Glycosaminoglycans and a newly purified aortic chondroitin proteoglycan block polycationic modulation of protein phosphatase activity.
Recently, we described a bovine aortic phosphatase which we called PCM-phosphatase (polycation modulable) because its activity in vitro can be modulated by polycations such as polylysine and histone-H1 (Di Salvo J, Gifford D, Kokkinakis A. Modulation of aortic protein phosphatase activity by polylysine. Proc Soc Exp Biol Med 177:24-32, 1984). We We suspected that polycationic modulation might be inhibited by polyanionic glycosaminoglycans. Accordingly, an aortic anionic substance was purified by sequential steps including (a) heating aortic extracts at 90 degrees C, (b) precipitation of protein with (NH4)2 SO4, and (c) anionic-exchange chromatography on a Mono Q HR 5/5 column using the Pharmacia fast protein liquid chromatography system. Electrophoresis (polyacrylamide-agarose) of the purified substance revealed one band which stained metachromatically with toluidine blue; however, no staining occurred with Coomassie blue. Electrophoretic mobility increased following proteolytic digestion of the substance with papain. The substance produced concentration-dependent reversal of polylysine-mediated inhibition of myosin light chain dephosphorylation, and it also reversed polylysine-mediated stimulation of phosphorylase phosphatase activity expressed by PCM-phosphatase. Its ability to inhibit or reverse polycationic modulation was abolished after incubation with either chondroitinase AC or chondroitinase ABC. Based on these properties the substance was identified as a chondroitin proteoglycan. Commercially available glycosaminoglycans (heparin and chondroitin sulfates) also reversed polycationic modulation. The results show that modulation of phosphatase activity may be significantly modified by naturally occurring glycosaminoglycans. These studies may also have an important bearing on the purported roles of phosphatase(s) and glycosaminoglycans in calcification of soft tissues.
D000818:Animals; D001011:Aorta / Q000032:analysis; D002417:Cattle; D002809:Chondroitin Sulfates / Q000302:isolation & purification / Q000494:pharmacology; D006025:Glycosaminoglycans / Q000494:pharmacology*; D006493:Heparin / Q000494:pharmacology; D009218:Myosins / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010762:Phosphorylase a / Q000378:metabolism; D011107:Polylysine / Q000037:antagonists & inhibitors*; D011509:Proteoglycans / Q000302:isolation & purification / Q000494:pharmacology*
D006025:Glycosaminoglycans; D011509:Proteoglycans; D011107:Polylysine; D006493:Heparin; D002809:Chondroitin Sulfates; D010762:Phosphorylase a; D010749:Phosphoprotein Phosphatases; D009218:Myosins