Purification and Characterisation of Amphiphilic Lactase/Phlorizin Hydrolase from Human Small Intestine

Abstract

Human intestinal lactase/phlorizin hydrolase (EC 3.2.1.23/62) was purified in its amphiphilic form by immunoadsorbent chromatography. The purification factor was .apprx. 600 and the recovery 14%. The enzyme was essentially free from other known brush-border peptidases and disaccharidases and appeared homogeneous in crossed immunoelectrophoresis and polyacrylamide gel electrophoresis in sodium dodecylsulphate. The purified enzyme hydrolyzed lactose (pH optimum 5.8-6.0, Km 21 mM), phlorizin (Km 0.44 mM) and other .beta.-galactosides and .beta.-glucosides. Tris inhibited the hydrolysis of lactose whereas phlorizin hydrolysis was almost unaffected. The activity against these 2 substrates also showed different thermal stability. It is suggested that the human enzyme has 2 different enzymatic sites: one for lactose hydrolysis, inhibited by phlorizin, and one for phlorizin hydrolysis. By gel filtration on Ultrogel AcA 34 the amphiphilic form of the enzyme had a MW of 320,000 while the hydrophilic form (papain-treated) had a MW of 280,000. This indicates that the anchoring segment(s) plus the bound detergent has a MW of .apprx. 40,000. In polyacrylamide gel electrophoresis in sodium dodecylsulphate the fully denatured enzyme had an apparent MW of 160,000. Apparently the human lactase/phlorizin hydrolase is composed of 2 monomers each with a MW of 160,000. The EM picture gives further evidence for this suggestion. In addition the possibility of a high MW, 1 polypeptide chain is discussed.