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.

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Water-soluble phospholipase B was purified to homogeneity from Torulaspora delbrueckii cell washings. The washings were concentrated by ultra filtration, and then a fraction with phospholipase B activity was precipitated with ammoniumsulfate, and purified by sequential column chromatographies on Octyl-Sepharose CL-4B, DEAE-Sephacel,and Sepharose 6B. The molecular weight of the enzyme was estimated to be 170,000~200,000 by SDS-polyacrylamide gel electrophoresis and by gel filtration with a Sephadex G-200 column. The isoelectric point of the enzyme was 4.0. The purified enzyme had two pH optima at pH 2.5 and pH 7.5. The activity at acidic pH was greatly stimulated by the divalent metal ions tested, but the activity at alkaline pH was stimulated mainly by Ca2+ and Fe2+. The purified enzyme had both lysophospholipase activity and phospholipase B activity in a ratio of37: 1 at acidic pH and 73: 1 at alkaline pH. The amino acid composition of the enzyme was characterized by high contents of Asp, Ser, Leu, and Gly.

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The purpose of this study was the cytotoxicity assay of dental material HEMA monomer to human dental pulp cell by MTT method and application of the flow cytometry to analyze effect of dental material on the cell cycle progression. The result of MTT method showed the inhibition of cell growth and 50% inhibitory concentration (IC50) of HEMA monomer in human dental pulp cell was 815.19 micrograms/ml. The result of the flow cytometry showed that there was a perturbation on human dental pulp cell cycle progression at the phases of Sand G2M with a dose-dependent manner. Biomaterials including dental materials should be safety to human bodies. Presently, many methods for testing the cytotoxicity of biomaterials were suggested. [1-2] MTT method is one of the cytotoxicity assay. It was provided by Monsmnn. [3] MTT is a kind of tetrazolium salt [3-(4,5-dimethylthiazol-2yi)-2,5-diphenyl tetrazolium bromide]. MTT method is the rapid, precision and quantitative colorimetric assay for cytotoxicity. It can be used to measure the proliferation, cytotoxicity or activation of living cells and is capable of handling large number of samples. Many investigators have used this advanced method.[4] Flow cytometry (FCM) analyzes the quantity of DNA bonded with dyes in each cell. It can provided the information of the cell cycle progression in detail. Currently, flow cytometry has been widely and successfully used in various fields of basic science research and clinical medicine. This FCM technology also can be used to study the cytotoxicity of dental materials and evaluate the biocompatibility of dental materials.[5-6] The contents of the study were (1) cytotoxicity assay on dental material HEMA monomer in human dental pulp cells by MTT method. (2) application flow cytometry to analyze the effect of dental material HEMA monomer on the cell cycle progression of the human dental pulp cells.