Tyrosine protein kinase activity of rat spleen and other tissues

Using a synthetic peptide (Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly) as a substrate, various normal tissues from the rat were probed for tyrosine protein kinase activity. Spleen was shown to contain much higher tyrosine protein kinase activity than other rat tissues (lung, brain, testes, liver, kidney, heart, and thymus, in decreasing order of specific activity). Most of the tyrosine protein kinase activity of the various rat tissues (greater than 80%) was present in the particulate fraction, and Nonidet P-40, a nonionic detergent, could activate the particulate form of the enzyme 2-20-fold in many of the tissues. Epidermal growth factor (1 microgram/ml), cyclic AMP, cyclic GMP, or Ca2+ did not increase spleen tyrosine protein kinase activity. Half-maximal enzyme activity was observed at 60-80 microM MgATP and at 2.2 mM peptide, and both Mg2+ (10 mM) and Mn2+ (0.5-1.0 mM) were effective divalent metal ions for the expression of activity. When the particulate fraction of spleen was incubated with [gamma-32P]ATP followed by polyacrylamide gel electrophoresis in the presence of Na dodecyl SO4, a number of alkali-stable bands were identified by autoradiography. Two major bands at Mr = 53,000 and 56,000 were shown to contain phosphotyrosine. Two similar alkali-stable bands containing phosphotyrosine but with lower amounts of 32P labeling were also observed in the particulate fractions of various other tissues (lung, brain, kidney, and testes). The particulate form of tyrosine protein kinase of rat spleen could be solubilized by using high concentrations of Nonidet P-40 (5%) at an alkaline pH (pH 9.0). Partial purification and subsequent filtration on Sephacryl S-200 yielded a peak of tyrosine protein kinase activity with an apparent molecular weight of 55,000. The two major phosphorylated bands of Mr = 53,000 and 56,000 co-migrated with the peak of enzyme activity. The solubilized and partially purified enzyme preparation phosphorylated only tyrosine residues when either endogenous proteins or casein were used as substrates. These results suggest that relatively high activities of tyrosine protein kinase exist in a normal tissue (rat spleen). Major endogenous substrates of the enzyme(s) appear to be represented by two proteins of Mr = 53,000 and 56,000; one or both of these substrates may be the tyrosine protein kinase itself.     

Affinity chromatography and properties of cGMP-dependent protein kinase from prawn tissues

Using affinity chromatography on 8-(2-aminoethyl)-amino-cAMP Sepharose, the cGMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) from tissues of the prawn Palaemon adspersus was purified to homogeneity as demonstrated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The degree of enzyme purification was 11 200, recovery--6.5%; the isoelectric point for the enzyme lies at 5.5. Data from gel filtration and centrifugation in sucrose density gradient suggest that the dimer of cGMP-dependent protein kinase has a molecular weight of 157 000, sedimentation coefficient of 7.2S and a Stokes' radius of 50 A. An active form of the enzyme with Mr = 76 500 (4.5S, 39 A) which apparently represents a subunit of the cGMP-dependent protein kinase was discovered. The activity of the both enzyme forms are stimulated by low concentrations of cGMP (Ka = 1.10(-7) M). The monomer and dimer molecules appear as prolate ellipsoids with axial ratios close to 7. The native cGMP-dependent protein kinase is probably made up of two subunits each of which contains a regulatory and a catalytic sites.     

Phosphatidylglycerol-modulated protein kinase activity from human spleen. I. Enzyme purification and properties

Protein kinase P (PK-P) is a phospholipid-modulated protein kinase activity previously described in human and murine cells. This paper details the 3300-fold, high yield purification to electrophoretic homogeneity of protein kinase P from human spleen by a three-step chromatographic process. Physical characterization disclosed a protein of Mr 27,000 (by electrophoresis) or 31,700 (by gel filtration and sedimentation) and pI 5.09. Protein kinase P activity was stimulated by phosphatidylglycerol or phosphatidylinositol, with maximal stimulation observed between 200 and 400 micrograms/ml phospholipid. No stimulation was noted using phosphatidic acid or phosphatidylserine. Histone H2B was the best substrate for demonstrating the protein kinase P phospholipid stimulation. Histone H1 was phosphorylated in a phospholipid independent manner. Vinculin and actin were not substrates. Optimum enzyme activity was observed at approximately 35 degrees C and pH 6.95. PK-P was relatively insensitive to the calmodulin and protein kinase C inhibitors W7 and H7, and to the cAMP-dependent protein kinase inhibitor. Kinetic analysis disclosed complex patterns including optimal rather than Michaelis-Menton kinetics for histone and phospholipid concentration, and a steep activation threshold with respect to histone concentration in the presence of phospholipid. Biphasic kinetics for Mg2+-ATP were observed, with the major stimulatory effect of phospholipid being on Vmax rather than Km. These data suggest a model for the mechanism of activation of protein kinase P by phospholipid entailing a direct three-way interaction between substrate, enzyme, and phospholipid micelles rather than allosteric activation by phospholipid.     

Purification, subunit structure and properties of a high-molecular-mass protein phosphatase capable of dephosphorylating mRNP of the brine shrimp Artemia sp

A phosphoprotein phosphatase active towards casein, phosphorylase a and mRNP proteins has been detected in the cytosol of cryptobiotic gastrulae of Artemia sp. This phosphatase has a relative molecular mass (Mr) of 225,000 as measured by gel filtration on Sephadex G-200 and has been purified to near homogeneity by ion-exchange chromatography on different DEAE-substituted matrices, affinity chromatography on polylysine-agarose, histone-Sepharose 4B and protamine-agarose, hydrophobic chromatography on phenyl-Sepharose 4B and gel filtration on Sephadex G-200. Sodium dodecyl sulphate gel electrophoresis of the final purification step revealed that the enzyme contains two types of subunits, alpha and beta, with Mr of 40,000 and 75,000, respectively. These values, in conjunction with the native Mr and the molar ratios of the subunits estimated by densitometric analysis of the gel, suggested that the subunit composition of the enzyme is alpha 2 beta 2. When treated with 1.7% (v/v) 2-mercaptoethanol at -20 degrees C or with ethanol, the enzyme released the catalytic alpha subunit of Mr 40,000. The protein phosphatase was activated by basic proteins e.g. protamine (A 0.5 = 1 microM), histone H1 (A 0.5 = 1.6 microM) and polylysine (A 0.5 = 0.2 microM) and inhibited by ATP (I 0.5 = 12 microM), NaF (I 0.5 = 3.1 mM) and pyrophosphate (I 0.5 = 0.6 mM). The enzyme is a polycation-stimulated protein phosphatase. Purified mRNP proteins, phosphorylated by the mRNP-associated casein kinase type II, are among the substrates used by the enzyme. The function of reversible phosphorylation-dephosphorylation of mRNP as a regulatory mechanism in mRNP metabolism is discussed.     

Purification and properties of a virion protein kinase.

The protein kinase associated with virions of frog virus 3 was purified to apparent homogeneity by ion exchange chromatography and gel filtration. The enzyme protein appeared as a single polypeptide of molecular weight 50,000 to 55,000 as determined by gel filtration, glycerol gradient sedimentation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and comprised approximately 0.4% of the total virion protein. The activity was classified as a cyclic nucleotide-independent protein kinase as it was not effected by cyclic adenosine 3':5'-monophosphate, cyclic guanosine 3':5'-monophosphate, or inhibited by a cyclic nucleotide-dependent protein kinase inhibitor protein, and utilized GTP as well as ATP as a phosphate donor. The greatest rates of phosphorylation were obtained with acidic phosphoprotein substrates such as casein or phosvitin, although potential physiological substrates for this activity included specific virion polypeptides of frog virus.     

Phosphorylation of rat kidney pyruvate kinase type L by cyclic 3',5'-AMP-dependent protein kinase.

Pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) type L was partly purified from rat kidney. During the last two purification steps, the incorporation of [32P]phosphate into protein on incubation with [32P]ATP and cyclic 3',5'-AMP-dependent protein kinase was found to parallel the pyruvate kinase activity. After phosphorylation of the enzyme, a major radioactive band with a molecular weight of 57 000 was found on polyacrylamide gel electrophoresis [32P]Phosphorylserine was isolated from the kidney pyruvate kinase. Immunological identity was found between the liver and kidney pyruvate kinases type L. By autoradiography of high-voltage electropherograms after partial acid hydrolysis of the phosphorylated rat liver and kidney pyruvate kinases type L, identical results were obtained. The affinity for phosphoenolpyruvate was found to be decreased by phosphorylation of the enzyme with a change in the apparent Km from 0.15 mM to 0.35 mM. After incubation of the phosphorylated kidney pyruvate kinase with phosphatase the phosphoenolpyruvate saturation curve was found to be identical to that for the unphosphorylated enzyme. Thus, the activity of the rat kidney pyruvate kinase type L is with all probability regulated by a reversible phosphorylation-dephosphorylation reaction, thereby indicating that hormonal regulation of gluconeogenesis via cyclic AMP may be of importance in the renal cortex.     

Identification of a ribosomal protein S6 kinase regulated by transformation and growth-promoting stimuli

A serine protein kinase specific for ribosomal protein S6 in 40 S subunits has been identified and purified greater than 15,000-fold (with 18% recovery) from developing chicken embryos. An analogous enzyme has also been detected in serum-stimulated chicken embryo fibroblasts. The S6 kinase was identified as a phosphoprotein of Mr approximately 65,000 based on (i) gel filtration, (ii) apparent autophosphorylation of a 65-kDa protein when several enzyme preparations were incubated with [gamma-32P]ATP in the absence of added substrate, (iii) comigration of S6 kinase activity with the autophosphorylating activity over a variety of chromatographic resins, and (iv) elution and renaturation of S6 kinase activity from the 65-kDa region of a sodium dodecyl sulfate-polyacrylamide gel. The purified protein kinase is highly specific for S6 in 40 S subunits and does not appreciably phosphorylate casein, histone H1, mixed histones, protamine, polyoma virus capsid protein, or phosphorylase a/b. These characteristics suggest that this enzyme is unrelated to other protein kinases believed to be activated in stimulated cells, including cAMP-dependent protein kinase, protein kinase C (Ca2+/phospholipid-dependent enzyme), or Ca2+/calmodulin-dependent protein kinases. In fibroblasts, S6 kinase is activated by a variety of mitogenic agents including the tyrosine-specific protein kinase of Rous sarcoma virus, pp60v-src, phorbol esters, and growth factors. The present identification and purification of the S6 kinase should facilitate future studies aimed at elucidating the molecular mechanisms by which signals from these diverse stimuli rapidly converge upon and activate this enzyme.     

A tyrosine-specific protein kinase from Ehrlich ascites tumor cells

A protein tyrosine kinase that phosphorylates both alpha and beta subunits of inactivated (Na+,K+)-ATPase from dog kidney was purified about 500-fold from Ehrlich ascites tumor cell membranes. The enzyme required divalent cations Mn2+, Mg2+, or Fe2+ but was inhibited by Cu2+ or Zn2+. The purified enzyme phosphorylated the beta subunit about five times faster than the alpha subunit of the (Na+,K+)-ATPase. The random polymer poly(Glu80Tyr20) was an excellent substrate while casein was only marginally phosphorylated. In contrast, the purified transforming gene product of Rous sarcoma virus phosphorylated all three substrates and the (Na+,K+)-ATPase was preferentially phosphorylated on the alpha subunit. The transforming gene product of Fujinami sarcoma visue and EGF receptor kinase from A431 cells phosphorylated (Na+,K+)-ATPase poorly whereas casein was an excellent substrate. The molecular weight of the partially purified protein tyrosine kinase from Ehrlich ascites tumor cells determined by gel filtration was about 60,000. One of two major phosphorylated phosphopeptides resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis had an Mr of 60 kDa, thus suggesting that it might be the autophosphorylated protein tyrosine kinase. A phosphatase that hydrolyzes phosphorylated histones or poly(Glu80Tyr20) was partially purified from the same membrane.     

Purification and characterization of myosin light-chain kinase from porcine myometrium and its phosphorylation and modulation by cyclic AMP-dependent protein kinase

Myosin light-chain kinase was purified from porcine myometrium to apparent homogeneity at about 262-fold with an Mr of 130 000 as determined by SDS-polyacrylamide gel electrophoresis and a sedimentation coefficient of 4.5 S. The approximate content of the soluble myosin light-chain kinase was estimated to be about 0.85 microM. The purified enzyme exhibited strict substrate specificity only for 20-kDa myosin light chain and Ka values of 0.6 nM and 0.3 microM for calmodulin and Ca2+, respectively. The enzyme was phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase, which resulted in a decrease in the affinity for calmodulin of 4-7-fold without effect on the Vmax. The maximal amount of phosphate incorporated into the enzyme was 0.5-0.8 and 1.0-1.4 mol per mol of the enzyme in the presence and absence of Ca2+ and calmodulin, respectively. In the presence of a subsaturating concentration of calmodulin, the enzyme showed a lower sensitivity for Ca2+ by phosphorylation.     

Purification and characterization fo a phosvitin kinase from the thyroid gland

1. Two cyclic AMP independent protein kinases phosphorylating preferentially acidic substrates have been identified in soluble extract from human, rat and pig thyroid glands/ Both enzymes were retained on DEAE-cellulose. The first enzyme activity eluted between 60 and 100 mM phosphate (depending on the species), phosphorylated both casein and phosvitin and was retained on phosphocellulose; this enzyme likely corresponds to a casein kinase already described in many tissues. The second enzyme activity eluted from DEAE-cellulose at phosphate concentrations higher than 3000 mM, phosphorylated only phosvitin and was not retained on phophocellulose. These enzymes were neither stimulated by cyclic AMP, cyclic GMP and calcium, nor inhbiited by the inhibitor of the cyclic AMP dependent protein kinases. 2. The second enzyme activity was purified from pig thyroid gland by the association of affinity chromatography on insolubilized phosvitin and DEAE-cellulose chromatography. Its specific activity was increased by 8400. 3. The purified enzyme (phosvitin kinase) was analyzed for biochemical and enzymatic properties. Phosvitin kinase phosphorylated phosvitin with an apparent K_m of 100 μg/ml; casein, histone, protamine and bovine serum albumin were not phosphorylated. The enzyme utilized ATP as well as GTP as phosphate donor with an apparent K_m of 25 and 28 μM, respectively. It had an absolute requirement for Mg²⁺ with a maximal activity at 4 mM and exhibited an optimal activity at pH 7.0. The molecular weight of the native enzyme was 110 000 as determined by Sephacryl S300 gel filtration. The analysis by SDS-polyacrylamide gel electrophoresis revealed a major band with a molecualr weight of 35 000 suggesting a polymeric structure of the enzyme.     

Cyclic AMP-dependent histone-specific nucleoplasmic protein kinase from rat liver.

A nucleoplasmic histone kinase activity was isolated from livers of adult rats and purified 39-fold compared with whole nuclei by ultracentrifugation of the nuclear extract and Sephadex G-200 gel filtration in the presence of cyclic AMP. Analysis by polyacrylamide-gel electrophoresis as well as by gel filtration indicates a mol.wt. of approx. 60,000 for the catalytic subunit and 130000-150000 for the cyclic AMP-binding activity. The purified enzyme displays a 20-fold greater preference for histone fractions 1 and 2b than for any non-histone substrate, including alpha-casein. Endogenous protein in the preparation is not appreciably phosphorylated. The unfractioned enzyme is stimulated significantly by cyclic GMP, cyclic IMP and dibutyryl cyclic AMP as well as by cyclic AMP. The catalytic reaction requires Mg2+ (Km 1.9mM) and ATP (Km 15.4 micron). Half-maximal activity of the enzyme is observed with histone 2b at 12micron and histone 1 at a higher substrate concentration. The pH optima are 6.1 and 6.5 with histones 2b and 1 respectively. This nuclear protein kinase appears to be distinct from other nuclear enzymes that have been reported, on the basis of histone specificity, univalent-salt-sensitivity, pH optima and nuclear location. However, the enzyme possesses many properties similar to those of the cytoplasmic kinases, including cyclic AMP-dependence, Mg2+ and ATP affinities and pH optima. It differs from cytoplasmic protein kinase type I, the major form in the liver, with respect to bivalent-cation effects and response to the heat-stable protein kinase inhibitor protein isolated from ox heart.     

Purification of catalytic subunit of low density lipoprotein receptor kinase and identification of heat-stable activator protein

Bovine adrenal cortex contains a high molecular weight casein kinase II-like enzyme (Mr 500,000) that phosphorylates a specific serine residue in the cytoplasmic domain of the low density lipoprotein (LDL) receptor (Kishimoto, A., Brown, M. S., Slaughter, C. A., and Goldstein, J. L. (1987) J Biol. Chem. 262, 1344-1351). In the current paper, we provide evidence to suggest that this 500-kDa kinase can be dissociated into two subunits, a catalytic subunit and an activator subunit, by treatment with 1 M NaCl. The catalytic subunit was purified to homogeneity (greater than 100,000-fold) using affinity chromatography on GTP-agarose plus several other chromatography steps. It had an Mr of 50,000 by gel filtration and 35,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The catalytic subunit phosphorylated casein actively, but it phosphorylated the LDL receptor with only low affinity. The affinity for the LDL receptor was increased 10-fold (saturation at 10 nM LDL receptor) by addition of a second protein that was released from a high molecular weight 500-kDa complex by 1 M NaCl. This activator protein (Mr 120,000 by gel filtration) was extremely heat stable but was destroyed by trypsin. It appeared to be required in stoichiometric amounts with relation to the LDL receptor. It did not increase the ability of the 50-kDa subunit to phosphorylate casein nor did it activate phosphorylation of the LDL receptor or casein by classic casein kinase II. The current data raise the possibility that the specificity of the 500-kDa LDL receptor kinase is attributable to a heat-stable activator subunit that binds to the LDL receptor and thereby renders it a better substrate for the catalytic subunit of the kinase.     

Purification and characterization of Novikoff ascites tumor protein kinase.

A protein kinase, designed KII, has been purified 5000-fold from Novikoff ascites tumor cells. The purification procedure also allows for the purification of a second major protein kinase, designated KI, as well as RNA polymerase I and II. Purified KII has a sedimentation constant of 7.6 S and a Stokes radius of 39 A, suggesting a molecular weight of about 122000. Polyacrylamide gel electrophoresis of the enzyme in the presence of sodium dodecyl sulfate suggests the enzyme is composed of subunits of molecular weights 44 000, 40 000, and 26 000 present in a molar ratio of 1:1:2. Incubation of the enzyme alone in the presence of [gamma-32P]ATP results in the phosphorylation of the 26 000-dalton subunit. Protein kinase II actively phosphorylates phosvitin, casein, and nonhistone chromosomal proteins but does not phosphorylate basic proteins such as histones or protamine to an appreciable extent. Km values of 3.6 micron for ATP and 6.5 micronM for GTP were determined in the presence of 4mM Mg2+. The enzyme is neither stimulated by cyclic adenosine 3',5'-monophosphate or cyclic guanosine 3', 5'-monophosphate nor inhibited by the regulatory subunit of rabbit muscle protein kinase. Its activity is stimulated by KCl at concentrations below 0.2 M and inhibited by higher concentrations.     

A cyclic AMP-independent protein kinase from Candida albicans.

A cyclic AMP-independent protein kinase which phosphorylates casein was purified to homogeneity from Candida albicans by affinity and ion-exchange chromatography. This protein kinase exhibits maximal activity with casein as substrate and is not stimulated by cyclic AMP or cyclic GMP. The Mr of the purified enzyme is 115,000, as determined by h.p.l.c. It migrates as a single band on gel electrophoresis and has three non-identical subunits, of Mr 44,000, 28,500 and 26,000, as determined by SDS/polyacrylamide-gel electrophoresis. This enzyme is insensitive to heparin, but is inhibited by polyamines. Furthermore, it is sensitive to thermal denaturation and to thiol reagents.     

Complete purification of the pseudorabies virus protein kinase

The recently described pseudorabies virus protein kinase has been purified from infected hamster fibroblasts by a combination of anion-exchange, hydrophobic-interaction and affinity chromatography. The purification resulted in enzyme with a specific activity in excess of 1,000 nmol phosphate mg-1 min-1 in relatively high yield. Gel electrophoresis of the purified enzyme under denaturing conditions revealed a single stained band at a position of migration corresponding to a Mr 38,000. Incubation of the purified enzyme with [gamma-32P]ATP in the absence of added substrate resulted in incorporation of 32P into this protein band, consistent with the 38-kDa protein being a protein kinase with a capacity for autophosphorylation. The phosphorylated form of the protein has an isoelectric point of approximately 4.9. Gel permeation chromatography of the purified enzyme indicated a native Mr 70,000, suggesting that the protein kinase has a homodimeric structure.     

Cyclic CMP phosphodiesterase: isolation, specificity and kinetic properties

Cyclic CMP phosphodiesterase activity was demonstrated in rat liver, heart, brain, kidney, intestine, skeletal muscle, blood, testes, ovaries, spleen and lung; that present in the liver was purified to homogeneity by a sequential process of ammonium sulphate fractionation, gel filtration, two ion-exchange chromatographic steps, preparative electrophoresis and two affinity chromatographic stages with selection at each stage for maximum specificity. The final enzyme preparation was confirmed as a single protein by HPLC and isoelectric focussing; the total yield obtained was 1.5% and the final specific activity of 48.6 mumol cyclic CMP hydrolysed/min/mg reflected a 88,000 fold purification. The phosphodiesterase had a Mr of 2.8 X 10(4), pH optimum 7.2-7.4, isoelectric point between 4.2 and 4.4 and a Km of 9.0 mM cyclic CMP. This enzyme differs from a previously isolated cyclic CMP phosphodiesterase in its amino acid composition and specificity. The absolute specificity for 3',5'-cyclic CMP as substrate distinguishes this cyclic CMP phosphodiesterase from all other reported phosphodiesterases and shows it to be a novel enzyme. Its potential as a research tool and the significance of its occurrence are discussed.     

Purification and biochemical characterization of alpha 2-adrenergic receptor from the rat adrenocortical carcinoma

The alpha 2-adrenergic receptor was purified from rat adrenocortical carcinoma 494 by an affinity chromatographic step using a novel para-aminoclonidine-sepharose resin followed by a gel-permeation high performance liquid chromatographic step. The iodinated receptor protein was homogeneous as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by high performance liquid chromatography. Both SDS-PAGE and high performance liquid chromatographic studies revealed that Mr of the protein was 64,000, suggesting the monomeric nature of the receptor protein. The purified protein showed the typical binding characteristics of alpha 2-adrenergic receptor.     

An adenosine 3':5' monophosphate dependent protein kinase from sea urchin spermatozoa.

A cyclic AMP dependent protein kinase (EC 2.7.1.37) from sea urchin sperm as purified to near homogeneity and characterized. A 68-fold purification of the enzyme was obtained. This preparation had a specific activity of 389 000 units/mg protein with protamine as the substrate. On the basis of the purification required, it may be calculated that the protein kinase constitutes as much as 1.5% of the soluble protein in sperm. There appeared to be a single form of the enzyme in sea urchin sperm, based on the behavior of the enzyme during DEAE-cellulose and Sephadex G-200 column chromatography. Magnesium ion was required for enzyme activity. The rate of phosphorylation of protamine was stimulated 2.5-fold by an optimal concentration of 0.9 M NaCl. The Km for ATP (minus cyclic AMP) was 0.119 +/- 0.013 (S.D.) and 0.055 mM +/- 0.009 (S.D.) in the presence of cyclic AMP. The specificity of the enzyme toward protein acceptors, in decreasing order of phosphorylation, was found to be histone f1 protamine, histone f2b, histone f3 and histone f2a; casein and phosvitin were not phosphorylated. The holoenzyme was found to have an apparent molecular weight of 230 000 by Sephadex G-200 chromatography. In the presence of 5 - 10(-6) M cyclic AMP, the holoenzyme was dissociated on Sephadex G-200 to a regulatory subunit of molecular weight 165 000 and a catalytic subunit of Mr 73 000. The dissociation could also be demonstrated by disc gel electrophoresis in the presence and absence of cyclic AMP.     

Cyclic AMP-dependent protein kinase activity in Trypanosoma cruzi.

A cyclic AMP-dependent protein kinase activity from epimastigote forms of Trypanosoma cruzi was characterized. Cytosolic extracts were chromatographed on DEAE-cellulose columns, giving two peaks of kinase activity, which were eluted at 0.15 M- and 0.32 M-NaCl respectively. The second activity peak was stimulated by nanomolar concentrations of cyclic AMP. In addition, a cyclic AMP-binding protein co-eluted with the second kinase activity peak. Cyclic AMP-dependent protein kinase activity was further purified by gel filtration, affinity chromatography on histone-agarose and cyclic AMP-agarose, as well as by chromatography on CM-Sephadex. The enzyme ('holoenzyme') could be partially dissociated into two different components: 'catalytic' and 'regulatory'. The 'regulatory' component had specific binding for cyclic AMP, and it inhibited phosphotransferase activity of the homologous 'catalytic component' or of the 'catalytic subunit' from bovine heart. Cyclic AMP reversed these inhibitions. A 'holoenzyme preparation' was phosphorylated in the absence of exogenous phosphate acceptor and analysed by polyacrylamide-gel electrophoresis. A 56 kDa band was phosphorylated. The same preparation was analysed by Western blotting, by using polyclonal antibodies to the regulatory subunits of protein kinases type I or II. Both antibodies reacted with the 56 kDa band.     

Purification and characterization of a type II casein kinase from Drosophila melanogaster

A cyclic nucleotide-independent protein kinase has been isolated from Drosophila melanogaster by chromatography on phosphocellulose and hydroxylapatite followed by gel filtration and glycerol gradient sedimentation. As determined by sodium dodecyl sulfate gel electrophoresis, the purified enzyme is greater than 95% homogeneous and is composed of two distinct subunits, alpha and beta, having Mr = 36,700 and 28,200, respectively. The native form of the enzyme is an alpha 2 beta 2 tetramer having a Stokes radius of 48 A, a sedimentation coefficient of 6.4 S, and Mr approximately 130,000. The purified kinase undergoes an autocatalytic reaction resulting in the specific phosphorylation of the beta subunit, exhibits a low apparent Km for both ATP and GTP as nucleoside triphosphate donor (17 and 66 microM, respectively), phosphorylates both casein and phosvitin but neither histones nor protamine, modifies both serine and threonine residues in casein, and is strongly inhibited by heparin (I50 = 21 ng/ml). These properties are remarkably similar to those of casein kinase II, an enzyme previously described in several mammalian and avian species. The strong similarities among the insect, avian, and mammalian enzymes suggest that casein kinase II has been highly conserved during evolution.