Ectoenzymes of the kidney microvillar membrane. Isolation and characterization of the amphipathic form of renal dipeptidase and hydrolysis of its glycosyl-phosphatidylinositol anchor by an activity in plasma.
Renal dipeptidase (EC 3.4.13.11) has been solubilized from pig kidney microvillar membranes with n-octyl-beta-D-glucopyranoside and then purified by affinity chromatography on cilastatin-Sepharose. The enzyme exists as a disulphide-linked dimer of two identical subunits of Mr 45,000 each. The purified dipeptidase partitioned into the detergent-rich phase upon phase separation in Triton X-114 and reconstituted into liposomes consistent with the presence of the glycosyl-phosphatidylinositol membrane anchor. The N-terminal amino acid sequence of the amphipathic, detergent-solubilized, form of renal dipeptidase was identical with that of the hydrophilic, phospholipase-solubilized, form, locating the membrane anchor at the C-terminus of the protein. The glycosyl-phosphatidylinositol anchor of both purified and microvillar membrane renal dipeptidase was a substrate for an activity in pig plasma which displayed properties similar to those of a previously described phospholipase D. The cross-reacting determinant of the glycosyl-phosphatidylinositol anchor was generated by incubation of purified renal dipeptidase with bacterial phosphatidylinositol-specific phospholipase c, whereas the anchor-degrading activity in plasma failed to generate this determinant.
Hooper
N M
NM
Department of Biochemistry, University of Leeds, U.K.
Turner
A J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D004150:Dipeptidases / Q000302:isolation & purification*; D006017:Glycolipids / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols; D006868:Hydrolysis; D007672:Kidney Cortex / Q000201:enzymology* / Q000648:ultrastructure; D008871:Microvilli / Q000201:enzymology; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000378:metabolism*; D010949:Plasma / Q000378:metabolism*; D013552:Swine
D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D010716:Phosphatidylinositols; D004150:Dipeptidases; C038937:dipeptidase