The intracellular location and multimolecular forms of α-galactosidase from leaves of Cucurbita pepo were studied. Leaf discs hydrolysed p-nitrophenyl-α-D-galactoside to approximately 60% of total in vitro activity. Less than 6% of total activity was released by treatment with 0.5 or 1.0 M NaCl. Protoplasts contained less than 0.1% of total activity.
α-Galactosidase was isolated and purified by pH fractionation, ammonium sulfate precipitation and affinity chromatography. A 9,700 fold purification was obtained with 55% recovery. The multimolecular forms were investigated by polyacrylamide disc electrophoresis, isoelectric focusing ion exchange and gel filtration chromatography. The multiple forms were composed of 30,000 dalton subunits. The aggregation of subunits was influenced by pH, buffer cation and ionic strength. A 60,000 dalton form occurred in 50 mM NaPi buffer, a 30,000 dalton form in 100 mM NaPi buffer. K+ ions in the buffer produced a 90,000 dalton form with increased activity.
There was no detectable transgalactosylation activity.