Studies on a cyclic AMP-dependent protein kinase from rat thyroid gland

Abstract

We have confirmed the presence of cyclic AMP-dependent protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) in the soluble protein fraction of rat thyroid homogenates, and have purified the enzyme with a 20% yield by gel filtration on Sephadex G-200 followed by ion exchange chromatography on DEAE-cellulose. Kinase activity was assayed in a standard system with [γ- 32P]ATP and a mixed fraction of calf thymus histones at pH 6.5, 30°C, in the presence and absence of cyclic AMP. Two peaks of cyclic AMP-dependent protein kinase activity were resolved by gel filtration and each was further purified on DEAE-cellulose and designated DK I and DK II: specific activities were, respectively, 5171 and 2080 units/mg protein, representing an approximate 700-fold purification of activity based on assay of the crude extracts. However, crude thyroid extracts reveal only 1/10 of their inherent kinase activity, probably due to the presence of ATPase activity, and in addition, to interaction of the large amounts of thyroglobulin with the histone phosphoryl acceptor. A unit of activity is defined as 1 pmol phosphate transferred from phosphoryl donor to acceptor per min. The two kinases were stable for up to 1 month when stored at 4°C. The K m with the respect to cyclic AMP is 6.1 · 10 −8 M for DK I and 3.1 · 10 −8 M for DK II, and with respect to ATP, in the presence and absence of cyclic AMP (1 · 10 −6 M), respectively, is 1.0 · 10 −5 M and 1.2 · 10 −5 M for DK I, and 0.93 · 10 −5 M and 0.75 · 10 −5 M for DK II. These values are similar to those reported for cyclic nucleotide dependent protein kinases from other tissues. Molecular weights estimated by gel filtration are 230 000 and 152 000 for DK I and DK II respectively. Sedimentation coefficients of 8.7 for DK I and 6.6 for DK II were estimated by centrifugation of the material in the linear sucrose density gradients. Enzymatic profiles of the gradients of the heavier kinase consistently showed a small peak corresponding to 6.6 S. Although the data might suggest that multiple forms of protein kinase exist in the rat thyroid, they are also consistent with the presence of a kinase in several states of aggregation peculiar to the purification procedure. Both DK I and DK II when incubated with cyclic AMP were converted to 4.8 S material that was essentially completely nucleotide independent, consistent with the well-described disaggregation of the catalytic subunit(s) from protein kinase holoenzyme.