic detergent, Triton x-100, in 1.0-% concentration activated the activity to 144%. The cationic detergent CTAB at 0.1% strongly inhibited PG activity by 70%. The sulfhydryl reagent, 2-ME, stimulated the PG activity. The growth regulators, 2,4-0 and IAA inactivated the enzyme, GA3 had no effect, but the NAA and ethylene could stimulate the activity. As estimated by SOS-PAGE the molecular weight of PG was 31500, lower than that of PG in other fruits. The most pH stability was at a range of pH 4.5-8.0. The most optimum ion strength was at 0.O4mol/L-0.O6mol/L. The U.V. spectrum of PG was about 267 nm.