Spontaneous release of glycosylphosphatidylinositol (GPI)-anchored renal dipeptidase from porcine renal proximal tubules.
The incubation of porcine renal proximal tubules (PTs) resulted in the release of the glycosylphosphatidylinositol (GPI)-anchored renal dipeptidase (RDPase, EC 3. 4. 13. 19) from the membrane after a lag period of approximately 6 hours. This spontaneous release of RDPase from the membrane was inhibited by antibiotics. When the incubation supernatant was added back to fresh PTs, both the antibiotic inhibition of RDPase release and the lag period disappeared. The released RDPase reacted with an anti-cross reacting determinant antibody indicating the presence of the Ins (1,2-cyc)P moiety. These results suggest that bacteria in the PTs, when incubated, grow and secrete a phosphatidylinositol-specific phospholipase C (PI-PLC). This enzyme then hydrolyses the GPI-anchored RDPase and is transferable. RDPase was purified following its release from the membrane by this simple and inexpensive method which may also be applied to other GPI-anchored proteins.
Park
Sung Wook
SW
Department of Pharmacy, College of Pharmacy, College of Natural Science, Chonnam National University, Kwangju, Korea.
Kang
Bok Yun
BY
Yoon
Hyun Joong
HJ
Park
Eun Mi
EM
Choi
Kyong
K
Lee
Hwanghee Blaise
HB
Hooper
Nigel M
NM
Park
Haeng Soon
HS
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D004150:Dipeptidases / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005839:Gentamicins / Q000494:pharmacology; D017261:Glycosylphosphatidylinositols / Q000737:chemistry*; D066298:In Vitro Techniques; D007687:Kidney Tubules, Proximal / Q000201:enzymology*; D011817:Rabbits; D013552:Swine
D000900:Anti-Bacterial Agents; D005839:Gentamicins; D017261:Glycosylphosphatidylinositols; D004150:Dipeptidases