Intracellular distribution of α-galactosidase in leaves of Cucurbita pepo

The intracellular distribution of α-galactosidase in leaves of Cucurbita pepo was studied at different developmental stages using tissue strips, homogenates, and isolated protoplasts. About 85% of the total activity was found in the 500 g supernatant after tissues were homogenized either in water, in buffer at pH 5.6 or at pH 7.0, or in buffer containing 0.8 M KCl. Isolated protoplasts contained less than 10% of the total activity which was confined to the 20 000 g supernatant after lysis. p-Nitrophenyl-α-D-galactoside was readily hydrolysed when incubated with leaf strips but less than 3% of α-galactosidase could be leached from strips held for 4 h in 100 mM phosophate buffer or in buffer containing either 0.8 M KCl, 1 mM EDTA, or 1 mM dithioerythritol. It is concluded that at all stages of leaf development a high proportion of α-galactosidase is located in the exocellular region, not strongly bound either to the outer surface of the plasmalemma or to the cell wall but prevented from diffusing through the wall matrix by some physical attribute such as molecular size. Enzyme release occurred only following breakage or enzymatic digestion of the wall. The in vivo properties of the exocellular enzyme in leaf strips were compared with those of three molecular forms of α-galactosidase (LI, LII, and LIII) which were partially purified from mature leaves. The exocellular enzyme was active over a broad pH range with optima at pH 3.0 and pH 6.0; this resembles a combination of pH optima for LI and LIII. Inhibition by Cu2+ and p-chloromercuribenzoate resembled that for LIII and LII, respectively. Galactose and galactinol at a 5 mM concentration were 25–30% inhibitory for all enzyme preparations; melibiose, raffinose, and stachyose were very weakly inhibitory. The function of an exocellular α-galactosidase and its bearing on the transport of galactosylsucrose oligosaccharides to and from the minor veins of C. pepo are discussed.