The human erythrocyte contains two forms of phosphatidylinositol-4-phosphate 5-kinase which are differentially active toward membranes.
A human erythrocyte cytosolic phosphatidylinositol-4-phosphate 5-kinase (PIP kinase) and a membrane-bound PIP kinase have been purified by phosphocellulose chromatography. Fractionation of the membrane-bound PIP kinase activities by phosphocellulose separated activity into two peaks, which eluted at 0.6 M NaCl (type I PIP kinase) and 1.0 M NaCl (type II PIP kinase). The cytosolic PIP kinase and the membrane-bound type II PIP kinase are 53 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, have indistinguishable 125I-peptide maps, and are immunochemically indistinguishable, suggesting that they are sequence identical. Antibodies raised to the cytosolic PIP kinase inhibit activity of both the membrane-bound type II and the cytosolic PIP kinases. The type I PIP kinase appears to be distinct from the cytosolic and membrane-bound type II PIP kinase; it is not immunocross-reactive, and antibodies toward type II PIP kinases do not inhibit type I PIP kinase. Further, membrane-bound type II PIP kinase can be removed from type I PIP kinase without loss of activity. Functional characterization of the PIP kinases demonstrates that the type I kinase has a 10-fold lower Km for PIP and a 5-fold higher Km for ATP compared with the type II enzymes. The type I and type II (membrane-bound or cytosolic) PIP kinases are modulated differentially by spermine and heparin. Finally, the type I PIP kinase phosphorylates intrinsic PIP on isolated erythrocyte membranes, whereas the type II PIP kinases have no activity toward native membranes.
Bazenet
C E
CE
Department of Pharmacology, University of Wisconsin Medical School, Madison 53706.
Ruano
A R
AR
Brockman
J L
JL
Anderson
R A
RA
D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000201:enzymology / Q000378:metabolism*; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008081:Liposomes; D008563:Membrane Lipids / Q000097:blood*; D008970:Molecular Weight; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols / Q000097:blood*; D010770:Phosphotransferases / Q000097:blood* / Q000302:isolation & purification; D017853:Phosphotransferases (Alcohol Group Acceptor); D013379:Substrate Specificity
D008081:Liposomes; D008563:Membrane Lipids; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; C037178:phosphatidylinositol 4-phosphate; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase