No abstract

No abstract

Galactolipase activity was first detected in extracts of Phaseolus multifloris primary leaves by Sastry and Kates (19). More recently Helmsing (9) has reported the purification of a galactolipase from bean leaf homogenates. In this communication, we report the isolation and partial purification of a galactolipase from the chloroplast fraction of the primary leaves of Phaseolus vulgaris var. Kentucky Wonder. The properties of this enzyme differ somewhat from those of previously reported galactolipases (9,19). Furthermore, we found that spinach subchloroplast particles which have the same membrane polarity as chloroplasts (13) were excellent substrates for the galactolipase, suggesting that the acyl ester linkages of the galactolipids in chloroplast membranes are exposed to the external medium. We have also characterized some of the effects of galactolipase treatment on light-dependent electron flow in subchloroplast particles. MATERIALS AND METHODSGalactolipase Purification. Pole bean seeds (Phaseolus vulgaris var. Kentucky Wonder) were soaked in tap water for 24 hr and were germinated in moist vermiculite at room temperature. Following germination, the seedlings were illuminated for 16 hr per day with fluorescent light (intensity, about 1.2 X 10' ergs/ cm2' sec). After 15 to 20 days, the primary leaves were harvested and stored at 4 C in the dark for 24 to 48 hr to remove starch. About 150 g of leaves were homogenized in a Waring Blendor for 30 sec with 800 ml of cold 0.4 M sucrose which also contained 0.1 M potassium phosphate buffer (pH 6.0). The homogenate was filtered first through three and then eight layers of cheesecloth. The filtrate was centrifuged at 3300g for 15 min at 4 C, and the resulting pellet, which was rich in chloroplasts, was resuspended in a small volume of sucrose-Pi buffer. The Chl concentration in the suspension was adjusted to 3 mg per ml with sucrose-Pi buffer.The chloroplast suspension was added dropwise to 17 volumes of rapidly stirred acetone at -17 C (21). The temperature of the acetone during addition was kept below -10 C. After the addition was completed, the stirring was stopped, and 699 www.plantphysiol.org on May 7, 2018 -Published by Downloaded from

No abstract

No abstract