Polyamine metabolism in McCoy cells: II. Cellular origin of excreted polyamine conjugated proteins.
Incubation of McCoy cultures with medium containing 14C-putrescine resulted in the incorporation of 14C-polyamine into intracellular proteins. A greater than 100,000 dalton 14C-polyamine conjugated protein was present in the McCoy cell lysate supernatant (CLSP). CLSP was heterogeneous containing proteins with pIs ranging from 4.55 to 5.50. The major proteins had pIs of 4.55 and 5.20. Electrophoresis of solubilized McCoy cell lysate pellet revealed a 14C-polyamine conjugated protein peak with Mr approximately or equal to 70,000 (CLPP). Both CLSP and CLPP contained bound polyamine. The major CLSP polyamine was spermidine while spermine exceeded the other two polyamines (putrescine and spermidine) in CLPP. About 25% of the polyamines associated with CLSP and CLPP were covalently bound with the exception of CLSP putrescine where 62.1% was covalently bound. Results suggested the presence of a polyamine protein isopeptide bond in CLSP. Sephadex gel filtration of cultured medium resulted in the identification of two macromolecular polyamine-containing fractions (MP1, Mr greater than 100,000 and MP2, M = 60,000-70,000). Antibody raised in rabbits against a membrane-organelle preparation cross-reacted with Sephadex gel filtration derived MP1 but not with peak MP2 suggesting that MP1 but not MP2 might be a membrane constituent. Antibody raised against medium polyamine conjugated protein peak 2 (MP2) cross-reacted with the cell lysate supernatant indicating that MP2 was present in the cytosol. It did not cross-react with the cell lysate pellet preparation. Antibody against MP2 also formed a precipitation band with MP1 indicating that there might be a common antigenic site on MP1 and MP2.
D002451:Cell Compartmentation; D006801:Humans; D007526:Isoelectric Point; D008970:Molecular Weight; D011073:Polyamines / Q000378:metabolism*; D011506:Proteins / Q000276:immunology / Q000378:metabolism*
D011073:Polyamines; D011506:Proteins