Identification and purification of a novel phospholipid/ganglioside-binding protein in rabbit serum.
ABSTRACT We have isolated a novel phospholipid/ganglioside-binding protein from rabbit sera or platelet-free plasma. Using an affinity chromatography of a commercial gel (Sephacryl S-series gel, Pharmacia) column and a preparative polyacrylamide gel electrophoresis, the protein can be easily purified. The protein agglutinates human red cells irrespective of the ABO blood types, and its hemagglutination reaction is specifically inhibited by some phospholipids (phosphatidylserine and phosphatidylglycerol) and ganglioside (N-acetylneuraminyl-galactosylglucosyl ceramide, GM3). The hemagglutination and its inhibition reactions are independent on any divalent cations (Ca2+, Mg2+, Mn2+, Ni2+). The protein seems to be assembled as multimers of disulfide-bonded molecular of 86 kDa and 59 kDa subunits.
- [show abstract] [hide abstract]
ABSTRACT: The enzymatic properties of glycosylphosphatidylinositol-specific phospholipase D (EC 188.8.131.52) were characterized using a 6,000-fold purified enzyme. This was obtained in 100 microg amounts from human serum with a recovery of 35%. Pure alkaline phosphatase containing one anchor moiety per molecule was used as substrate. The enzyme is stimulated by n-butanol, but in contrast to other phospholipases this activation is not produced by a transphosphatidylation reaction. The previously reported non-linearity of the specific activity with respect to phospholipase concentration in the test was no longer observed upon purification, indicating inhibitor removal. The serum inhibitor(s) co-chromatograph with serum proteins and lipoproteins. The main part of the inhibitory activity was found in the lipid fraction after protein denaturation and can be subfractionated into acid phospholipids, cholesteryl esters and triacylglycerides. Added phosphatidyl-serine, phosphatidylinositol, phosphatidylglycerol, gangliosides, cholesteryl esters, and sphingomyelins turned out to be strong inhibitors, as well as phosphatidic acid. Phosphatidylethanolamine and various monoacylglycerols were found to be activators. The low glycosylphosphatidylinositol-specific phospholipase activity found in native serum did not increase significantly upon 90% removal of phospholipids by n-butanol. High serum concentrations of strongly inhibiting compounds, complex kinetic interactions among aggregates of these substances, and compartmentalization effects are discussed as possible reasons for the observed inactivity.Biological Chemistry 01/2000; 381(5-6):471-85. · 2.96 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: We have reported that rabbit serum contains a phospholipid (PL)/ganglioside-binding protein which adsorbs to Sephacryl S-400 gel and agglutinates human red blood cells. A new protein similar to the PL/ganglioside-binding protein was simply purified from normal human plasma using Sephacryl S-400, Sepharose CL-4B and DEAE-Sepharose CL-6B columns. The purified protein was found to agglutinate rabbit red blood cells. The hemagglutination was specifically inhibited by two PL, phosphatidylserine and phosphatidylinositol, but not by any other PL, gangliosides, saccharides or glycoproteins tested. From analyses of the N-terminal amino acid sequence and immunological specificity, the protein was identified to be a human immunoglobulin M.Experimental and Clinical Immunogenetics 01/1997; 14(4):281-5.
- Biochimica et Biophysica Acta 05/1998; 1391(3):287-306. · 4.66 Impact Factor