Zhizhuang Zhao

Continuous spectrophotometric assay of protein tyrosine phosphatase using phosphotyrosine

A continuous activity assay for protein tyrosine phosphatases (PTPs), employing phosphotyrosine (P-Tyr) as a substrate, has been developed and applied to measure the activities of two purified enzymes, namely, the full length T-cell protein tyrosine phosphatase (TC PTP) and its truncated form (TC delta C11 PTP). The reaction was followed by changes in ultraviolet absorption and fluorescence resulting from the dephosphorylation of P-Tyr. Both enzymes obey Michaelis-Menten kinetics, with Km = 304 microM, Vmax = 62,000 units/mg for TC PTP and Km = 194 microM, Vmax = 73,000 units/mg for TC delta C11 PTP. The D- and L-forms of P-Tyr are equally effective as substrates. The optimum pH for both enzymes is 4.75. The known effectors of PTPs have the predicted effects on catalytic activity.

Preparation and functional characterization of a catalytically active fragment of phosphorylase kinase

Limited proteolysis of rabbit muscle phosphorylase kinase catalyzed by chymotrypsin generates a 33 kD product whose kinase activity is independent of both calcium and pH over the range of 6.8 to 8.3 (Malencik, D.A. & Fischer, E.H.Calcium and Cell Function III: 161–188, 1982). This active preparation consists of three related species containing residues 1–290, 1–296, and 1–298 of the 44.7 kD γ-subunit of phosphorylase kinase (Harris, W.R., Malencik, D.A., Johnson, C.M., Carr, S.A., Roberts, G.D., Byles, C.E., Anderson, S.R., Heilmeyer, L.M.G., Fischer, E.H. & Crabb, J.W.J. Biol. Chem. 265:11740–11745, 1991). Good recoveries of catalytic activity — with varying degrees of calcium dependence — result upon the digestion of phosphorylase kinase with assorted proteases. However, especially high yields of the chymotryptic fragment are obtainable, with purification on an Ultrogel-34 column and a DEAE Sepharose CL-6B column giving 23% of the maximum possible protein.Physical characterization shows that the 33 kD chymotryptic fragment is globular, withs20,w=2.9S, and that it has an isoelectric point of 5.3. Our continuous catalytic assay, based on differences in the binding of the fluorescent dye 1-anilinonaphthalene-8-sulfonate by phosphorylasea andb, shows that, on a molar basis, the activity of the fragment is 2.8 fold greater than that of phosphorylase kinase (Malencik, D.A., Zhao, Z. and Anderson, S.R.Biochem. Biophys. Res. Comm. 174: 344–350, 1991). The active fragment also undergoes autophosphorylation. Incubation with Mg[γ-P32] ATP results in the reaction of 0.7 mol32P/mol fragment. When the catalytic subunit of the cAMP-dependent protein kinase is also present, the amount of32P incorporated increases to 1.1 mol/mol. In the former case, phosphorylation occurs primarily at Ser30 while in the latter an additional reaction takes place at Ser81. The phosphopeptides correspond to sequences occurring in the γ-subunit of phosphorylase kinase.