RESUMEN
We have isolated heparan sulfate proteoglycans (HSPGs) from cloned rat microvascular endothelial cells using a combination of ion-exchange chromatography, affinity fractionation with antithrombin III (AT III), and gel filtration in denaturing solvents. The anticoagulantly active heparan sulfate proteoglycans (HSPGact) which bind tightly to AT III bear mainly anticoagulantly active heparan sulfate (HSact) whereas the anticoagulantly inactive heparan sulfate proteoglycans (HSPGinact) possess mainly anticoagulantly inactive heparan sulfate (HSinact). HSact and HSinact were also isolated by a combination of ion-exchange chromatography, treatment with protease and chondroitin ABC lyase, and affinity fractionation with AT III. HSact and HSinact have molecular sizes of about 25-30 kDa with the same overall composition of monosaccharides except that HSact exhibits about nine glucuronsyl 3-O-sulfated glucosamines/chain whereas HSinact possesses about three glucuronsyl 3-O-sulfated glucosamines/chain. Direct isolation of the AT III-binding site of HSact by exposing carbohydrate chains to Flavobacterium heparitinase in the presence of protease inhibitor revealed only a single interaction site which contained two to three glucuronsyl 3-O-sulfated glucosamine residues. The core proteins of HSPGact and HSPGinact were isolated by treatment with Flavobacterium heparitinase and purification by ion-exchange chromatography. The molecular sizes of the core proteins were established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and their primary structures were examined by cleavage with trypsin or endopeptidase Glu-C as well as separation of peptides by reverse-phase high performance liquid chromatography. The results showed that both sets of core proteins exhibited three major components with molecular sizes of 50, 30, and 25 kDa, respectively. The 25-kDa species appears to be a proteolytic degradation product of the 30-kDa species. The peptide mapping revealed that HSPGact and HSPGinact possess extremely similar core proteins.
Asunto(s)
Células Clonales/metabolismo , Endotelio Vascular/metabolismo , Heparitina Sulfato/aislamiento & purificación , Proteoglicanos/aislamiento & purificación , Animales , Antitrombina III/metabolismo , Autorradiografía , Sitios de Unión , Western Blotting , Cromatografía Liquida , Electroforesis en Gel de Agar , Electroforesis en Gel de Poliacrilamida , Endotelio Vascular/citología , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/biosíntesis , Heparitina Sulfato/metabolismo , Mapeo Peptídico , Proteoglicanos/biosíntesis , Proteoglicanos/metabolismo , RatasRESUMEN
The murine lymphoma cell line LSTRA expresses high levels of a membrane-associated tyrosine protein kinase, which we now show to be acylated by [3H]myristate in vivo. This [3H]myristate-labeled tyrosine protein kinase is immunoprecipitated from detergent extracts of postnuclear particulate fractions with an antibody directed against its single site of tyrosine phosphorylation. This site has an amino acid sequence also found in the transforming proteins of the Rous sarcoma and Y73 viruses. Preincubation of the antibody with a peptide containing the same sequence completely blocks this immunoprecipitation. The [3H]myristate linkage to the protein is stable in boiling 2% NaDodSO4/0.125 M Tris Cl, pH 6.7/5% 2-mercaptoethanol, which suggests an amide rather than an ester linkage. Analogous attempts to label with [3H]palmitate show negligible incorporation into either nonnuclear particulate proteins or immunoprecipitated proteins. Chemical characterization of the immunoprecipitated protein isolated by NaDodSO4/PAGE verifies that the 3H label is in protein-associated myristate. Sonicated 5% NaDodSO4 extracts of LSTRA and YAC-1 (another murine lymphoma line) cells contain quite different distributions of myristoylated proteins.