Purification and Properties of a High Specific Activity Protein Kinase from Wheat Germ

Abstract

A protein kinase was extensively purified to near-homogeneity from wheat germ by a procedure involving affinity chromatography on caseinSepharose 4B, gel filtration, and repeated chromatography on carboxymethyl-Sepharose CL-6B. The protein kinase preparations have the highest specific activities (up to 656 nanomoles phosphate incorporated per minute per mill of protein) yet reported for plant protein kinases. The major polypeptides in purified preparations were revealed as two barely-resolved bands (molecular weight 31,000) on polyacrylamide gel electrophoresis in subunit-dissociating conditions. The molecular size of the protein kinase as determined from gel filtration is 30,000. The protein kinase catalyzes the phosphorylation of casein, phosvitin, and the wheat germ cyclic AMPbinding protein cABPII but not of bovine serum albumin and histones nor of the wheat germ cytokinin-binding protein CBP. The protein kinase has a pH optimum of 7.9 and a K. value for ATP of 10 micromolar. The protein kinase differs from wheat germ CBP kinase in molecular weight, differential sensitivity to inhibitors, and in substrate specificity.Protein phosphorylation-dephosphorylation represents an important means of enzyme regulation in eukaryote cells and a key process involved in hormone action in animal cells through the modulation of specific protein kinases by cyclic AMP, cyclic GMP, or Ca2' (7). Proteins known to be phosphorylated in higher plants include chromosomal proteins (13; see 27), thylakoid proteins (1, 26), ribosome-associated proteins (21, 24, 25; see 27), RNA polymerase (10), and pyruvate dehydrogenase (18). Phosphorylation of pyruvate dehydrogenase results in inactivation of the enzyme complex (18), and phosphorylation of wheat embryo protein synthesis initiation factor eIF-2 causes inactivation of the factor and inhibition of translation (19,24). The phosphorylation of the chloroplast light-harvesting Chl a/b-binding protein appears to be involved in regulation of the distribution of absorbed excitation energy between the two photosystems, and the phosphorylation of this protein is light dependent (1). The mechanism of regulation of other plant protein phosphorylation reactions is unknown.Protein kinases have been resolved from higher plants (2,5,6,8,10,12,16,17,22) but no cyclic nucleotide-dependent or Ca2+_ calmodulin-activated plant protein kinases have been reported. Highly purified plant protein kinase preparations have been isolated with specific activities in the range of approximately 10 to 200 nmol phosphate transferred/min mg of protein (5,6,8,10,12,16,22). Two casein-phosphorylating protein kinases (mol wt 39,000 and 120,000) have been purified to apparent homogeneity from soybean cotyledons (6), and electrophoretic homogeneity has ' Supported by a grant from the Australian Research Grants Committee. also been obtained for a wheat germ protein kinase (22). This latter enzyme preparation phosphorylates casein and phosvitin, contains an approximately 20,000 D subunit, and has a specific activity ...

Protein Kinase Activities in Tonoplast and Plasmalemma Membranes from Corn Roots

“…These pH optima are lower than those previously reported for plant protein kinase activities. pH maxima about 9.5 and 7.5 were observed for soybean histone H1 kinase (13) and chromatin-associated casein kinase (18), respectively, and an optimum pH of 7.9 was reported for casein kinase from wheat germ (3). However, the high pH optimum for casein kinase may have resulted from increased solubility of casein at high pH ( 18).…”

“…On the other hand, very little information is available about protein phosphorylation in plants. Protein kinase and protein phosphatase activities have been described in plants, associated with chromatin (3,18), chloroplasts (1), soluble fractions (3,13,20,26,27), and microsomes (8,23,26). Of these enzymes, only histone kinase (13), LHCP,,-kinase (1), and cytokinin-binding protein kinase (20) have been characterized with respect to their substrates.…”

“…Casein kinases have also been characterized (3,18,27); however, their physiological substrates are unknown. As a consequence, the physiological role of these enzymes is still not known.…”

“…These results indicate that a regulatory cycle of protein phosphorylation and dephosphorylation may operate in the plasmalemma. The activity in the tonoplast appears to originate from plasmalemma contamination.It is well established that protein phosphorylation is a major mechanism by which second messengers produce their physiological responses in a variety of animal tissues (2,19,24 (3,13,20,26,27), and microsomes (8,23,26). Of these enzymes, only histone kinase (13), LHCP,,-kinase (1), and cytokinin-binding protein kinase (20) have been characterized with respect to their substrates. '…”

Possible involvement of protein phosphorylation/dephosphorylation in the modulation of Ca2+ channel in tonoplast-free cells ofNitellopsis

“…However, the Km of higher plant protein kinases for ATP is assumed to be around 10/xM (cf. Davies & Polya, 1983). Thus protein kinases in ATP-depleted twice-perfused cells are assumed to be operating although the electrogenic H + is inhibited.…”