Human intestinal lactase/phlorizin hydrolase (EC 22.214.171.124/62) was purified in its amphiphilic form by immunoadsorbent chromatograpgy. The purification factor was approximately 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 21mM), phlorizin (Km 0.44mM) and other β-galactosides and β-glucosides. Tris inhibited the hydrolysis of lactose whereas phlorizin hydrolysis was almost unaffected. The activity against these two substrates also showed different thermal stability. It is suggested that the human enzyme has two 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 molecular weight of 320 000 while the hydrophilic form (papain-treated) had a molecular weight of 280 000. This indicates that the anchoring segment(s) plus the bound detergent has a molecular weight of approximately 40 000. In polyacrylamide gel electrophoresis in sodium dodecylsulphate the fully denatured enzyme had an apparent molecular weight of 160000. It is therefore suggested that the human lactase/phlorizin hydrolase is composed of two monomers each with a molecular weight of 160 000. The electromicroscopic picture gives further evidence for this suggestion. In addition the possibility of a high molecular weight, one polypeptide chain is discussed.