Erwin M. Reimann

Phosphorylation of protein B-50 (GAP-43) from adult rat brain cortex by casein kinase II

The phosphoprotein B-50 (GAP-43) was purified from adult rat brain cortex and phosphorylated by casein kinase II. Phosphorylation of B-50 by casein kinase II approached 1.2 mol phosphate/mol B-50. The apparent Km of casein kinase II for B-50 was 4 microM with an apparent Vmax of 13 nmol.min-1.mg-1. A tryptic phosphopeptide map on reversed phase HPLC and phosphoamino acid analysis of [32P]B-50 showed that casein kinase II phosphorylated in serine residue(s) which were located in a single tryptic peptide. Phosphorylation of B-50 by casein kinase II was inhibited more than 90% by 5 micrograms heparin/ml or 2.4 mM peptide substrate specific for casein kinase II (RRREEETEEE). The initial phosphorylation rate was increased about 2-fold by 1 mM spermine.

Selective precipitation of 32Pi onto filter papers. Application to ATPase and cyclic AMP phosphodiesterase determination

A simple and selective method is described for the isolation of 32Pi based on the precipitation of the phosphomolybdate complex with triethylamine. Precipitation takes place on filter paper, which are then washed with a solution containing ammonium molybdate and triethylamine to remove other radioactive phosphates. Very large numbers of samples and very small sample volumes can be accommodated easily. The use of this method to measure ATPase and cyclic AMP phosphodiesterase activity is demonstrated.

The effect of radioactive contaminants on the estimation of binding parameters by Scatchard analysis.

Abstract 1. 1.|When protein-ligand interactions are measured with a radioactive ligand, the presence of a radioactive contaminant that is not bound can lead to errors in the determination of the concentration of bound and unbound ligand, and consequently to errors in estimation of the dissociation constant (Kd). The extent of errors caused by a non-binding contaminant was determined by computer simulation for five types of protein-ligand interaction: first order dissociation, two classes of binding sites with different dissociation constants, displacement, negative cooperativity and positive cooperativity. For each type of reaction several values of Kd and several concentrations of binding sites were assumed. 2. 2.|The presence of a contaminant results in Scatchard plots which are convex upward instead of linear for the first order dissociation reaction. Scatchard plots normally are concave upward with three of the reactions studied: two classes of binding sites, displacement reactions and negative cooperativity. The contaminant causes a reduction in the concavity and in certain instances the plots may appear linear; with larger amounts of a contaminant they may be convex upward. The shape of the Scatchard plots for positive cooperativity is convex upward in either the presence or absence of a contaminant. Except for the displacement reaction the distortion of the shape of the Scatchard plots increases as the concentration of binding sites increases. 3. 3.|A contaminant, therefore, can cause distortions of the Scatchard plots which can lead to: (1) misinterpretations of the type of protein-ligand interaction, (2) overestimation of the dissociation constants, and (3) errors in calculation of the concentration of binding sites. In some instances as little as 1% contaminant may have a profound effect on the apparent affinity and number of binding sites.

Isolation and characterization of cyclic AMP-independent glycogen synthase kinase from rat skeletal muscle.

Glycogen synthase kinase was isolated from rat skeletal muscle. This kinase, which is cyclic nucleotide-independent and calcium-independent, was separated from phosphorylase kinase, cyclic AMP-dependent protein kinase and phosvitin kinase by phosphocellulose chromatography. Gel filtration on Sephadex G-100 resolved the glycogen synthase kinase into two fractions with apparent molecular weights of 68 000 (peak I) and 52 000 (peak II). This step also separated glycogen synthase kinase from the catalytic subunit of the cyclic AMP-dependent protein kinase, which had an apparent molecular weight of 39 000. Peak II glycogen synthase kinase activity was not affected by the addition of calcium, EGTA or a number of cyclic nucleotides. In addition to ATP, dATP would serve as the phosphate donor. Other trinucleotides tested were either poor or ineffective substrates. Activity was about 5-fold greater with Mg2+ than with Mn2+. Glycogen stimulated activity about 25%. Modifications of the methods of Soderling et al. ((1970) J. Biol. Chem. 245, 6317--6328) and Nimmo et al. ((1976) Eur. J. Biochem. 68, 21--30) were developed for purification of glycogen synthease (UDPglucose:glycogen 4-alpha D-glucosyltransferase, EC 2.4.1.11) to specific activity of 35 units/mg of protein. Using this preparation of glycogen synthase as substrate, the phosphorylation and inactivation catalyzed by glycogen synthase kinase was compared to that catalyzed by cyclic AMP-dependent protein kinase or phosphorylase kinase. Each of the kinases had different specificities for phosphorylation sites on glycogen synthase.

Phosphorylation of membranes from the rat gastric mucosa

Gastric mucosal membranes derived primarily from parietal cells were found to contain endogenous protein kinase systems as well as several phosphate-accepting substrates. One specific membrane protein with a molecular weight of 88 000 was phosphorylated only in the presence of calcium, while the degree of phosphorylation of three other membrane proteins was similarly increased. The activity of the calcium-dependent protein kinase was found to be totally inhibited in the presence of trifluoperazine, a phenothiazine known to specifically inactivate calmodulin. These results suggest that a calmodulin- and calcium-dependent phosphorylation system may be a component of the parietal cell membrane. Phosphorylation of the membrane proteins was not affected by either cyclic AMP or cyclic GMP. The heat-stable inhibitor protein of cyclic AMP-dependent protein kinase did not inhibit the endogenous protein kinase activity suggesting that the membrane enzyme is not similar to the cytosolic protein kinase. However, the catalytic subunit of the soluble enzyme was capable of phosphorylating a number of membrane proteins indicating that after maximal autophosphorylation of the gastric membranes, phosphate-acceptor sites are still available to the cytosolic cyclic AMP-dependent protein kinase.

Identification of the phosphoserine residue in histone H1 phosphorylated by protein kinase C

The site‐specific phosphorylation of bovine histone H1 by protein kinase C was investigated in order to further elucidate the substrate specificity of protein kinase C. Protein kinase C was found to phosphorylate histone H1 to 1 mol per mol. Using N‐bromosuccinimide and thrombin digestions, the phosphorylation site was localized to the globular region of the protein, containing residues 71–122. A tryptic peptide containing the phosphorylation site was purified. Modification of the phosphoserine followed by amino acid sequence analysis demonstrated that protein kinase C phosphorylated histone H1 on serine 103. This sequence, Gly97‐Thr‐Gly‐Ala‐Ser‐Gly‐Ser(PO4)‐Phe‐Lys105, supports the contention that basic amino acid residues C‐terminal to the phosphorylation site are sufficient determinants for phosphorylation by protein kinase C.

Inactivation of glycogen synthase a by the catalytic subunit of cyclic AMP-dependent protein kinase. Kinetics of inactivated forms

Rabbit skeletal muscle glycogen synthase was phosphorylated to varying degrees with [γ-32P]ATP and the catalytic subunit of the cyclic AMP-dependent protein kinase. Phosphorylation of glycogen synthase up to 1 mol phosphate per mol subunit had very little effect on the activity ratio (activity measured in the absence of glucose 6-phosphate divided by activity measured in the presence of glucose 6-phosphate), the A0.5 for glucose 6-phosphate (concentration of glucose 6-phosphate which gives half maximal activation), or the Km for UDPGlc measured in the absence of glucose 6-phosphate. Phosphorylation to the extent of 1.8 mol phosphate per mol subunit resulted in partial inactivation of glycogen synthase (activity ratio = 0.6) due primarily to an increase in the Km for UDPGlc measured in the absence of glucose 6-phosphate. Phosphorylation to the extent of 2.6–2.8 mol phosphate per mol subunit resulted in further inactivation (activity ratio = 0.05–0.13) due primarily to a decrease in V (maximal velocity) measured in the absence of glucose 6-phosphate and partly to an additional increase in the Km for UDPGlc measured in the absence of glucose 6-phosphate. The form of glycogen synthase containing 2.6–2.8 mol phosphate per mol subunit was unique in that activation by glucose 6-phosphate showed little or no positive cooperativity, A0.5 for glucose 6-phosphate was relatively high, and V measured in the absence of glucose 6-phosphate was reduced. Phosphorylation of glycogen synthase had very little effect on either the Km for UDPGlc or the V measured in the presence of glucose 6-phosphate. We conclude from these studies that inactivation of glycogen synthase a by the catalytic subunit of cyclic AMP-dependent protein kinase in vitro is complex, and that no single kinetic parameter is the best index of inactivation.

Differences between glycogen synthases from rat and rabbit skeletal muscle as indicated by phosphopeptide maps

Glycogen synthase I was purified from rat skeletal muscle. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, the enzyme migrated as a major band with a subunit Mr of 85,000. The specific activity (24 units/mg protein), activity ratio (the activity in the absence of glucose-6-P divided by the activity in the presence of glucose-6-P X 100) (92 +/- 2) and phosphate content (0.6 mol/mol subunit) were similar to the enzyme from rabbit skeletal muscle. Phosphorylation and inactivation of rat muscle glycogen synthase by casein kinase I, casein kinase II (glycogen synthase kinase 5), glycogen synthase kinase 3 (kinase FA), glycogen synthase kinase 4, phosphorylase b kinase, and the catalytic subunit of cAMP-dependent protein kinase were similar to those reported for rabbit muscle synthase. The greatest decrease in rat muscle glycogen synthase activity was seen after phosphorylation of the synthase by casein kinase I. Phosphopeptide maps of glycogen synthase were obtained by digesting the different 32P-labeled forms of glycogen synthase by CNBr, trypsin, or chymotrypsin. The CNBr peptides were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the tryptic and chymotryptic peptides were separated by reversed-phase HPLC. Although the rat and rabbit forms of synthase gave similar peptide maps, there were significant differences between the phosphopeptides derived from the N-terminal region of rabbit glycogen synthase and the corresponding peptides presumably derived from the N-terminal region of rat glycogen synthase. For CNBr peptides, the apparent Mr was 12,500 for rat and 12,000 for the rabbit. The tryptic peptides obtained from the two species had different retention times. A single chymotryptic peptide was produced from rat skeletal muscle glycogen synthase after phosphorylation by phosphorylase kinase whereas two peptides were obtained with the rabbit enzyme. These results indicate that the N-terminus of rabbit glycogen synthase, which contains four phosphorylatable residues (Kuret et al. (1985) Eur. J. Biochem. 151, 39-48), is different from the N-terminus of rat glycogen synthase.

Characterization of GSK-M, a glycogen synthase kinase from rat skeletal muscle

A form of glycogen synthase kinase designated GSK-M3 was purified 4000-fold from rat skeletal muscle by phosphocellulose, Affi-Gel blue, Sephacryl S-300 and carboxymethyl-Sephadex column chromatography. Separation of GSK-M from the catalytic subunit of the cAMP-dependent protein kinase was facilitated by converting the catalytic subunit to the holoenzyme form by addition of the regulatory subunit prior to the gel filtration step. GSK-M had an apparent Mr 62,000 (based on gel filtration), an apparent Km of 11 microM for ATP, and an apparent Km of 4 microM for rat skeletal muscle glycogen synthase. The kinase had very little activity with 0.2 mM GTP as the phosphate donor. Kinase activity was not affected by the addition of cyclic nucleotides, EGTA, heparin, glucose 6-P, glycogen, or the heat-stable inhibitor of cAMP-dependent protein kinase. Phosphorylation of glycogen synthase from rat skeletal muscle by GSK-M reduced the activity ratio (activity in the absence of Glc-6-P/activity in the presence of Glc-6-P X 100) from 90 to 25% when approximately 1.2 mol of phosphate was incorporated per mole of glycogen synthase subunit. Phosphopeptide maps of glycogen synthase obtained after digestion with CNBr or trypsin showed that this kinase phosphorylated glycogen synthase in serine residues found in the peptides containing the sites known as site 2, which is located in the N-terminal CNBr peptide, and site 3, which is located in the C-terminal CNBr peptide of glycogen synthase. In addition to phosphorylating glycogen synthase, GSK-M phosphorylated inhibitor 2 and activated ATP-Mg-dependent protein phosphatase. Activation of the protein phosphatase by GSK-M was dependent on ATP and was virtually absent when ATP was replaced with GTP. GSK-M had minimal activity toward phosphorylase b, casein, phosvitin, and mixed histones. These data indicate that GSK-M, a major form of glycogen synthase kinase from rat skeletal muscle, differs from the known glycogen synthase kinases isolated from rabbit skeletal muscle.

Partial purification and characterization of an adenosine 3′,5′-monophosphate-dependent protein kinase from rabbit gastric mucosa

Abstract An adenosine 3′,5′-monophosphate (cyclic AMP)-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) from rabbit gastric mucosa was partially purified and characterized. Purification by (NH 4 ) 2 SO 4 precipitation, DEAE-cellulose chromatography, hydroxylapatite chromatography, and gel filtration increased the specific activity of the enzyme 60-fold. This protein kinase could be separated into a cyclic AMP-binding (regulatory) subunit and a catalytic subunit by chromatography on hydroxylapatite in the presence of cyclic AMP. The isolated catalytic subunit did not require cyclic AMP for activity. Recombination of the subunits in the presence of MgATP and bovine serum albumin restored cyclic AMP dependency to the catalytic subunit. Stokes radii of the catalytic subunit, the regulatory subunit, and the holoenzyme were found to be 2.6, 2.7 and 3.9 nm, respectively. The respective values for sedimentation coefficient were found to be 3.7, 3.5 and 5.1 S. Molecular weights calculated from these data gave values of 39 000 for both the catalytic and regulatory subunits and 82 000 for the holoenzyme. The enzyme phosphorylated histone, protamine, casein and endogeneous protein of the gastric mucosa. The apparent K m for ATP was 2·10 −5 M in the presence or absence of cyclic AMP. The concentration of cyclic AMP required for half maximal stimulation was 10 −8 M.