Tomato polygalacturonase extractability
1995
Jackman, R.L. | Gibson, H.J. | Stanley, D.W.
The extractability of polygalacturonase (PG) activity from alcohol-insoluble solids (AIS) prepared from red-ripe tomato pericarp tissue was investigated using acetate (pH 4.5), citrate (pH 4.5) and MES (pH 6.0) buffers. The acetate buffer was also investigated with increasing concentrations of NaCl, CaCl2, MgCl2, ethylenediaminetetraacetic acid (EDTA) or ethyleneglycol-bis (beta-aminoethylether)-N,N'-tetraacetic acid (EGTA). Heat treatment of AIS to prepare control material suppressed PG extractability but not in situ PG activity, as evidenced by the release of significant quantities of soluble uronides into extraction media. With only a single extraction, acetate buffer containing > 1 M NaCl yielded the greatest amount of PG activity (> 50% of maximum extractability), with MES buffer being only marginally less efficient. However, 3 successive extractions over a 2 h period yielded significantly greater amounts of PG activity. The greatest yields of PG activity were obtained by successive extraction with parent MES and acetate buffers. There appeared to be little benefit in adding NaCl or a chelating agent to the extraction medium. Use of these extractants is suggested to have led to losses of PG activity during dialysis, via coprecipitation of PG protein with otherwise soluble uronide material that was released in greater quantities when these extractants were used. Increasing CaCl2 and MgCl2 concentrations reduced the amount of extracted PG activity similarly, to about 50% of maximum levels with successive extractions.
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