Chiun-Jye Yuan

Expression, purification, characterization, and deletion mutations of phosphorylase kinase γ subunit: identification of an inhibitory domain in the γ subunit

A catalytic fragment,γ1-298, derived from limited chymotryptic digestion of phosphorylaseb kinase (Harris, W.R.et al., J. Biol. Chem., 265: 11740–11745, 1990), is reported to have about six-fold greater specific activity than does the γ subunit-calmodulin complex. To test whether there is an inhibitory domain located outside the catalytic core of the γ subunit, full-length wild-type and seven truncated forms of γ were expressed inE. coli. Recombinant proteins accumulate in the inclusion bodies and can be isolated, solubilized, renatured, and purified further by ammonium sulfate precipitation and Q-Sepharose column. Four out of seven truncated mutants show similar (γ1-353 andγ1-341) or less (γ1-331 andγ1-276) specific activity than does the full-length wild-type γ,γ1-386. Three truncated forms,γ1-316,γ1-300, andγ1-290 have molar specific activities approximately twice as great as those of the full-length wild-type γ and the nonactivated holoenzyme. All recombinant γs exhibit similarKm values for both substrates, i.e., about 18μM for phosphorylaseb and about 75 μM for MgATP. Three truncated γs,γ1-316,γ1-300, andγ1-290, have a 1.9- to 2.5-fold greater catalytic efficiency (Vmax/Km) than that of the full-length wild-type γ and a 3.5- to 4.5-fold greater efficiency than that of the truncatedγ1-331. This evidence suggests that there is at least one inhibitory domain in the C-terminal region of γ, which is located atγ301-331·γ1-290, but notγ1-276, which contains the highly conserved kinase domain, is the minimum sequence required for the γ subunit to exhibit phosphotransferase activity. Bothγ1-290 andγ1-300 have several properties similar to full-length wild-type γ, including metal ion responses (activation by free Mg2+ and inhibition by free Mn2+) pH dependency, and substrate specificities.

Ca2+-independent interaction of the γ subunit of phosphorylase kinase with dansyl-calmodulin☆

Abstract A strong Ca 2+ -independent interaction between the isolated, active γ subunit of phosphorylase kinase and dansyl-calmodulin (dansyl-CaM) was observed by monitoring changes in fluorescence intensity in the absence of calcium ion. The pure, active γ subunit of phosphorylase kinase was simply prepared by dialyzing the HPLC-purified, inactive γ subunit against 8 m urea, containing 0.1 m m DTT, 0.1 m Hepes at pH 6.8 or 0.1 m Tris at pH 8.2, followed by dilution of urea with pH 6.8 or 8.2 buffer. The dissociation constants determined by fluorescence spectroscopy for the γ subunit to dansyl-CaM are 25.7 ± 0.6 and 104 ± 12 n m at pH 6.8 in the presence and absence of CaCl 2 . At pH 8.2, these values are 4.9 ± 0.3 and 29 ± 8 n m in the presence and absence of CaCl 2 . As the free Ca 2+ decreases to as low as 10 −9 m , the fluorescence intensity and the fluorescence polarization of the γ subunit and dansyl-CaM complex do not decrease in parallel, indicating that the complex does not come apart at low Ca 2+ concentration. The presence of Mg 2+ affects the interaction between dansyl-CaM and the γ subunit, as indicated by the increase in the polarization of fluorescence of dansyl-CaM. Mn 2+ interferes with the interaction of the γ subunit and dansyl-CaM. Free ATP has little effect.

[36] High-performance liquid chromatographic separation and renaturation of protein kinase subunits: application to catalytic subunit of phosphorylase kinase

Publisher Summary This chapter describes various methods used for the separation of phosphorylase kinase subunits under denaturing conditions and subsequent reactivation of the γ subunit and for renaturation of the bacterial expressed form of the subunit of phosphorylase kinase. For high-performance liquid chromatographic (HPLC) separation of phosphorylase kinase subunits, the subunits are eluted according to size, with the δ subunit eluting first between 43 and 50% acetonitrile and α subunit eluting last at about 58% acetonitrile. Phosphorylase kinase is injected directly to the column previously equilibrated in 0.1% TFA and the γ subunit is eluted at approximately 50% acetonitrile and 0.09% TFA, with a pH of 2.5. The γ subunit prepared in this way is pure, judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). For renaturation of HPLC-isolated γ subunit of phosphorylase kinase, the HPLC-purified γ subunit is diluted to 1–5 μ g/ml in an ice-cold solution containing one part assay buffer and four parts buffer A plus 60–100 μ g/ml calmodulin and 1.5 mg/ml phosphorylase b or bovine serum albumin. This results in a final pH of 8.2 and final concentrations of no more than 5% acetonitrile and 0.01% TFA. The reactivation mixture is kept on ice for several days to attain optimum catalytic activity.