Purification and characterization of a novel calcium-dependent protein kinase from soybean

A novel calcium-dependent protein kinase (CDPK) previously reported to be activated by the direct binding of Ca2+, and requiring neither calmodulin nor phospholipids for activity [Harmon, A.C., Putnam-Evans, C.L., & Cormier, M.J. (1987) Plant Physiol. 83, 830-837], was purified to greater than 95% homogeneity from suspension-cultured soybean cells (Glycine max, L. Wayne). Purification was achieved by chromatography on DEAE-cellulose, phenyl-Sepharose, Sephadex G-100, and Blue Sepharose. The purified enzyme (native molecular mass = 52,200 Da) resolved into two immunologically related protein bands of 52 and 55 kDa on 10% SDS gels. Enzyme activity was stimulated 40-100-fold by micromolar amounts of free calcium (K0.5 = 1.5 microM free calcium) and was dependent upon millimolar Mg2+. CDPK phosphorylated lysine-rich histone III-S and chicken gizzard myosin light chains but did not phosphorylate arginine-rich histone, phosvitin, casein, protamine, or Kemptide. Phosphorylation of histone III-S, but not autophosphorylation, was inhibited by KCl. CDPK displayed a broad pH optimum (pH 7-9), and kinetic studies revealed a Km for Mg2(+)-ATP of 8 microM and a Vmax of 1.7 mumol min-1 mg-1 with histone III-S (Km = 0.13 mg/mL) as substrate. Unlike many other protein kinases, CDPK was able to utilize Mg2(+)-GTP, in addition to Mg2(+)-ATP, as phosphate donor. The enzyme phosphorylated histone III-S exclusively on serine; however, CDPK autophosphorylated on both serine and threonine residues. These properties demonstrate that CDPK belongs to a new class of protein kinase.     

Assay of protein kinase C with an N-bromosuccinimide-cleavage fragment of histone H1.

N-Bromosuccinimide cleavage of a lysine-rich histone fraction (histone III-S) yields a peptide substrate, purified by reverse-phase h.p.l.c., for the Ca2+ + phospholipid-dependent protein kinase (protein kinase C). This substrate displays no reactivity with the cyclic AMP-dependent protein kinase, and may prove useful for the detection of protein kinase C activity in crude tissue extracts.     

Purification and characterization of a membrane-bound protein kinase from spinach thylakoids

A protein kinase was isolated from spinach thylakoid membranes by solubilization with octyl glucoside and cholate. The enzyme was purified to apparent homogeneity by ammonium sulfate precipitation, gel filtration, and sucrose density centrifugation, followed by affinity chromatography on either Affi-Gel blue (yielding denatured enzyme) or on histone cross-linked to Sepharose (yielding active enzyme). Electrophoresis on denaturing polyacrylamide gels, followed by staining with silver, revealed the kinase as a single band corresponding to an apparent molecular mass of 64 kDa. The active enzyme underwent autophosphorylation and could be detected by autoradiography following incubation with [gamma-32P]ATP and Mg2+ ion. The specific phosphotransferase activity of purified kinase was approximately 30 nmol of phosphate min-1 (mg protein)-1 with lysine-rich histone (III-S or V-S) as substrate; casein was phosphorylated at approximately 30% of this rate. The physiological substrate for the kinase is presumed to be light-harvesting chlorophyll a/b protein complex. In solubilized form, this was phosphorylated at approximately 10% of the rate observed with histone III-S as substrate, or 10-100 times slower than the estimated rate of phosphorylation of the light-harvesting complex in situ. Possible reasons for this shortfall are considered. The kinase is proposed as the principal effector of thylakoid protein phosphorylation and associated State transition phenomena.     

Purification and characterization of a calcium-regulated protein kinase from beet root (Beta vulgaris) plasma membranes

A calcium-regulated protein kinase (CRPK) associated with the plasma membrane of beet root cells was purified by deoxycholate extraction of plasma membrane proteins followed by chromatography in DEAE- and phenyl-Sepharose columns. This procedure rendered a purified CRPK preparation with a specific activity higher than 2.2 μmol mg^-1min^-1 using histone III-S as substrate. The molecular mass of CRPK, estimated by SDS-PAGE, was 58 kDa. Gel filtration and glycerol gradient centrifugation indicated that the native enzyme is a monomer. Phosphorylation of histone by CRPK displayed a broad pH optimum between 7.0 and 8.2, Syntide-2, a CaM-kinase II substrate, was phosphorylated much more efficiently than kemptide, Ac-MBP (4–14), CKII substrate or poly-(Glu, Tyr) 4:1. The kinetics of this enzyme can be described by a steady-state ordered mechanism in which four bound Ca²⁺ ions are required for the phosphorylation of the peptide substrate. After Ca²⁺ binding, the affinity of the enzyme for ATP was not affected while that for syntide-2 or histone decreased. Curves of kinase activity vs ATP at different syntide-2 concentrations indicated that the K_m values for ATP or syntide-2 increased with increasing cosubstrate concentrations. Similar curves of kinase activity vs ATP at different histone concentrations indicated that the apparent affinities of CRPK for ATP or histone were not affected by the cosubstrate concentration. The purified CRPK was not recognized by antibodies against soybean or Arabidopsis calcium-dependent protein kinase (CDPK). These results suggest that CRPK and CDPKs could be different enzymes, although they show some similar properties.     

Stimulation of the ATPase activity of rat brain protein kinase C by phospho acceptor substrates of the enzyme

We recently reported that autophosphorylated rat brain protein kinase C (PKC) catalyzes a Ca2(+)- and phosphatidylserine- (PS-) dependent ATPase reaction. The Ca2(+)- and PS-dependent ATPase and histone kinase reactions of PKC each had a Km app(ATP) of 6 microM. Remarkably, the catalytic fragment of PKC lacked detectable ATPase activity. In this paper, we show that subsaturating concentrations of protein substrates accelerate the ATPase reaction catalyzed by PKC and that protein and peptide substrates of PKC induce ATPase catalysis by the catalytic fragment. At subsaturating concentrations, histone III-S and protamine sulfate each accelerated the ATPase activity of PKC in the presence of Ca2+ and PS by as much as 1.5-fold. At saturating concentrations, the protein substrates were inhibitory. Poly(L-lysine) failed to accelerate the ATPase activity, indicating that the acceleration observed with histone III-S and protamine sulfate was not simply a result of their gross physical properties. Furthermore, histone III-S induced the ATPase activity of the catalytic fragment of PKC, at both subsaturating and saturating histone concentrations. The induction of ATPase activity was also elicited by the peptide substrate Arg-Arg-Lys-Ala-Ser-Gly-Pro-Pro-Val, when the peptide was present at concentrations near its Km app. The induction of the ATPase activity by the nonapeptide provides strong evidence that the binding of phospho acceptor substrates to the active site of PKC can stimulate ATP hydrolysis. Taken together, our results indicate that PKC-catalyzed protein phosphorylation is inefficient, since it is accompanied by Pi production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glyoxalase I from Brassica juncea is a calmodulin stimulated protein.

Brassica juncea glyoxalase I (S-lactoylglutathione-lyase, EC 4.4.1. 5) is a 56 kDa, heterodimeric protein. It requires magnesium (Mg2+) for its optimal activity. In this report we provide biochemical evidence for modulation of glyoxalase I activity by calcium/calmodulin (Ca2+/CaM). In the presence of Ca2+ glyoxalase I showed a significant (2.6-fold) increase in its activity. It also showed a Ca2+ dependent mobility shift on denaturing gels. Its Ca2+ binding was confirmed by Chelex-100 assay and gel overlays using 45CaCl2. Glyoxalase I was activated by over 7-fold in the presence of Ca2+ (25 microM) and CaM (145 nM) and this stimulation was blocked by the CaM antibodies and a CaM inhibitor, trifluroperazine (150 microM). Glyoxalase I binds to a CaM-Sepharose column and was eluted by EGTA. The eluted protein fractions also showed stimulation by CaM. The stimulation of glyoxalase I activity by CaM was maximum in the presence of Mg2+ and Ca2+; however, magnesium alone also showed glyoxalase I activation by CaM.     

Effects of suramin, an anti-human immunodeficiency virus reverse transcriptase agent, on protein kinase C. Differential activation and inhibition of protein kinase C isozymes.

Suramin inhibited protein kinase C (PKC) type I-III activity in a concentration-dependent manner. Similar inhibitory effects were observed with M-kinase, the constitutively active catalytic fragment of PKC, and autophosphorylation of PKC types I-III. Kinetic experiments indicated that suramin competitively inhibits activity with respect to ATP (Ki = 17, 27, and 31 microM, respectively) and that it can also inhibit by interaction with the substrate histone III-S. With protamine as the Pi acceptor, suramin inhibition was dependent on lipid, being approximately 4-fold less sensitive to inhibition in the absence of phosphatidylserine and diacylglycerol than in their presence. Suramin at low concentrations (10-40 microM), in the presence of Ca2+ and absence of lipid, was able to stimulate kinase activity (approximately 200-400%) in a type-dependent manner and at higher concentrations inhibited activity with histone III-S as substrate. These results indicate that suramin, a hexa-anionic hydrophobic compound, can act as a negatively charged phospholipid analog in activating PKC in the presence of Ca2+ and absence of lipid and can inhibit Ca2+/phosphatidylserine/diacylglycerol-stimulated kinase activity at higher concentrations by competing with ATP or by interaction with the exogenous substrate. Suramin inhibited cAMP-dependent protein kinase much less potently (IC50 = 656 microM) than PKC. The ability of suramin to inhibit PKC-mediated processes in intact cells was tested using the phorbol ester-stimulated respiratory burst of neutrophils as a model system. The respiratory burst of human neutrophils, when preincubated with suramin and then stimulated with phorbol ester, was inhibited in a concentration-dependent manner, suggesting that suramin may also be able to inhibit PKC-mediated processes in intact cells.     

Protamine induces autophosphorylation of protein kinase C: stimulation of protein kinase C-mediated protamine phosphorylation by histone.

Protein kinase C (PKC), a protein phosphorylating enzyme, is characterized by its need for an acidic phospholipid and for activators such as Ca2+ and diacylglycerol. The substrate commonly used in experiments with PKC is a basic protein, histone III-S, which needs the activators mentioned. However, protamine, a natural basic substrate for PKC, does not require the presence of cofactor/activator. We report here that protamine can induce the autophosphorylation of PKC in the absence of any PKC-cofactor or activator; this may represent a possible mechanism of cofactor-independent phosphorylation of this protein. It was investigated if protamine itself can act as a PKC-activator and stimulate histone phosphorylation in the manner of Ca2+ and phospholipids. Experiments however showed that protamine is not a general effector of PKC. On the contrary, histone stimulated PKC-mediated protamine phosphorylation and protamine-induced PKC-autophosphorylation. Histone alone did not induce PKC-autophosphorylation. Kinetic studies suggest that histone increases the maximal velocity (Vmax) of protamine kinase activity of PKC without affecting the affinity (Km). Other polycationic proteins such as polyarginine serine and polyarginine tyrosine were not found to influence PKC-mediated protamine phosphorylation, indicating that the observed effects are specific to histone, and are not general for all polycationic proteins. These results suggest that histone can modulate the protamine kinase activity of PKC by stimulating protamine-induced PKC-autophosphorylation.     

Heterologous expression and biochemical characterization of two calcium-dependent protein kinase isoforms CaCPK1 and CaCPK2 from chickpea

In plants, calcium-dependent protein kinases (CPKs) constitute a unique family of enzymes consisting of a protein kinase catalytic domain fused to carboxy-terminal autoregulatory and calmodulin-like domains. We isolated two cDNAs encoding calcium-dependent protein kinase isoforms (CaCPK1 and CaCPK2) from chickpea. Both isoforms were expressed as fusion proteins in Escherichia coli. Biochemical analyses have identified CaCPK1 and CaCPK2 as Ca(2+)-dependent protein kinases since both enzymes phosphorylated themselves and histone III-S as substrate only in the presence of Ca(2+). The kinase activity of the recombinant enzymes was calmodulin independent and sensitive to CaM antagonists W7 [N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide] and calmidazoilum. Phosphoamino acid analysis revealed that the isoforms transferred the gamma-phosphate of ATP only to serine residues of histone III-S and their autophosphorylation occurred on serine and threonine residues. These two isoforms showed considerable variations with respect to their biochemical and kinetic properties including Ca(2+) sensitivities. The recombinant CaCPK1 has a pH and temperature optimum of pH 6.8-8.6 and 35-42 degrees C, respectively, whereas CaCPK2 has a pH and temperature optimum of pH 7.2-9 and 35-42 degrees C, respectively. Taken together, our results suggest that CaCPK1 and CaCPK2 are functional serine/threonine kinases and may play different roles in Ca(2+)-mediated signaling in chickpea plants.     

Biochemical characterization of Ca2+/calmodulin dependent protein kinase from Candida albicans

A multifunctional Ca²⁺/calmodulin dependent protein kinase was purified approximately 650 fold from cytosolic extract of Candida albicans. The purified preparation gave a single band of 69 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis with its native molecular mass of 71 kDa suggesting that the enzyme is monomeric. Its activity was dependent on calcium, calmodulin and ATP when measured at saturating histone IIs concentration. The purified Ca²⁺/CaMPK was found to be autophosphorylated at serine residue(s) in the presence of Ca²⁺/calmodulin and enzyme stimulation was strongly inhibited by W-7 (CaM antagonist) and KN-62 (Ca²⁺/CaM dependent PK inhibitor). These results confirm that the purified enzyme is Ca²⁺/CaM dependent protein kinase of Candida albicans. The enzyme phosphorylated a number of exogenous and endogenous substrates in a Ca²⁺/calmodulin dependent manner suggesting that the enzyme is a multifunctional Ca²⁺/calmodulin-dependent protein kinase of Candida albicans.     

Molecular cloning, overexpression, and characterization of autophosphorylation in calcium-dependent protein kinase 1 (CDPK1) from Cicer arietinum

In plants, calcium-dependent protein kinases (CDPKs) are key intermediates in calcium-mediated signaling that couple changes in Ca²⁺ levels to a specific response. In the present study, we report the high-level soluble expression of calcium-dependent protein kinase1 from Cicer arietinum (CaCDPK1) in Escherichia coli. The expression of soluble CaCDPK1 was temperature dependent with a yield of 3–4 mg/l of bacterial culture. CaCDPK1 expressed as histidine-tag fusion protein was purified using Ni–NTA affinity chromatography till homogeneity. The recombinant CaCDPK1 protein exhibited both calcium-dependent autophosphorylation and substrate phosphorylation activities with a V _max and K _m value of 13.2 nmol/min/mg and 34.3 μM, respectively, for histone III-S as substrate. Maximum autophosphorylation was seen only in the presence of calcium. Optimum temperature for autophosphorylation was found to be 37 °C. The recombinant protein showed optimum pH range of 6–9. The role of autophosphorylation in substrate phosphorylation was investigated using histone III-S as exogenous substrate. Our results show that autophosphorylation happens before substrate phosphorylation and it happens via intra-molecular mechanism as the activity linearly depends on enzyme concentrations. Autophosphorylation enhances the kinase activity and reduces the lag phase of activation, and CaCDPK1 can utilize both ATP and GTP as phosphodonor but ATP is preferred than GTP.     

NMR derived solution structure of an EF-hand calcium-binding protein from Entamoeba Histolytica.

We present the three-dimensional (3D) solution structure of a calcium-binding protein from Entamoeba histolytica (EhCaBP), an etiologic agent of amoebiasis affecting millions worldwide. EhCaBP is a 14.7 kDa (134 residues) monomeric protein thought to play a role in the pathogenesis of amoebiasis. The 3D structure of Ca(2+)-bound EhCaBP has been derived using multidimensional nuclear magnetic resonance (NMR) spectroscopic techniques. The study reveals the presence of two globular domains connected by a flexible linker region spanning 8 amino acid residues. Each domain consists of a pair of helix-loop-helix motifs similar to the canonical EF-hand motif of calcium-binding proteins. EhCaBP binds to four Ca(2+) with high affinity (two in each domain), and it is structurally related to calmodulin (CaM) and troponin C (TnC) despite its low sequence homology ( approximately 29%) with these proteins. NMR-derived structures of EhCaBP converge within each domain with low RMSDs and angular order-parameters for backbone torsion angles close to 1.0. However, the presence of a highly flexible central linker region results in an ill-defined orientation of the two domains relative to one other. These findings are supported by backbone (15)N relaxation rate measurements and deuterium exchange studies, which reveal low structural order parameters for residues in the central linker region. Earlier, biochemical studies showed that EhCaBP is involved in a novel signal transduction mechanism, distinct from CaM. A possible reason for such a functional diversity is revealed by a detailed comparison of the 3D structure of EhCaBP with that of CaM and TnC. The studies indicate a more open C-terminal domain for EhCaBP with larger water exposed total hydrophobic surface area as compared to CaM and TnC. Further dissimilarities between the structures include the presence of two Gly residues (G63 and G67) in the central linker region of EhCaBP, which seem to impart it a greater flexibility compared to CaM and TnC and also play crucial role in its biological function. Thus, unlike in CaM and TnC, wherein the length and/or composition of the central linker have been found to be crucial for their function, in EhCaBP, both flexibility as well as amino acid composition is required for the function of the protein.     

Cloning and functional characterization of NtCPK4, a new tobacco calcium-dependent protein kinase

A cDNA clone, encoding calcium (Ca2+)-dependent protein kinase (CDPK or CPK), was isolated from tobacco (Nicotiana tabacum). The full-length cDNA of 2360 bp contains an open reading frame for NtCPK4 consisting of 572 amino acid residues. Sequence alignment indicated that NtCPK4 shared high similarities with other CPKs and some CPK-related protein kinases (CRKs). Biochemical analyses showed that NtCPK4 phosphorylated itself and calf thymus histones fraction III-S (histone III-S) in a calcium-dependent manner, and the K0.5 of calcium activation was 0.29 microM or 0.25 microM with histone III-S or syntide-2 as substrates, respectively. The Vmax and Km were 588 nmol min-1 mg-1 and 176 microg ml-1, respectively, when histone III-S was used as substrate, while they were 2415 nmol min-1 mg-1 and 58 microM, respectively, with syntide-2 as substrate. In addition, the phosphorylation of NtCPK4 occurred on threonine residue, as shown by capillary electrophoresis analyses. All of these data demonstrated that NtCPK4 was a serine/threonine protein kinase. NtCPK4 as a low copy gene was expressed in all tested organs including the root, leaf, stem, and flower of tobacco, while its expression was temporally and spatially modulated in both productive and vegetative tissues during tobacco growth and development. NtCPK4 expression was also increased in response to the treatment of gibberellin or NaCl. Our study suggested that NtCPK4 might play vital roles in plant development and responses to environmental stimuli.     

Purification of a protein histidine kinase from the yeast Saccharomyces cerevisiae. The first member of this class of protein kinases

An enzyme of molecular weight 32,000 comprising a single subunit has been isolated from whole cell extracts of the yeast Saccharomyces cerevisiae. In vitro, the enzyme transfers the gamma phosphate of ATP to a protein substrate, histone H4, to produce an alkali-stable phosphorylation. Modification of the substrate histidine with diethylpyrocarbonate prevented phosphorylation. Phosphoamino acid analysis of the phosphorylated substrate showed the presence of 1-phosphohistidine. Hence, the isolated enzyme is a protein histidine kinase. A novel assay for acid-labile alkali-stable protein phosphorylation was used in the purification of the kinase activity to a final specific activity of 2,700 nmol/15 min/mg. The purified enzyme phosphorylates specifically histidine 75 in histone H4 and does not phosphorylate histidine 18 nor histidine residues in any other core histone. Steady state kinetic data are consistent with an ordered sequential reaction with Km values for Mg-ATP and histone H4 of 60 and 17 microM, respectively. The protein histidine kinase requires a divalent cation such as Mg2+, Co2+, or Mn2+ but will not use Ca2+, Zn2+, Cu2+, Fe2+, spermine, or spermidine. This is the first purification of an enzyme that catalyzes N-linked phosphorylation in proteins.     

Existence of endogenous substrate proteins for Ca2+/calmodulin-dependent and Ca2+/phospholipid-dependent protein kinases in rat adrenal glomerulosa cells

In rat adrenal glomerulosa cells, endogenous substrate proteins for Ca2+/calmodulin (CaM)-dependent protein kinase (glomerulosa CaM kinase) and Ca2+/phospholipid-dependent protein kinase (protein kinase C) were investigated. In a 105,000 g-supernatant fraction (cytosol), the Mr 100,000 protein was phosphorylated in the presence of calcium (calculated free Ca2+ concentration, 460 microM) alone or calcium and CaM, and the phosphorylation of this protein was completely inhibited by the CaM antagonists pimozide (500 microM) and melittin (5 microM) in the presence of calcium alone, respectively. These results indicate that the Mr 100,000 protein is a major substrate for glomerulosa CaM kinase, and considerable amounts of endogenous CaM might be present in the cytosol. In the presence of phospholipids (the micelles of 8 micrograms of phosphatidyl serine and 1 microgram of diacylglycerol), at least twelve proteins of Mr 127,000, 80,000, 70,000, 36,000, 35,000, 33,000, 32,000, 30,000, 27,000, 22,000, 19,000 and 17,000 were phosphorylated, and the phosphorylation of these proteins was enhanced by the addition of calcium, indicating that these proteins are substrates for protein kinase C. No endogenous protein phosphorylation was found in a 105,000 g-particulate fraction. Thus, these findings demonstrate that adrenal glomerulosa cells have specific substrate proteins for glomerulosa CaM kinase and protein kinase C, respectively.     

Involvement of a Ca(2+)-dependent protein kinase component downstream to the gibberellin-binding phosphoprotein, RuBisCO activase, in rice.

Previously, we reported the identification of a gibberellin (GA)-binding protein in rice using ligand binding assay that was homologous to RuBisCO activase (Komatsu et al., FEBS Lett. 384, 167-171, 1996). Here, we provide an evidence for the involvement of protein kinases components downstream to the GA-binding phosphoprotein, RuBisCO activase in rice. Ca(2+)-dependent protein kinase activity was studied in subcellular fractions of leaf sheath from transgenic rice containing sense and antisense constructs of RuBisCO activase. In-gel kinase assay using histone III-S as a substrate showed constitutive induction of a 46- and 48-kDa Ca(2+)-dependent protein kinase activity in the sense transgenic plants. Kinase activities of these proteins were significantly reduced in the presence of uniconazole, a potent GA biosynthesis inhibitor, but one of them was strongly promoted by GA(3) treatment in transgenic plants carrying a smaller subunit of RuBisCO activase (OsrcaA1) compared to the larger subunit OsrcaA2. Also, in vitro phosphorylation studies using two-dimensional polyacrylamide gel showed changes in the degree of phosphorylation of several proteins in OsrcaA1- and OsrcaA2-sense transgenic rice. These studies suggest the presence of two independent cytosolic Ca(2+)-dependent protein kinase signaling components downstream to the GA-binding protein in rice suggesting their role in GA signaling.     

Elongation factor 2 as the major substrate for Ca2+/calmodulin-dependent protein kinase in rat adrenal glomerulosa cells

We have previously shown the existence of the major substrate protein of Mr 100,000 (substrate 100 K protein) for Ca2+/calmodulin (CaM)-dependent protein kinase in rat adrenal glomerulosa cells. In the present study, the identity of the substrate 100 K protein to elongation factor 2 (EF-2) was investigated. In a 105,000 g-supernatant fraction (cytosol), the protein of Mr 100,000 with the pI (isoelectric point) value of 6.7 was phosphorylated in the presence of calcium and CaM. The optical densities of this phosphorylated band were greatly enhanced in the presence of the EF-2 purified from pig liver (1 microgram) [20-23-fold, n = 5] when compared with those in the absence of the component. In the presence of the purified EF-2, the phosphorylation of Mr 100,000 was detected only in the presence of calcium alone or calcium plus CaM. This phosphorylation in the presence of calcium alone was completely inhibited in the presence of the CaM antagonist pimozide (500 microM), showing the existence of endogenous CaM in the cytosol. In the same fraction, the ADP-ribosylated protein of Mr 100,000 was detected in the presence of diphtheria toxin (fragment A) and (adenylate-32P) NAD, indicating the presence of EF-2 in the cytosol from rat adrenal glomerulosa cells. These results suggest that the substrate 100 K protein may be identical to EF-2 in rat adrenal glomerulosa cells.     

Global Forebrain Ischemia Induces a Posttranslational Modification of Multifunctional Calcium- and Calmodulin-Dependent Kinase II

The activity of multifunctional calcium/calmodulin-dependent protein kinase II (CaM kinase II) has recently been shown to be inhibited by transient global ischemia. To investigate the nature of ischemia-induced inhibition of the enzyme, CaM kinase II was purified to greater than 1,000-fold from brains of control and ischemic gerbils. The characteristics of CaM kinase II from control and ischemic preparations were compared by numerous parameters. Kinetic analysis of purified control and ischemic CaM kinase II was performed for autophosphorylation properties, ATP, magnesium, calcium, and calmodulin affinity, immunoreactivity, and substrate recognition. Ischemia induced a reproducible inhibition of CaM kinase II activity, which could not be overcome by increasing the concentration of any of the reaction parameters. Ischemic CaM kinase II was not different from control enzyme in affinity for calmodulin, Ca2+, Mg2+, or exogenously added substrate or rate of autophosphorylation. CaM kinase II isolated from ischemic gerbils displayed decreased immunoreactivity with a monoclonal antibody (immunoglobulin G3) directed toward the beta subunit of the enzyme. In addition, ischemia caused a significant decrease in affinity of CaM kinase II for ATP when measured by extent of autophosphorylation. To characterize further the decrease in ATP affinity of CaM kinase II, the covalent-binding ATP analog 8-azido-adenosine-5'-[alpha-32P]triphosphate was used. Covalent binding of 25 microM azido-ATP was decreased 40.4 +/-12.3% in ischemic CaM kinase II when compared with control enzyme (n = 5; p less than 0.01 by paired Student's t test). Thus, CaM kinase II levels for ischemia and control fractions were equivalent by protein staining, percent recovery, and calmodulin binding but were significantly different by immunoreactivity and ATP binding. The data are consistent with the hypothesis that ischemia induces a posttranslational modification that alters ATP binding in CaM kinase II and that results in an apparent decrease in enzymatic activity.     

Ca2+/calmodulin dependent protein kinase from Mycobacterium smegmatis ATCC 607

A soluble Ca²⁺/calmodulin dependent protein kinase has been partially purified (~400 fold) from Mycobacterium smegmatis ATCC 607 using several purification steps like ammonium sulphate precipitation (30-60%), Sepharose CL-6B gel filtration, DEAE-cellulose and finally calmodulin-agarose affinity chromatography. On SDS-PAGE, this enzyme preparation showed a major protein band of molecular mass 35 kD and its activity was dependent on calcium, calmodulin and ATP when measured under saturating histone IIs (exogenous substrate) concentration. Phosphorylation of histone IIs was inhibited by W-7 (calmodulin inhibitor) and KN-62 (CaM-kinase inhibitor) with IC₅₀ of 1.5 and 0.25 m respectively, but was not affected by inhibitors of PKA (Sigma P5015) and PKC (H-7). All these results confirm that purified enzyme is Ca²⁺/ calmodulin dependent protein kinase of M. smegmatis. The protein kinase of M. smegmatis demonstrated a narrow substrate specificity for both exogenous as well as endogenous substrates. These results suggest that purified CaM-kinase must be involved in regulating specific function(s) in this organism.     

Endogenous modulators of glucocorticoid receptor function also regulate purified protein kinase C

Modulator-1 and -2, proposed to be novel ether-linked aminophosphoglycerides, were originally identified as regulators of glucocorticoid receptor function (Bodine, P. V., and Litwack, G. (1990) J. Biol. Chem. 265, 9544-9554). We now demonstrate that these modulators are also potent new stimulators of protein kinase C activity in vitro. These endogenous biomolecules regulate purified protein kinase C activity in a biphasic and dose-dependent pattern, as determined by histone phosphorylation. Modulators, at concentrations within their apparent cellular range, stimulate protein kinase C-catalyzed histone phosphorylation 2-4-fold when added separately, or 10-12-fold when added together. This enhancement of kinase activity apparently is specific for protein kinase C, since neither protein kinase M, nor cAMP-dependent protein kinase A are stimulated by the modulators. The stimulation of purified protein kinase C occurs only when the enzyme has been initially activated by calcium, phosphatidylserine, and diacylglycerol, indicating that the modulators do not simply substitute for one of the enzyme cofactors. In addition, the modulators appear to interact directly with protein kinase C, perhaps with the regulatory domain of the enzyme, since these biomolecules inhibit the binding of phorbol ester to purified protein kinase C. Finally, time-course studies of protein kinase C-catalyzed histone phosphorylation indicate that the velocity of the enzyme reaction is increased by the modulators. Taken together, these results suggest that the modulators are a new class of regulators of protein kinase C.