Purification and characterization of a 40 S ribosomal protein S6 kinase from vanadate-stimulated Swiss 3T3 cells.
Recently we have identified a mitogen-activated S6 kinase from Swiss 3T3 cells (Jenö, P., Ballou, L. M., Novak-Hofer, I., and Thomas, G. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 406-410). Here we describe the detailed purification of this enzyme from high-speed supernatants (400,000 x g) of vanadate-treated cell extracts. The enzyme is purified through six sequential steps including cation- and anion-exchange, sizing, and affinity chromatography. At each step, the enzyme behaves as one entity and, on the final step of purification, is revealed on silver-stained sodium dodecyl sulfate-polyacrylamide gels as a single protein of Mr 70,000. As reported earlier, the overall purification factor is 3,000-fold, and the specific activity of the homogeneously purified enzyme is 0.6 mumol/min/mg of protein. However, recovery of total activity is only 0.2%. This large loss of activity appears to be due to freeze-thawing the enzyme between each step of purification. The purified kinase does not phosphorylate casein, histones 2A and 3S, or phosvitin. It has a Km for ATP of 28 microM and a broad optimum for Mg2+ between 5 and 20 mM. Mn2+ does not affect the basal level of kinase activity, and at concentrations as low as 1 mM, it completely suppresses the effect of 20 mM Mg2+ on kinase activity. The relationship of this enzyme to two other purified S6 kinases is discussed.
Jenö
P
P
Friedrich Miescher-Institut, Basel, Switzerland.
Jäggi
N
N
Luther
H
H
Siegmann
M
M
Thomas
G
G
D000818:Animals; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D019893:Ribosomal Protein S6 Kinases; D014638:Vanadates / Q000494:pharmacology*
D014638:Vanadates; D011494:Protein Kinases; D019893:Ribosomal Protein S6 Kinases