Purification and some properties of membrane-bound phospholipase B from Torulaspora delbrueckii.
Membrane-bound phospholipase B was purified to a homogeneous state from Torulaspora delbrueckii cell homogenate. Cell homogenate was extracted with Triton X-100, and the enzyme was precipitated with acetone. The acetone powder was washed repeatedly with Tris-HCl buffer (pH 8.0) until no phospholipae B activity was detected in the soluble fraction. The enzyme was extracted with Triton X-100 from the final residue and purified about 1,390-fold by sequential chromatofocusing, Sepharose 6B, and DEAE-Sephadex A-50 column chromatography. The final preparation showed a single broad protein band on SDS-polyacrylamide gel electrophoresis when stained with silver stain reagent and PAS-reagent. The molecular weight of phospholipase B was about 390,000 and 140,000-190,000 as estimated by gel filtration on Sepharose 6B and SDS-polyacrylamide gel electrophoresis, respectively, suggesting that phospholipase B is an oligomeric protein. The isoelectric point was at pH 4.5. Phospholipase B has two pH optima, one acidic (pH 2.5-3.0) and the other alkaline (pH 7.2-8.0). At acidic pH the phospholipase B activity was greatly increased in the presence of divalent metal ions, although metal ions are not a factor for enzyme activity. On the other hand, at alkaline pH the enzyme required Ca2+ or Mn2+ for activity. The pH- and thermal-stabilities at both pHs were similar. The phospholipase B hydrolyzed all diacylphospholipids tested at acidic pH, but hydrolyzed only phosphatidylcholine at alkaline pH. The hydrolysis rates of lysophospholipids were much higher (about 10-fold) than those of diacylphospholipids at both pHs.
Kuwabara
Y
Y
Department of Agricultural Chemistry, College of Agriculture, Ehime University.
Maruyama
M
M
Watanabe
Y
Y
Tanaka
S
S
Takakuwa
M
M
Tamai
Y
Y
D002413:Cations, Divalent; D002462:Cell Membrane / Q000032:analysis*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D008245:Lysophospholipase / Q000032:analysis / Q000302:isolation & purification*; D008970:Molecular Weight; D010740:Phospholipases / Q000302:isolation & purification*; D012440:Saccharomyces / Q000201:enzymology*; D013379:Substrate Specificity
D002413:Cations, Divalent; D010740:Phospholipases; D008245:Lysophospholipase