Purification and properties of protein kinase C from bovine polymorphonuclear leucocytes

A calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was purified to near homogeneity from bovine polymorphonuclear leucocytes. The purified enzyme had a specific activity of 10 000 U/mg protein and on SDS gelelectrophoresis the Mr was 88 000. The enzyme activity was almost totally dependent upon phosphatidylserine and could be strongly activated by Ca2+ concentrations in the micromolar range. At lower concentrations of calcium (less than 1 X 10(-7) M) the enzyme was only activated by the simultaneous presence of phosphatidylserine and diolein. Phorbol 12-myristate 13-acetate mimicked the effect of diolein and partially activated the enzyme. Protein kinase C activity and the phorbolester binding protein co-purified throughout all the purification steps.     

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.     

Phosphorylation by calcium calmodulin-dependent protein kinase II and protein kinase C modulates the activity of nitric oxide synthase.

Nitric oxide synthase purified from rat brain, which is Ca2+ and calmodulin dependent, was phosphorylated by calcium calmodulin-dependent protein kinase II as well as protein kinase C. Phosphorylation by calcium calmodulin-dependent protein kinase II resulted in a marked decrease in enzyme activity (33% of control) without changing the co-factor requirements, whereas a moderate increase in enzyme activity (140% of control) was observed after phosphorylation by protein kinase C. These findings indicate that brain nitric oxide synthase activity may be regulated not only by Ca2+/calmodulin and several co-factors, but also by phosphorylation.     

Activation of adipocyte adenylate cyclase by protein kinase C

Adenylate cyclase activity in purified rat adipocyte membranes is stimulated by the calcium- and phospholipid-dependent enzyme protein kinase C. Over the concentration range of 100-1000 milliunits/ml, both highly purified (approximately 3000 units/mg of protein) protein kinase C from rat brain and partially purified (14 units/mg of protein) protein kinase C from guinea pig pancreas stimulate cyclase activity. The actions of both protein kinase C preparations on adenylate cyclase activity are dependent on added calcium, which is effective at concentrations less than 10 microM. Exogenous phospholipids are not required for stimulation of adenylate cyclase by protein kinase C; but, under typical cyclase assay conditions, the adipocyte membranes satisfy the lipid requirement for protein kinase C phosphorylation of histone. The tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate enhances the kinase action on cyclase, and the phorbol ester is effective at concentrations equimolar with the kinase (less than 10 nM). With the brain protein kinase C, 12-O-tetradecanoylphorbol-13-acetate effects are especially evident at limiting calcium concentrations. Inhibitors of protein kinase C activity, such as chlorpromazine, palmitoylcarnitine, and polymyxin B, inhibit selectively that adenylate cyclase activity which is stimulated by protein kinase C plus calcium. It is concluded that protein kinase C acts directly on the adipocyte adenylate cyclase system.     

Ethoxylated alcohol (Neodol-12) and other surfactants in the assay of protein kinase C

Most commonly used surfactants were found to be inhibitors of partially purified rat brain protein kinase C at or above their critical micellar concentrations (CMC). These include sodium lauryl sulfate, deoxycholate, octyl glucoside, dodecyl trimethylammonium bromide, linear alkylbenzene sulfonate and Triton X-100. Several detergents, including the nonionic surfactants digitonin and Neodol-12 (ethoxylated alcohol), did not inhibit protein kinase C activity, even at concentrations greater than their CMC, while the anionic surfactant, AEOS-12 (ethoxylated alcohol sulfate), inhibited enzyme activity only slightly (less than 8%). Since these latter surfactants have little or no inhibitory effect on protein kinase C, they may be of value in solubilizing cells and tissues for the determination of enzyme activity in crude extracts. Among the detergents tested, sodium lauryl sulfate and linear alkylbenzene sulfonate significantly stimulated protein kinase C activity in the absence of phosphatidylserine and calcium. This was found to be dependent on the presence of histone in the protein kinase C assay. These detergents failed to stimulate protein kinase C activity when endogenous proteins in the partially purified rat brain extracts were used as the substrate. Our results indicate that activity of protein kinase C can be modified by the conditions of the assay and by the detergents used to extract the enzyme.     

Autophosphorylation-dependent inactivation of plant chimeric calcium/calmodulin-dependent protein kinase.

Chimeric calcium/calmodulin dependent protein kinase (CCaMK) is characterized by the presence of a visinin-like Ca(2+)-binding domain unlike other known calmodulin- dependent kinases. Ca(2+)-Binding to the visinin-like domain leads to autophosphorylation and changes in the affinity for calmodulin [Sathyanarayanan P.V., Cremo C.R. & Poovaiah B.W. (2000) J. Biol. Chem. 275, 30417-30422]. Here, we report that the Ca(2+)-stimulated autophosphorylation of CCaMK results in time-dependent loss of enzyme activity. This time-dependent loss of activity or self-inactivation due to autophosphorylation is also dependent on reaction pH and ATP concentration. Inactivation of the enzyme resulted in the formation of a sedimentable enzyme due to self-association. Specifically, autophosphorylation in the presence of 200 microm ATP at pH 7.5 resulted in the formation of a sedimentable enzyme with a 33% loss in enzyme activity. Under similar conditions at pH 6.5, the enzyme lost 67% of its activity and at pH 8.5, 84% enzyme activity was lost. Furthermore, autophosphorylation at either acidic or alkaline reaction pH lead to the formation of a sedimentable enzyme. Transmission electron microscopic studies on autophosphorylated kinase revealed particles that clustered into branched complexes. The autophosphorylation of wild-type kinase in the presence of AMP-PNP (an unhydrolyzable ATP analog) or the autophosphorylation-site mutant, T267A, did not show formation of branched complexes under the electron microscope. Autophosphorylation- dependent self-inactivation may be a mechanism of modulating the signal transduction pathway mediated by CCaMK.     

Ca2+ and phorbol ester activation of protein kinase C at intracellular Ca2+ concentrations and the effect of TMB-8

A calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was purified to near homogeneity from human polymorphonuclear leukocytes and shown to be identical to bovine protein kinase C. The Ca2+ activation of the enzyme was studied and the Ca2+ concentrations required to activate the enzyme were compared to free cytosolic Ca2+ concentrations in resting and activated polymorphonuclear leukocytes. The free calcium concentrations in the cytosol and in the enzyme assay mixture were determined using the calcium indicator quin 2. The enzyme activity was almost totally dependent upon phosphatidylserine and could be strongly activated by Ca2+ concentrations in the micromolar range, but was not activated by phosphatidylserine at Ca2+ concentrations corresponding to the intracellular free Ca2+ concentration under resting conditions. However, at similar Ca2+ concentrations (less than 2.5 X 10(-7) M) the enzyme was highly activated by phorbol 12-myristate 13-acetate (PMA) or diolein in the presence of phosphatidylserine. It was demonstrated that PMA stimulation of human polymorphonuclear leukocytes did not induce any increase in the level of the intracellular free calcium concentration. It was concluded that PMA activation of protein kinase C occurred independently of a rise in the intracellular Ca2+ concentration. K0.5 (half-maximal activation) for the PMA activation of purified protein kinase C was shown to be equivalent to the K0.5 for PMA stimulation of superoxide (O-2) production in human polymorphonuclear leukocytes, suggesting that protein kinase C is involved in activation of the NADPH oxidase. The presumed intracellular Ca2+ antagonist TMB-8 inhibited the PMA-induced superoxide production, but neither by an intracellular Ca2+ antagonism nor by a direct inhibition of protein kinase C activity.     

A novel protein kinase from Brassica juncea stimulated by a protozoan calcium binding protein. Purification and partial characterization.

A novel protein kinase (BjCCaBPk) from etiolated Brassica juncea seedlings has been purified and partially characterized. The purified enzyme migrated on SDS/PAGE as a single band with an apparent molecular mass of 43 kDa. The optimum pH for the kinase activity was 8.0. It was stimulated more than sixfold by the protozoa Entamoeba histolytica calcium binding protein EhCaBP (10.5 nM) but not by calmodulin (CaM) when used at equimolar concentration. Moreover the kinase also did not bind CaM-Sepharose. There was neither inhibition of the kinase activity in the presence of W-7 (a CaM antagonist), KN-62 (a specific calcium/CaM kinase inhibitor) and anti-CaM Ig, nor any effect on BjCCaBPk activity of staurosporine (a protein kinase C inhibitor). Furthermore a CaM-kinase specific substrate, syntide-2, proved to be a poor substrate for the BjCCaBPk compared with histone III-S. The phosphorylation of histone III-S involved serine residues. Southern and Northern blot analysis showed the presence of EhCaBP homologues in Brassica. The data suggest that BjCCaBPk may be a novel protein kinase with an affinity towards a calcium binding protein like EhCaBP.     

Calmodulin-dependent NAD kinase of human neutrophils

NAD kinase from human neutrophils has been partially purified by sequential application of Red Agarose, ion-exchange, and gel-filtration chromatography. The enzyme has a broad pH optimum, 7.0-9.5, is strictly dependent upon the presence of Mg2+, and in the absence of calcium exhibits Km values of 0.6 and 0.9 mM for NAD and ATP, respectively. NAD kinase activity is extremely sensitive to free calcium concentration, with half-maximal activity observed at free calcium concentrations of approximately 0.4 microM. In cellular extracts calcium-dependent activation of NAD kinase increases the maximum velocity of the reaction from 2- to 5-fold while not affecting Km values for NAD and ATP. The activity of the partially purified NAD kinase is stimulated 3.5-fold by the addition of calmodulin in the presence of calcium. This stimulation is inhibited by the addition of 20 microM trifluoperazine to the incubation. These data are interpreted as implicating calmodulin in NAD kinase regulation. The total concentration of NADP + NADPH in the human neutrophil used increased 2.2-fold in response to activation by phorbol myristic acetate. Finally, neutrophil NAD kinase has a Mr, based upon gel filtration, of 169,000.     

Lipid vesicles which can bind to protein kinase C and activate the enzyme in the presence of EGTA.

Maximal protein kinase C activity with vesicles of phosphatidic acid and 1,2-dioleoyl-sn-glycerol is observed in the absence of added Ca2+. Addition of phosphatidylcholine to these vesicles restores some calcium dependence of enzyme activity. 1,2-Dioleoyl-sn-glycerol eliminates the Ca(2+)-dependence of protein kinase C activity found with phosphatidic acid alone. Phorbol esters do not mimic the action of 1,2-dioleoyl-sn-glycerol in this respect. This suggests that the 1,2-dioleoyl-sn-glycerol effect is a result of changes it causes in the physical properties of the membrane rather than to specific binding to the enzyme. The effect of 1,2-dioleoyl-sn-glycerol on the phosphatidic-acid-stimulated protein kinase C activity is dependent on the molar fraction of 1,2-dioleoyl-sn-glycerol used and results in a gradual shift from Ca2+ stimulation at low 1,2-dioleoyl-sn-glycerol concentrations to calcium inhibition at higher concentrations of 1,2-dioleoyl-sn-glycerol. Phosphatidylserine-stimulated activity is also shown to be largely independent of the calcium concentration at higher molar fractions of 1,2-dioleoyl-sn-glycerol. Thus, with certain lipid compositions, protein kinase C activity becomes independent of the calcium concentration or requires only very low, stoichiometric binding of Ca2+ to high affinity sites on the enzyme. Protein kinase C can bind to phosphatidic acid vesicles more readily than it can bind to phosphatidylserine vesicles in the absence of calcium. Addition of 1,2-dioleoyl-sn-glycerol to phosphatidylserine vesicles promotes the partitioning of protein kinase C into the membrane in the absence of added Ca2+. There is no isozyme specificity in this binding. These results suggest that a less-tightly packed headgroup region of the bilayer causes increased insertion of protein kinase C into the membrane. This is a necessary but not sufficient condition for activation of the enzyme in the presence of EGTA.     

Calmodulin-stimulated protein kinase activity from rat pancreas

Previous work from our laboratory has demonstrated that neurohumoral stimulation of the exocrine pancreas is associated with the phosphorylation of the Mr 29,000 ribosomal protein S6. In a cell-free system using pancreatic postmicrosomal supernatant as the kinase donor, we found that the following co-factors stimulate the phosphorylation of the Mr 29,000 ribosomal protein: calcium with calmodulin, calcium with phosphatidyl serine, and cAMP. These findings suggest that the pancreas contains a calcium-calmodulin-dependent protein kinase (CaM-PK) that can phosphorylate the Mr 29,000 ribosomal protein. A CaM-PK activity was partially purified sequentially by ion exchange, gel filtration, and calmodulin-affinity chromatography. Phosphorylation of the Mr 29,000 ribosomal protein by the partially purified CaM-PK was dependent on the presence of both calcium and calmodulin and not on the other co- factors. The CaM-PK fraction contained a phosphoprotein of Mr 51,000 whose phosphorylation was also dependent on calcium and calmodulin. When 125I-calmodulin-binding proteins from the CaM-PK fraction were identified using electrophoretic transfers of SDS-polyacrylamide gels, a single Mr 51,000 protein was labeled. The preparation enriched in CaM- PK activity contained an Mr 51,000 protein that underwent phosphorylation in a calcium-calmodulin-dependent manner and an Mr 51,000 calmodulin-binding protein. It is therefore possible that the CaM-PK may comprise a calmodulin-binding phosphoprotein component of Mr 51,000.     

Ca2+-dependent protein kinase from alfalfa (Medicago varia): Partial purification and autophosphorylation

A calcium-dependent protein kinase (CDPK) was purified to 1400-fold from the soluble fraction of alfalfa (Medicago varia) cells by ammonium sulfate fractionation, Sephacryl-300, DEAE-Sephacel, Phenyl-Sepharose and Hydroxylapatite column chromatography. The enzyme is mainly monomeric. During the course of the purification steps a 50 kDa phosphoprotein doublet and a 56 kDa phosphoprotein copurified with the CDPK activity. Mobility shift of these proteins have been shown by SDS PAGE in Ca²⁺ free conditions. Tests on enzyme activity after separation by native gel electrophoresis revealed two protein kinase activities in our enzyme preparation and the phosphorylation of the 50 kDa and 56 kDa proteins. We suggest that these proteins are the autophosphorylated forms of calcium dependent protein kinases. Preincubation of the CDPK in ATP resulted in a marked increase in enzyme activity, but did not alter the Ca²⁺ sensitivity of the protein kinase.     

A calcium-dependent but calmodulin-independent protein kinase from soybean

A calcium-dependent protein kinase activity from suspension-cultured soybean cells (Glycine max L. Wayne) was shown to be dependent on calcium but not calmodulin. The concentrations of free calcium required for half-maximal histone H1 phosphorylation and autophosphorylation were similar (≈2 micromolar). The protein kinase activity was stimulated 100-fold by ≥10 micromolar-free calcium. When exogenous soybean or bovine brain calmodulin was added in high concentration (1 micromolar) to the purified kinase, calcium-dependent and -independent activities were weakly stimulated (≤2-fold). Bovine serum albumin had a similar effect on both activities. The kinase was separated from a small amount of contaminating calmodulin by sodium dodecyl sulfate polyacrylamide gel electrophoresis. After renaturation the protein kinase autophosphorylated and phosphorylated histone H1 in a calcium-dependent manner. Following electroblotting onto nitrocellulose, the kinase bound ⁴⁵Ca²⁺ in the presence of KCl and MgCl₂, which indicates that the kinase itself is a high-affinity calcium-binding protein. Also, the mobility of one of two kinase bands in SDS gels was dependent on the presence of calcium. Autophosphorylation of the calmodulin-free kinase was inhibited by the calmodulin-binding compound N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), showing that the inhibition of activity by W-7 is independent of calmodulin. These results show that soybean calcium-dependent protein kinase represents a new class of protein kinase which requires calcium but not calmodulin for activity.     

Dictyostelium myosin light chain kinase. Purification and characterization

A Dictyostelium myosin light chain kinase has been purified approximately 15,000-fold to near homogeneity. The purified kinase is a single polypeptide of approximately 34 kDa that phosphorylates only the 18-kDa Dictyostelium myosin regulatory light chain and itself among substrates tested. The enzyme was purified largely by ammonium sulfate fractionation and hydrophobic (butyl) interaction chromatography. Analysis using polyclonal antibodies raised against the purified 34-kDa protein confirms that this protein is responsible for myosin light chain kinase activity. Protein microsequence of the 34-kDa protein reveals conserved protein kinase sequences. The purified Dictyostelium myosin light chain kinase exhibits a Km for Dictyostelium myosin of 4 microM and a Vmax of 8 nmol/min/mg. Unlike other characterized myosin light chain kinases, this enzyme is not regulated by calcium/calmodulin. Western blot analysis demonstrates that the purified kinase is not a proteolytic fragment that has lost calcium/calmodulin regulation. The Dictyostelium myosin light chain kinase activity is not directly regulated by cyclic nucleotides. However, this kinase undergoes an intramolecular autophosphorylation that activates the enzyme.     

Regulatory properties of rabbit liver phosphorylase kinase

• 1.1. Purified native rabbit liver phosphorylase kinase becomes activated during the assay of its activity while low molecular weight forms of the same enzyme do not.
• 2.2. The activation requires ATP and maganesium ions, suggesting the phosphorylation of the enzyme by a protein kinase as the mechanism involved.
• 3.3. The activation of the enzyme can be reverted by the action of a type 1 protein phosphatase isolated from the same tissue.
• 4.4. The activation can also be catalyzed by the catalytic subunit of cAMP-dependent protein kinase in a process that requires a much lower ATP concentration to proceed.
• 5.5. The activation is believed to be due to an autocatalytic phosphorylation of phosphorylase kinase itself. In support of this hypothesis are the regulation of the process through calcium ions, the low levels of endogenous protein kinase detected in the purified preparation, the high ATP concentrations required in the absence of cAMP dependent protein kinase and the fact that the process cannot be blocked by an excess of the heat stable inhibitor specific for the later enzyme.
• 6.6. The low molecular weight forms of the enzyme on their side are not affected by the action of neither protein phosphatase 1 nor cyclic AMP dependent protein kinase.
• 7.7. Both activated and nonactivated phosphorylase kinase are partially dependent on calcium ions, the affinity of the former being higher than that of the latter. The low molecular forms do not require calcium ions to express their activity.

     

Nitric oxide synthase regulatory sites. Phosphorylation by cyclic AMP-dependent protein kinase, protein kinase C, and calcium/calmodulin protein kinase; identification of flavin and calmodulin binding sites.

Nitric oxide (NO) is an important molecular messenger accounting for endothelial-derived relaxing activity in blood vessels, mediating cytotoxic actions of macrophages, and functioning as a neurotransmitter in the brain and periphery. NO synthase (NOS) from brain has been purified to homogeneity and molecularly cloned. We now report that NOS is stoichiometrically phosphorylated by cAMP dependent protein kinase, protein kinase C, and calcium/calmodulin-dependent protein kinase, with each kinase phosphorylating a different serine site on NOS. Activation of PKC in transfected cells reduces NOS enzyme activity by approximately 77% in intact cells and by 50% in protein homogenates from these cells. Utilizing fluorescence spectroscopy we find that purified monomer NOS contains 1 molar equivalent of both FMN and FAD. This stoichiometry is supported by enzymatic digestion of the flavins with phosphodiesterase, and titration of the FMN with a specific FMN binding protein. We demonstrate that purified NOS is labeled by a photoaffinity derivative of calmodulin. These recognition sites on NOS provide multiple means for regulation of NO levels and "cross-talk" between second messenger systems.     

Phosphorylation of partially purified 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase from rat spleen.

A new improved method for purification of the enzyme 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine: acetyl-CoA acetyltransferase (EC 2.3.1.67) from rat spleen is described. The catalytic subunit of cyclic AMP-dependent protein kinase in the presence of MgATP stimulated about 3-fold the activity of this partially purified enzyme activity. When [gamma-32P]ATP was included in the assay mixture, the analysis of phosphoprotein products by SDS/polyacrylamide-gel electrophoresis and autoradiography showed the incorporation of [32P]phosphate into a single protein band of about 30 kDa. Analysis of the phosphorylated amino acids indicated that the phosphate was incorporated into a serine residue. Activation of the acetylation reaction by the protein kinase was reversible. The reversal of the activation was coincident with the loss of the [32P]phosphate incorporated into the 30 kDa protein band, which suggests that the acetyltransferase is regulated by a phosphorylation-dephosphorylation mechanism dependent on cyclic AMP.     

Cyclic nucleotide dependent protein kinase and the phosphorylation of endogenous proteins of retinal rod outer segments.

Cyclic nucleotide dependent protein kinase has been extracted wiht Tris or Lubrol PX from purified rod outer segments (ROS) of bovine retina. The activity of the enzyme is unaffected by light but is stimulated by either cyclic guanosine 3',5'-monophosphate (cGMP) or cyclic adenosine 3',5'-monophosphate (cAMP). Most of the solubilized enzyme elutes from DEAE-cellulose with about 0.18 M NaCl (type II protein kinase). An endogenous 30,000 molecular weight protein of the soluble fraction of ROS as well as exogenous histone are phosphorylated by the protein kinase in a cyclic nucleotide dependent manner. The Tris-extracted enzyme can be reassociated in the presence of Mg2+ with ROS membranes that are depleted of protein kinase activity. The reassociated protein kinase is insensitive to exogenous cyclic nucleotides, and it catalyzes the phosphorylation of the membrane protein, bleached rhodopsin. While the soluble and membrane-associated protein kinases may be interchangeable, they appear to be modulated by different biological signals; soluble protein kinase activity is increased by cyclic nucleotides whereas membrane-bound activity is enhanced when rhodopsin is bleached by light.     

Phosphorylation of endogenous and exogenous peptides by rat heart sarcolemma

Rat heart plasma membranes contain a calcium-dependent protein kinase which phosphorylates endogenous protein substrates as well as added histones. The major endogenous protein phosphorylated is of 17 kDa on SDS-polyacrylamide gel electrophoresis. Proteins of 85 kDa and 60 kDa were also phosphorylated. Treatment of a rat heart homogenate with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate increased the recovery of kinase activity in the sarcolemmal membranes by up to 10-fold. The activity in such membranes was no longer calcium dependent. Although several histones were effective substrates for the enzyme, myosin light chain and phosvitin were not phosphorylated. These membranes contain a very active ATP hydrolysing activity which necessitated very brief incubation times to avoid loss of substrate. The membranes also contain cyclic AMP dependent protein kinase activity which is not active unless cyclic AMP is added to the incubations. The calcium dependent endogenous kinase, which is not inhibited by the heat stable inhibitor protein of cyclic AMP-dependent kinase, or by trifluoperazine, has several properties in common with protein kinase C. Preincubation of the sarcolemmal membranes with a high concentration of insulin caused inhibition of the phosphorylation of the endogenous 17 kDa and 85 kDa bands. There was no effect on the phosphorylation of the 60 kDa peptide. This effect of insulin was specific for the hormone and required preincubation of the hormone with the membranes for 20 min.     

The Dendritic Peptide Neurogranin Can Regulate a Calmodulin-Dependent Target

Abstract: Neurogranin, a peptide capable of binding the calcium-poor form of calmodulin, was tested in vitro for its ability to modulate a typical calmodulin target. The target employed was the calcium/calmodulin-dependent form of nitric oxide synthase, which is produced by several different types of neurons. Neurogranin for the study was purified from perchloric acid-soluble calf brain proteins by a combination of calmodulin-Sepharose affinity chromatography and reverse-phase HPLC. The protocol yielded highly purified neurogranin that was active in assays using purified nitric oxide synthase. The titration of the enzyme activity with neurogranin demonstrated a concentration-dependent effect of the peptide on enzyme activation. Subsequent analysis of the ability of increased calcium concentrations to activate the enzyme was performed in the presence of different amounts of neurogranin. The effect of neurogranin on the calcium-dependent activation of the enzyme was to depress enzyme activity in the range of 0.2 to ∼1 µ M calcium. Treatment of the neurogranin peptide with protein kinase C eliminated its inhibition on nitric oxide synthase activation. Treatment of the protein kinase C-phosphorylated peptide with calcineurin did not restore the ability of neurogranin to inhibit enzyme activity, whereas treatment with alkaline phosphatase did restore this ability. These results suggest that neurogranin may serve as a member of a unique class of endogenous calmodulin inhibitor that functions to regulate the activation of calmodulin-requiring targets in neurons.