Purification and characterisation of amphiphilic lactase/phlorizin hydrolase from human small intestine
- PMID:6786877
- DOI: 10.1111/j.1432-1033.1981.tb05193.x
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 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 21 mM), phlorizin (Km 0.44mM) and other beta-galactosides and beta-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 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 320000 while the hydrophilic form (papain-treated) had a molecular weight of 280000. This indicates that the anchoring segment(s) plus the bound detergent has a molecular weight of approximately 40000. 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 160000. The electromicroscopic picture gives further evidence for this suggestion. In addition the possibility of a high molecular weight, one polypeptide chain is discussed.
Similar articles
- Further characterization of intestinal lactase/phlorizin hydrolase.Skovbjerg H, Norén O, Sjöström H, Danielsen EM, Enevoldsen BS.Skovbjerg H, et al.Biochim Biophys Acta. 1982 Sep 22;707(1):89-97. doi: 10.1016/0167-4838(82)90400-9.Biochim Biophys Acta. 1982.PMID:6814489
- Isolation and characterization of the proximal and distal forms of lactase-phlorizin hydrolase from the small intestine of the suckling rat.Cousineau J, Green JR.Cousineau J, et al.Biochim Biophys Acta. 1980 Sep 9;615(1):147-57. doi: 10.1016/0005-2744(80)90018-2.Biochim Biophys Acta. 1980.PMID:6775701
- Glycosylation of lactase-phlorizin hydrolase in rat small intestine during development.Büller HA, Rings EH, Pajkrt D, Montgomery RK, Grand RJ.Büller HA, et al.Gastroenterology. 1990 Mar;98(3):667-75. doi: 10.1016/0016-5085(90)90287-b.Gastroenterology. 1990.PMID:2105255
- Lactose intolerance and the genetic regulation of intestinal lactase-phlorizin hydrolase.Montgomery RK, Büller HA, Rings EH, Grand RJ.Montgomery RK, et al.FASEB J. 1991 Oct;5(13):2824-32. doi: 10.1096/fasebj.5.13.1916106.FASEB J. 1991.PMID:1916106Review.
- Hypolactasia and lactase persistence. Historical review and the terminology.Sahi T.Sahi T.Scand J Gastroenterol Suppl. 1994;202:1-6. doi: 10.3109/00365529409091739.Scand J Gastroenterol Suppl. 1994.PMID:8042014Review.
Cited by
- Preparation of lactose-free pasteurized milk with a recombinant thermostable β-glucosidase from Pyrococcus furiosus.Li B, Wang Z, Li S, Donelan W, Wang X, Cui T, Tang D.Li B, et al.BMC Biotechnol. 2013 Sep 21;13:73. doi: 10.1186/1472-6750-13-73.BMC Biotechnol. 2013.PMID:24053641Free PMC article.
- Mechanistic inference of the metabolic rates underlyingC breath test curves.Brouwer AF, Lee GO, Schillinger RJ, Edwards CA, Van Wyk H, Yazbeck R, Morrison DJ.Brouwer AF, et al.J Pharmacokinet Pharmacodyn. 2023 Jun;50(3):203-214. doi: 10.1007/s10928-023-09847-x. Epub 2023 Feb 15.J Pharmacokinet Pharmacodyn. 2023.PMID:36790613Free PMC article.
- Disposition of the carboxy-terminus tail of rabbit lactase-phlorizin hydrolase elucidated by phosphorylation with protein kinase A in vitro and in tissue culture.Keller P, Semenza G, Shaltiel S.Keller P, et al.FEBS Lett. 1995 Jul 24;368(3):563-7. doi: 10.1016/0014-5793(95)00679-4.FEBS Lett. 1995.PMID:7635222Free PMC article.
- Processing of human pro-lactase-phlorizin hydrolase at reduced temperatures: cleavage is preceded by complex glycosylation.Naim HY.Naim HY.Biochem J. 1992 Jul 1;285 ( Pt 1)(Pt 1):13-6. doi: 10.1042/bj2850013.Biochem J. 1992.PMID:1637291Free PMC article.
- Repetitive and zonal expression profiles of absorption-related genes in the gastrointestinal tract of ascidian Ciona intestinalis type A.Iguchi R, Nakayama S, Sasakura Y, Sekiguchi T, Ogasawara M.Iguchi R, et al.Cell Tissue Res. 2023 Nov;394(2):343-360. doi: 10.1007/s00441-023-03828-9. Epub 2023 Sep 6.Cell Tissue Res. 2023.PMID:37670165