Characterization of the glycosyl-phosphatidylinositol-anchored human renal dipeptidase reveals that it is more extensively glycosylated than the pig enzyme.
Renal dipeptidase (EC 3.4.13.11) has been purified from human kidney cortex by affinity chromatography on cilastatin-Sepharose following solubilization with either n-octyl-beta-D-glucopyranoside or bacterial phosphatidylinositol-specific phospholipase C (PI-PLC). Phase separation in Triton X-114 revealed that the detergent-solubilized form was amphipathic and retained the glycosyl-phosphatidylinositol membrane anchor whereas the phospholipase solubilized form was hydrophilic. Both forms of the enzyme existed as a disulphide-linked dimer of two identical subunits of Mr 59,000 each. The glycosyl-phosphatidylinositol anchor of purified human renal dipeptidase was hydrolysed by a range of bacterial PI-PLCs and by a plasma phospholipase D. Mild acid treatment and nitrous acid deamination of the hydrophilic form revealed that the cross-reacting determinant, characteristic of the glycosyl-phosphatidylinositol anchor, was due exclusively to the inositol 1,2-cyclic phosphate ring epitope. The N-terminal amino acid sequences of the amphipathic and hydrophilic forms were identical, locating the membrane anchor at the C-terminus. The N-terminal sequence of human renal dipeptidase showed a high degree of similarity with that of the pig enzyme, and enzymic deglycosylation revealed that the difference in size of renal dipeptidase between these two species is due almost entirely to differences in the extent of N-linked glycosylation.
Hooper
N M
NM
Department of Biochemistry, University of Leeds, U.K.
Keen
J N
JN
Turner
A J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D003902:Detergents; D004150:Dipeptidases / Q000302:isolation & purification* / Q000378:metabolism; D004220:Disulfides; D006017:Glycolipids / Q000032:analysis*; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols; D006801:Humans; D007425:Intracellular Membranes / Q000201:enzymology; D007672:Kidney Cortex / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010716:Phosphatidylinositols / Q000032:analysis*; D012689:Sequence Homology, Nucleic Acid; D013552:Swine; D010738:Type C Phospholipases
D003902:Detergents; D004220:Disulfides; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D046911:Macromolecular Substances; D010716:Phosphatidylinositols; D010738:Type C Phospholipases; D004150:Dipeptidases; C038937:dipeptidase