牛肠碱性磷酸酶是二聚体膜源性糖蛋白，至少存在三种同工亚型。每个单体通常含有两个 N - 连接聚糖以及一个或多个 O - 连接聚糖，该酶的活性发挥需要锌离子、镁离子或钙离子参与。 此糖蛋白碳水化合物含量约 12%，其中己糖占 6%，其余中性糖类占 6%。每个碱性磷酸酶分子含有 4 个锌原子与 4 个二硫键，镁离子可使其活性达到峰值。该酶能够催化磷酸单酯水解，底物包括对硝基苯磷酸、苯磷酸、酚酞磷酸、α- 甘油磷酸、β- 甘油磷酸、2 - 磷酸甘油酸、磷酸丙糖、6 - 磷酸葡萄糖、1 - 磷酸葡萄糖、1 - 磷酸果糖、6 - 磷酸果糖、5 - 磷酸腺苷、3 - 磷酸腺苷、磷酸烯醇式丙酮酸以及 β- 烟酰胺腺嘌呤二核苷酸磷酸等物质。砷酸盐、半胱氨酸、碘单质、无机磷酸盐、焦磷酸盐、磷酸二异丙酯、磷酸三苯酯、二异丙基氟磷酸与 L - 苯丙氨酸均为该酶强效抑制剂。
应用
该酶可用于检测磷脂酰肌醇 4 - 磷酸与磷脂酰肌醇 4,5 - 二磷酸，也可结合萘酚修饰电极开展碱性磷酸酶活性测定。 可对酪蛋白等蛋白质进行去磷酸化处理；还能脱去 DNA、RNA5' 端磷酸基团，规避核酸自连现象。核酸经该酶去磷酸化后，可借助 T4 多核苷酸激酶标记放射性磷酸基团。 可与抗体及其他蛋白偶联，应用于酶联免疫吸附试验、蛋白免疫印迹、组织化学检测；常规用于蛋白质与核酸去磷酸化，高灵敏度实验中也可用于蛋白标记。此产品可用于鸟苷三磷酸水解相关研究。 高比活规格适用于抗体与蛋白偶联实验。
生化生理特性
牛肠黏膜来源的碱性磷酸酶，在 pH7.5~9.5 区间稳定性最佳。该酶底物谱广泛，可作用于醇类、胺类、焦磷酸类及酚类磷酸酯，酶活性依赖锌、镁或钙离子。
制备说明
亲和纯化，原料取自牛肠黏膜。稀释液组分：10 mM Tris-HCl（pH8.0）、1~5 mM MgCl₂、0.1~0.2 mM ZnCl₂、50% 甘油，产品于 2~8℃条件下储存。
Bovine intestinal alkaline phosphatase is a dimeric, membrane-derived glycoprotein. At least three isoforms exist, which typically possess two N-linked and one or more O-linked glycans per monomer. The enzyme requires zinc, and magnesium or calcium divalent ions for activity.
The enzyme is a glycoprotein containing approximately 12% carbohydrate (6% hexoses and 6% other neutral sugars). Each molecule of alkaline phosphatase contains four zinc atoms and four disulfide bridges. Maximal activity with alkaline phosphatase is achieved in the presence of magnesium. It catalyzes the hydrolysis of phosphate monoesters such as p-nitrophenyl phosphate, phenyl phosphate, phenolphthalein phosphate, α-glycerol phosphate, β-glycerol phosphate, 2-phosphorylglycerate, triosephosphate, glucose-6-phosphate, glucose 1-phosphate, fructose 1-phosphate, fructose 6-phosphate, adenosine 5-phosphate adenosine 3-phosphate, phosphoenolpyruvate, and β-nicotinamide adenine dinucleotide phosphate. Arsenate, cysteine, iodine, inorganic phosphate, pyrophosphate, diisopropyl phosphate, triphenylphosphate, diisopropyl fluorophosphate, and L-phenylalanine are some of the strong inhibitors of alkaline phosphatase.
Application:
The enzyme from Sigma has been used in the assay of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. It has also been used for alkaline phosphatase assays using a Nafion™-modified electrode.
Alkaline phosphatase can be used to dephosphorylate casein and other proteins. Alkaline phosphatase may be also be used to dephosphorylate the 5′-termini of DNA or RNA to prevent self-ligation. DNA or RNA can also be tagged with radiolabeled phosphate (via T4 polynucleotide kinase) after dephosphorylation with alkaline phosphatase..
Alkaline phosphatase is used for conjugation to antibodies and other proteins for ELISA, Western blotting, and histochemical detection. It is routinely used to dephosphorylate proteins, such as casein, and nucleic acids. It may be used for protein labeling when high sensitivity is required. Alkaline phosphatase may be also be used to dephosphorylate the 5′-termini of DNA or RNA to prevent self-ligation. DNA or RNA can also be tagged with radiolabeled phosphate (via T4 polynucleotide kinase) after dephosphorylation with alkaline phosphatase. Product P5521 has been used to study GTP hydrolysis
High specific activity grade recommended for antibody and protein conjugation.
Biochem/physiol Actions
Alkaline phosphatase, from bovine intestinal mucosa, is most stable in the pH range
7.5-9.5. The enzyme has a broad specificity for phosphate esters of alcohols, amines, pyrophosphate, and phenols and it requires zinc, and magnesium or calcium divalent ions for activity.
The enzyme has a broad specificity for phosphate esters of alcohols, amines, pyrophosphate, and phenols. It is routinely used to dephosphorylate proteins and nucleic acids.
Preparation Note
Affinity purified
This product is prepared from bovine intestinal mucosa. Dilutions should be made in 10 mM Tris-HCl, pH 8.0, 1-5 mM magnesium chloride,
0.1-0.2 mM zinc chloride, and 50% glycerol and stored at 2-8 °C.
ZJ26F0636372
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