牛肝及牛乳鹼性磷酸?糖組成的分析

Abstract

本論文利用Con A Sepharose親和管柱，Superose?12 FPLC膠體過濾管柱，MonoQ FPLC離子交換管柱及Superdex 200 FPLC膠體過濾管柱自牛肝與牛乳中純化出鹼性磷酸?(Alkaline phosphatase; AP)，並以人類胎盤 AP作為比較。 純化所得的AP利用1．膠體過濾層析分子量估算法(Gel filtration calibration) 2. SDS-膠體電泳法(SDS-PAGE)及3. SDS-毛細管電泳法(SDS-CE)決定分子量。牛肝AP由方法1．所得分子量為113,000，方法2．及3．所得分子量為64,000;牛乳AP由方法1. 所得分子量為122,000，方法2．及3．所得分子量為60,000。等電點的測量使用了1. Immobiline等電集焦(Immobiline isoelectric focusing；IEF)及2．毛細管等電集焦(CE-IEF)兩種方法。牛肝AP的pI為5.34-5.70，牛乳AP的pI為5.52-7.70。 由lectin stainings的結果，牛肝AP，牛乳AP及人類胎盤AP均為糖蛋白；牛乳AP在galatose, mannose,fucose 及sialic acid的含量上較牛肝AP高。在使用對N-glycan有專一性的PNGase F進行酵素去糖(Enzymatic deglycosylation)後，牛肝AP與牛乳AP的分子量減少2.4 KDa，而牛乳酵素仍可被具有galactose專一辨識性的 RCA120 lectin染出，顯示牛乳AP可能具有O-glycan。而使用trifluoromethanesulfonic acid (TFMSA)會造成牛肝 AP與牛乳AP的嚴重水解。人類胎盤AP經酵素去糖後，分子量改變為6.4 KDa；使用TFMSA進行化學去糖處理所造成的分子量變化為2.0 KDa。

Secretory and membrane-bound alkaline phosphatases (AP) were purified respectively from bovine milk and liver. Alkaline phosphatase from human placenta was included for comparison. Affinity column chromatography on ConA-Sepharose, gel filtration on Superose?l2, Superdex-200, and ion- exchange column chromatography on Mono Q were conducted consecutively to isolate the enzyme. The final specific activity (SA) of alkaline phosphatase from bovine liver and milk were 0.26 units/mg of protein, 33 units/mg of protein respectively (human placental AP, 340 units/mg of protein). Both alkaline phosphatases from bovine milk and liver share with similar molecular weights and pIs. The molecular weights of alkaline phosphatase were estimated by using gel filtration, with a Mr of 113,000 for bovine liver AP and 122,000 for bovine milk and by using SDS-PAGE and SDS-CE, with a Mr of 60,000 for bovine milk and a Mr of 64,000 for bovine liver. The pI of alkaline phosphatase was analyzed by appling immobiline IEF, and CE-IEF with a pI range of 5.52-7.70 for bovine milk and a pI of 5.30 - 5.73 for bovine liver. Both alkaline phosphatases from bovine milk and liver are dimeric glycoproteins as demonstrated by SDS-PAGE plus gel giltration, and lectin staining on nitrocellulose membrane blots. Alkaline phosphatase from milk is enriched with mannose, galactose, fucose and sialic acid, in comparison with the enzyme from liver. The carbohydrate moiety of alkaline phosphatase from bovine milk or liver is with a difference of Mr 24,000 as revealed by SDS-PAGE of alkaline phosphatase after enzymatic deglycosylation by N-glycan specific PNGase F. The result of RCA120 lectin staining indicated that alkaline phosphatase from bovine milk may contain O-glycan. Both enzymes are labile to the deglycosylation by chemical treatment with TFMSA, but not the human alkaline phosphatase from placenta. The human alkaline phosphatase was resistant to the deglycosylation by chemical treatment with TFMSA in contrast to the deglycosylation by PNGase as demonstrated by the value of carbohydrate moiety of Mr 2,000 vs Mr 6,400.