Phosphotyrosyl-protein phosphatases. I. Separation of multiple forms from bovine brain and purification of the major form to near homogeneity.
Seven Tyr-protein phosphatase activities were isolated from bovine brain using phosphotyrosyl-casein as a model substrate. The activities were resolved from the cytosolic fraction by a three-step procedure employing successive DEAE-cellulose, phosphocellulose, and gel permeation chromatography steps. The seven activities accounted for 70% of the Tyr-protein phosphatase activity in bovine brain extracts and were distinct from type 1 and type 2 Ser/Thr-protein phosphatases and from the major alkaline phosphatase activities. Apparent molecular weights of the activities by gel permeation chromatography were: phosphotyrosyl-protein phosphatase (PTP)-1A (Mr 86,000), PTP-1B (Mr 24,000), PTP-2 (Mr 88,000), PTP-3 (Mr 90,000), PTP-4 (Mr 80,000), PTP-5 (Mr 48,000), and PTP-6 (Mr 104,000). PTP-5 was the major activity accounting for 26% of total while the remaining activity was divided rather evenly among the other six activities. PTP-5 was further purified to near homogeneity by additional chromatographies on Affi-Gel Blue, heparin-agarose, and Mono S giving an overall purification of 50,000-fold and a yield of 5.8%. One of two major polypeptides (Mr 46,000) in the preparation was identified as PTP-5 since it alone expressed protein phosphatase activity when protein-staining bands were eluted from sodium dodecyl sulfate-polyacrylamide gels and renatured. PTP-5 had a neutral pH optimum, and using phosphotyrosyl-casein as substrate it had a Km of 130 nM and a Vmax of 10 mumol Pi released.min-1.mg protein-1. These kinetic parameters are well within the range of values obtained for other pure protein phosphatases. PTP-5 also dephosphorylated pp60v-src (autophosphorylated at Tyr-416) at 10% of the rate observed with phosphotyrosyl-casein. Additionally the ratio of phosphotyrosyl-casein/pp60v-src phosphatase activity was relatively constant throughout the PTP-5 purification procedure. These results indicate that PTP-5 is able to bind and efficiently dephosphorylate phosphotyrosyl-proteins and suggest that it is a physiologically relevant Tyr-protein phosphatase.
Jones
S W
SW
Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138.
Erikson
R L
RL
Ingebritsen
V M
VM
Ingebritsen
T S
TS
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D019000:Phosphotyrosine; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D007527:Isoenzymes; D019000:Phosphotyrosine; D014443:Tyrosine; D000469:Alkaline Phosphatase; D010749:Phosphoprotein Phosphatases