Rapid purification of phospholamban by monoclonal antibody immunoaffinity chromatography

Monoclonal antibodies have been raised against canine phospholamban purified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). Four of twenty-four antibodies were purified to close to homogeneity from mouse ascites. All four antibodies could react with isolated bovine cardiac sarcoplasmic reticulum (SR) to result in the stimulation of ATP-dependent Ca2+ pump activity and blocking of phospholamban phosphorylation by cAMP-dependent protein kinase. Relative efficiencies of antibodies in Ca2+ pump stimulation and on phospholamban phosphorylation were not correlated. An immunoabsorbent prepared by conjugating antibody Al to Affi-Gel 10 was used for the purification of phospholamban. Isolated bovine cardiac SR was solubilized in a buffer containing deoxycholate and the soluble fraction was applied to the immunoaffinity column. After washing the column with a series of detergent-containing buffer solutions, the column-bound protein which contained essentially pure phospholamban was eluted by a buffer containing 2.8 M MgCl2. The phospholamban recovery from the immunoaffinity column was close to 100%; the overall yield of purification from SR vesicles was about 70%. SDS-PAGE analysis showed that purified phospholamban consisted of a 25 and 5 kilodalton (kDa) protein species. Upon brief boiling (20 s) of the sample in SDS-PAGE sample buffer, five molecular species ranging from 5 to 25 kDa could be detected by immunotransblotting following SDS-PAGE. This observation supports the notion that phospholamban is composed of five 5-kDa polypeptides. The pure phospholamban could be phosphorylated maximally by cAMP-dependent protein kinase to 1-1.5 mol phosphate/mol phospholamban (25,000 g). This stoichiometry of phosphorylation could be increased to about 5 upon addition of the immunoaffinity column flow through fraction.     

Immunoaffinity purification and reconstitution of human alpha(2)-adrenergic receptor subtype C2 into phospholipid vesicles

Large quantities of correctly folded, pure alpha(2)-adrenergic receptor protein are needed for structural analysis. We report here the first efficient method to purify human alpha(2)-adrenergic receptor subtype C2 to homogeneity from recombinant yeast Saccharomyces cerevisiae by one-step purification using a monoclonal antibody column (specific for alpha(2)C2). We show that the adrenoceptor antagonist phentolamine stabilized the receptor during purification. We used a very effective chaotropic agent, NaSCN, to elute the receptor from the immunoaffinity column with an overall yield of 34% before reconstitution. Ligand binding of detergent-solubilized, immunoaffinity-purified receptors could not be demonstrated, but partial recovery of ligand binding activity was achieved when purified receptors were reconstituted into phospholipid vesicles. The reconstituted receptors still bound radioligand after storage on ice for 4 weeks. This purification procedure can be easily scaled-up and thus demonstrates the utility of a monoclonal antibody column and NaSCN elution to purify large quantities of G-protein-coupled receptors.     

Immunoaffinity purification of the epidermal growth factor receptor. Stoichiometry of binding and kinetics of self-phosphorylation

Epidermal growth factor (EGF) receptor protein has been purified in a single high-yield step by immunoaffinity chromatography of extracts of A431 cells. A monoclonal antibody directed against the EGF binding site of the receptor was immobilized to Sepharose 4B as a specific immune absorbent and competitive elution with EGF was used to obtain purified EGF receptor protein with tyrosine kinase activity. The stoichiometry of EGF binding was determined by comparing 125I-EGF binding to A431 cells with the mass of EGF receptor protein in those cells as measured by immunoaffinity chromatography, radioimmunoassay, and immune precipitation. Each measurement indicated one EGF binding site/EGF receptor protein molecule. Study of the kinetics of autophosphorylation revealed rapid incorporation of 1 mol of phosphate/mol of enzyme followed by slower incorporation of additional phosphate groups. The autophosphorylation reaction has a Km for ATP (0.2 microM) which is about 10-fold lower than that for phosphorylation of exogenous substrates. The kinetically preferred autophosphorylation is an intramolecular reaction.     

Monoclonal antibodies against the human epidermal growth factor receptor from A431 cells. Isolation, characterization, and use in the purification of active epidermal growth factor receptor

A431 cells have been used as an immunogen for generating monoclonal antibodies against the epidermal growth factor (EGF) receptor. Two immunoglobulin M and eight immunoglobulin G3 anti-EGF receptor antibodies were cloned. All ten antibodies immunoprecipitated biosynthetically labeled mature A431 cell EGF receptor and were able to recognize the receptor in Western blotting. However, none of the antibodies immunoprecipitated precursor polypeptides of the A431 cell EGF receptor, neither did they recognize EGF receptors from human foreskin fibroblasts, human placenta, nor a human-mouse hybrid cell expressing EGF receptor. The antibodies were found to bind to glycolipids from A431 cells and it was shown that the determinant involved was the blood group A antigen. It appears that this determinant is present on both the EGF receptor and glycolipids of A431 cells but is not expressed on EGF receptors from other human cells tested. One of the monoclonal antibodies raised was used for immunoaffinity purification of the EGF receptor. The procedure took advantage of the carbohydrate nature of the antigenic determinant by employing sugar-specific elution. The mild conditions permitted the purification of A431 cell EGF receptor (70-80% pure) that possessed an intrinsic EGF-stimulated tyrosine kinase activity with a specific activity of about 20 nmol/min/mg.     

Utilization of Arg-elution method for FLAG-tag based chromatography

FLAG-tag is one of the commonly used purification technologies for recombinant proteins. An antibody, M2, specifically binds to the FLAG-tag whether it is attached to N- or C-terminus of proteins to be purified. The bound proteins are generally eluted by competition with a large excess of free FLAG peptide. This requires synthetic FLAG peptide and also removal of bound FLAG peptide for M2 column regeneration. We have shown before that arginine at mild pH can effectively dissociate protein–protein or protein–ligand interactions, e.g. in Protein-A, antigen and dye-affinity chromatography. We have tested here elution of FLAG-fused proteins by arginine for columns of M2-immobilized resin using several proteins in comparison with competitive elution by FLAG peptide or low pH glycine buffer. Active and folded proteins were successfully and effectively eluted using 0.5–1 M arginine at pH 3.5–4.4, as reported in this paper.     

Immunoaffinity purification of the calpains

A monoclonal antibody to the small subunit common to both mu- and m-calpains can be used in an immunoaffinity column to purify either mu- or m-calpain in a proteolytically active form. Extracts in 150 mM NaCl, pH 7.5, are loaded onto a column containing the anti-28-kDa antibody; the column is washed with 500 mM NaCl, pH 7.5, and the bound calpain is eluted with 150 mM NaCl, 50 mM Tris-HCl, pH 9.5, and 1 mM EDTA. These elution conditions do not affect the proteolytic activity of either mu- or m-calpain. It is most efficient to reduce the volume and to remove any proteolytic activity from crude extracts by using successive phenyl Sepharose and ion-exchange columns before loading onto the immunoaffinity column. The column purifies m-calpain more effectively than mu-calpain; m-calpain is greater than 90% pure after a single pass through this column, whereas mu-calpain can be purified to >70% purity. The epitope for the monoclonal antibody is between amino acids 92 and 104 (numbers for human calpain) in the 28-kDa subunit. Evidently, this area is shielded in the calpain molecule in a way that affects binding of the antibody to the native molecule.     

Secreted forms of human neutrophil collagenase

Collagenase in human neutrophils is found within intracellular granules which can be stimulated to be secreted with phorbol myristic acetate. This extracellular secreted form of neutrophil collagenase was isolated by immunoaffinity chromatography using a monoclonal antibody previously shown to specifically recognize neutrophil collagenase. The enzyme efficiently bound to this column and was eluted with NaSCN as three major species of 75, 57, and 22 kDa, respectively. These proteins were closely related immunologically since, after radiolabeling and separation by gel filtration, each of the three proteins was precipitated by the monoclonal antibody. Also, the 75- and 57-kDa proteins exhibited collagenase activity after elution from polyacrylamide gels run under nondenaturing conditions. Further, the 57-kDa protein autodegraded into a 22-kDa protein with time. Polyclonal antibody, prepared to the 57-kDa enzyme, also recognized the 75- and 22-kDa proteins using an immunoblot technique. When crude neutrophil supernatants containing latent collagenase were immunoblotted, both the 75- and the 57-kDa enzymes were present. Our immunoaffinity purified active enzymes, although activated during the course of purification, resemble the latent enzymes in crude neutrophil supernatants. The multiple forms of secreted collagenase from degranulated leukocytes may resemble more closely that seen in inflammation.     

One-step immunoaffinity purification of active progesterone receptor. Further evidence in favor of the existence of a single steroid binding subunit

A very high capacity immunoaffinity matrix for the purification of progesterone receptor was prepared by cross-linking a monoclonal antireceptor antibody to protein A-Sepharose through the Fc fragment. The monoclonal antibody was selected for its property of losing affinity for the receptor at pH 10.5, i.e., in conditions where the receptor remains stable for extensive periods of time. This made it possible to elute active receptor form the immunosorbent. From crude rabbit uterine cytosol the steroid-receptor complexes were purified in a single step. A 1-mL column (containing 7 mg of monoclonal antibody) bound 1600 pmol of steroid-receptor complexes of which 79.5% were eluted. The overall yield of purification was 49%. The specific activity of the purified steroid-receptor complexes was 6.71 +/- 0.79 nmol of bound steroid/mg of protein (mean +/- SE of four experiments). The purified receptor consisted of a mixture of 110 000- and 79 000-dalton forms. The latter appeared to be produced by proteolysis of the larger form during purification since immunoblot experiments showed that, at the start of purification, the 110 000-dalton form was present in overwhelming majority (80-95%) in the uterine cytosol and that the 79 000-dalton form only appeared during purification. This conclusion was also supported by the peptide analysis of both forms of receptor: the purified receptor was denatured and labeled with 125I; the 110 000- and 79 000-dalton forms were isolated by gel electrophoresis in denaturing conditions and electroelution and were then submitted to mild or extensive digestions by trypsin, chymotrypsin, and protease V8 from Staphylococcus aureus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of the purified protein tyrosine kinase domain of the epidermal growth factor receptor

Biological responses to epidermal growth factor (EGF) depend on the ligand-stimulated protein tyrosine kinase activity of its receptor. To further characterize the enzymatic activity of the EGF receptor, the baculovirus expression system was used to express the cytoplasmic protein tyrosine kinase domain of the EGF receptor. Spodoptera frugiperda (Sf9) cells infected with recombinant baculovirus correctly expressed an active tyrosine kinase domain of the EGF receptor as demonstrated by 35S metabolic labeling, immunoblotting with anti-EGF receptor and anti-phosphotyrosine antibodies, and autophosphorylation analysis. The kinase domain (Mr 66,000) was purified to near homogeneity using a monoclonal anti-phosphotyrosine antibody column, providing 0.5 mg of kinase domain/liter of Sf9 cells (23% yield). The purified kinase domain exhibited a strong preference for Mn2+ compared to Mg2+. The specific activity of the kinase domain was low compared to purified, EGF-activated EGF receptor. However, the addition of sphingosine or ammonium sulfate greatly increased the activity of the kinase domain to equal or exceed the activity of ligand-activated holo EGF receptor. These results indicate that the addition of sphingosine or ammonium sulfate to the purified kinase domain can mimic the effect of EGF to induce a conformation of the holo EGF receptor which is optimal for tyrosine kinase activity. Deletion of the ligand binding domain, analogous to that which occurs in erb B, is not sufficient to fully activate the kinase, implying that EGF causes conformational changes additional to removal of an inhibitory constraint.     

Calmodulin inhibits the epidermal growth factor receptor tyrosine kinase.

We demonstrate in this report that the epidermal growth factor (EGF) receptor from rat liver can be isolated by calmodulin affinity chromatography by binding in the presence of Ca2+ and elution with a Ca(2+)-chelating agent. The bulk of the EGF receptor is not eluted by a NaCl gradient in the presence of Ca2+. We ascertained the identity of the isolated receptor by immunoblot and immunoprecipitation using a polyclonal antibody against an EGF receptor from human origin. The purified receptor is autophosphorylated in tyrosine residues in an EGF-stimulated manner, and EGF-dependent phosphorylation of serine residues was also detected. Both the EGF and the transforming growth factor-alpha stimulate the tyrosine-directed protein kinase activity of the isolated receptor with similar affinities. Furthermore, we demonstrate that calmodulin inhibits the EGF-dependent tyrosine-directed protein kinase activity associated to the receptor in a concentration-dependent manner. This inhibition is partially Ca2+ dependent and is not displaced by increasing the concentration of EGF up to an EGF/calmodulin ratio of 10 (mol/mol). In addition, calmodulin was phosphorylated in an EGF-stimulated manner in the presence of a basic protein (histone) as cofactor and in the absence, but not in the presence, of Ca2+.     

Steroid-affinity purification of the rat liver glucocorticoid hormone receptor complex

The molybdate-stabilized GHRC was isolated from rat liver cytosol with a 9000-fold purification and 46% yield. The major purification step was achieved using an affinity matrix consisting of an agarose support coupled to a dexamethasone ligand via an aliphatic spacer arm. Spacer arms containing disulfide bridges were found to be unsuitable due to their instability in cytosol. To reduce the non-specific binding properties of the affinity matrix, underivatized amino groups were acetylated, since the receptor was found to bind avidly to such groups thus evading elution by the ligand. Sodium molybdate present during biospecific elution from the gel stabilized the steroid-binding activity of the receptor. The use of denaturing and sulfhydryl modifying reagents (NaSCN, DMSO, Mersalyl) during elution led to partial or complete irreversible loss of steroid-binding activity of the unoccupied receptor. Efficient biospecific elution occurred at competing concentration of high affinity steroid in the presence of sodium molybdate. The ligand specific eluate was further purified by DEAE-Sephacel chromatography resulting in additional purification of 3.2-fold. The GHRC eluted from the DEAE-Sephacel column at a salt concentration characteristic of the untransformed GHRC. Molybdate was removed from the purified untransformed GHRC in the ligand eluate by DEAE-Sephacel chromatography in the absence of molybdate, for subsequent heat transformation.     

Purification from goat antiserum of immunoglobulins against G6PD from rabbits]

DEAE Affi-Gel Blue (Bio-Rad) provides an efficient and rapid fractionation of human serum proteins by a single chromatographic step. When goat serum is applied to the matrix and chromatography is performed following the procedure utilized for the human serum proteins, the elution pattern changes and the Ig purification is not satisfactory. We achieved a better Ig purification from goat serum by the following improved procedure. We performed first an AS-40 fractionation followed by extensive dialysis in 50 mM Na-citrate pH 5.7. The sample was then loaded onto a P11 column equilibrated in the same buffer. The fraction eluted at Vo contained total IgG and the other serum proteins, except beta-globulins which were eluted with 0.24 M phosphate. Peak 1 concentrated and dialyzed in 20 mM phosphate buffer pH 8 was then applied to a DEAE Affi-Gel Blue column, equilibrated in the same buffer. Two protein peaks were eluted from this column and electrophoretically characterized as: peak 1, containing a pure Ig fraction (70% yield), peak 2 with albumin and other contaminating serum proteins. When goat antiserum is obtained against a specific protein, our technique may be suitably employed to purify polyclonal antibodies for immunoprecipitation studies.     

Immunoaffinity Purification of Epitope-Tagged Human β2-Adrenergic Receptor to Homogeneity

To obtain large quantities of pure human β₂-adrenergic receptor (β₂-AR) needed for structural studies, an efficient method for β₂-AR purification was developed using a recombinant receptor with an eight amino acid epitope at its C-terminus. This epitope is recognized by KT3-monoclonal antibody. The epitope tagged β₂-AR was expressed in Sf9 cells with a specific activity of 5–20 pmol/mg of membrane protein. The epitope-tagged and wild-type receptors had identical ligand binding properties. The tagged receptor was solubilized using dodecyl-β-maltoside with a quantitative yield. Solubilized epitope-tagged receptors were partially purified by KT3-mAb immunoaffinity in 60–70% yield. Further purification of the receptors on an alprenolol-affinity column resulted in a homogenous preparation with an overall yield of >30%. The purified receptor was concentrated to >1 mg/ml without loss of ligand binding activity.     

Arylamine N-acetyltransferase from chicken liver. I. Monoclonal antibodies, immunoaffinity purification, and amino acid sequences

Five monoclonal antibodies against arylamine acetyltransferase (EC 2.3.1.5) from the chicken liver were established by immunizing a mouse with a partially purified enzyme preparation. None of the antibodies cross-reacted with arylamine N-acetyltransferase from the livers of cow, rabbit, and rat, nor with arylalkylamine N-acetyltransferase from the chicken pineal gland, indicating a high specificity of the antibodies. By using the antibodies, two immunoaffinity purification procedures were elaborated: A partially purified enzyme preparation was incubated with the monoclonal antibody, and the resulting enzyme-IgG complex was separated by a protein A-Sepharose column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein band with a molecular mass of 34 kDa in addition to the heavy and light chains of IgG. Secondly, an immunoaffinity column was prepared by immobilizing a monoclonal antibody to Sepharose 4B. After a partially purified enzyme preparation was absorbed on the column, N-acetyltransferase activity was eluted with 1 M NaCl and 1 M urea. The eluted sample contained a single 34-kDa protein. The purified enzyme preferred arylamines to arylalkylamines as substrates, indicating that it was arylamine N-acetyltransferase. The purified protein was subjected to digestion by lysylendopeptidase and separated by high performance liquid chromatography. Partial amino acid sequences of three peptides were determined by a gas-phase sequence analyzer.     

Affinity chromatography of Ankistrodesmus braunii nitrate reductase using blue dextran-sepharose (author's transl)

Blue Dextran has been coupled covalently to Sepharose-4B to purify the enzymatic complex NAD(P)H-nitrate reductase (EC 1.6.6.2) from the green alga Ankistrodesmus braunii by affinity chromatography. The optimum conditions for the accomplishment of the chromatographic process have been determined. The adsorption of nitrate reductase on Blue Dextran Sepharose is optimum when a phosphate buffer of low ionic strength and pH 6.5-7.0 is used. Once the enzyme has been bound to Blue Dextran Sepharose, it can be specifically eluted by addition of NADH and FAD to the washing buffer. However, none of the nucleotides added separately is able to promote the elution of the enzyme from the column. The elution can be also achieved, but not specifically, by increasing the ionic strength of the buffer with KCl. These results have made possible a procedure for the purification of A. braunii nitrate reductase which led to electrophoretic homogeneity, with an overall yield of 70% and a specific activity of 49 units/mg of protein.     

The use of affinity chromatography in purification of cyclic nucleotide receptor proteins.

Biospecific affinity chromatography has been used to purify specific cyclic AMP and cyclic GMP receptor proteins. Several variables are important for successful purification of the cyclic AMP receptor protein, the most critical being the length of the aliphatic spacer side arm. 8-(2-Aminoethyl)-amino-cyclic AMP coupled to the aliphatic spacer side arm. 8-(2-Aminoethyl)-amino-cyclic AMP coupled to agarose specifically retains the cyclic AMP receptor protein by interaction with the immobilized nucleotide. Binding of the cyclic AMP receptor subunit of cyclic AMP-dependent protein kinase to the immobilized nucleotide results in dissociation of the catalytic protein phosphokinase subunit which is not retained. The retained cyclic AMP receptor protein is subsequently eluted by cyclic AMP. Homogeneous cyclic AMP receptor protein prepared from rabbit skeletal muscle by affinity chromatography has been characterized. The molecular weight of the native protein as determined by analytical ultracentrifugation and polyacrylamide gel electrophoresis at varying acrylamide concentrations is 76 800 and 82 000, respectively. The protein is asymmetric with frictional and axial ratios of 1.64 and 12. SDS and urea polyacrylamide gel electrophoresis indicate that the native cyclic AMP receptor is composed of two identical subunits of 42 700 molecular weight. The native protein dimer binds 2 moles of cyclic AMP per mole of protein and is active in suppressing activity of isolated catalytic subunits of cyclic AMP-dependent protein kinase. Cyclic GMP receptor protein from bovine lung has been purified using the same affinity chromatography media. Since cyclic nucleotide binding to cyclic GMP-dependent protein kinase does not result in dissociation of regulatory receptor and catalytic phosphotransferase subunits, the cyclic GMP-dependent protein kinase holoenzyme is retained on the column and can be subsequently specifically eluted with cyclic GMP.     

Isolation of a receptor protein involved in attachment of human rhinoviruses

Human rhinoviruses can be classified into major and minor groups on the basis of receptor specificity. Recently, a mouse monoclonal antibody was isolated which selectively blocked the attachment of the major group of human rhinoviruses to cells. Using this monoclonal antibody, the cellular receptor for the major group of human rhinoviruses was isolated. A radioimmunoassay was developed by using the receptor antibody to specifically detect rhinovirus receptor during isolation. Solubilized receptor from detergent-treated HeLa cell membrane extracts eluted from gel filtration columns with an apparent molecular weight of 440,000. A cellular receptor protein, which had a molecular weight of 90,000 when analyzed on sodium dodecyl sulfate-polyacrylamide gels, was purified from solubilized extracts on an immunoaffinity column containing receptor antibody. Polyclonal rabbit antiserum, resulting from immunization with the isolated receptor protein, specifically blocked the attachment of the major group of human rhinoviruses and indicated that the 90-kilodalton protein plays a functional role in attachment. Prolonged exposure of HeLa cell monolayers with the receptor antibody showed no inhibition of cell growth and division.     

两步串联层析法纯化鼠抗人CD80单克隆抗体4E5

采用阴离子交换与凝胶过滤两步串联层析法,纯化了小鼠腹水来源的CD80阻断型单克隆抗体4E5。腹水样品经离心、过滤预处理后,在Tris-HCl缓冲溶液(pH8.0, 50mmol/L)条件下上阴离子交换柱对目的单抗进行捕集,采用0-0.5 mol/L NaCl浓度分步洗脱;含目的单抗的洗脱馏分再上凝胶过滤柱纯化,用PB缓冲溶液（pH7.2, 20mmol/L）洗脱,获得目的单抗4E5,其生物学活性高、纯度大于95%,抗体总回收率达61%。     

One-step immunopurification and lectinochemical characterization of the Duffy atypical chemokine receptor from human erythrocytes

Duffy antigen/receptor for chemokines (DARC) is a glycosylated seven-transmembrane protein acting as a blood group antigen, a chemokine binding protein and a receptor for Plasmodium vivax malaria parasite. It is present on erythrocytes and endothelial cells of postcapillary venules. The N-terminal extracellular domain of the Duffy glycoprotein carries Fy(a)/Fy(b) blood group antigens and Fy6 linear epitope recognized by monoclonal antibodies. Previously, we have shown that recombinant Duffy protein expressed in K562 cells has three N-linked oligosaccharide chains, which are mainly of complex-type. Here we report a one-step purification method of Duffy protein from human erythrocytes. DARC was extracted from erythrocyte membranes in the presence of 1% n-dodecyl-β-D-maltoside (DDM) and 0.05% cholesteryl hemisuccinate (CHS) and purified by affinity chromatography using immobilized anti-Fy6 2C3 mouse monoclonal antibody. Duffy glycoprotein was eluted from the column with synthetic DFEDVWN peptide containing epitope for 2C3 monoclonal antibody. In this single-step immunoaffinity purification method we obtained highly purified DARC, which migrates in SDS-polyacrylamide gel as a major diffuse band corresponding to a molecular mass of 40-47?kDa. In ELISA purified Duffy glycoprotein binds anti-Duffy antibodies recognizing epitopes located on distinct regions of the molecule. Results of circular dichroism measurement indicate that purified DARC has a high content of α-helical secondary structure typical for chemokine receptors. Analysis of DARC glycans performed by means of lectin blotting and glycosidase digestion suggests that native Duffy N-glycans are mostly triantennary complex-type, terminated with α2-3- and α2-6-linked sialic acid residues with bisecting GlcNAc and α1-6-linked fucose at the core.     

Isolation and characterization of two growth factor-stimulated protein kinases that phosphorylate the epidermal growth factor receptor at threonine 669.

A growth factor-stimulated protein kinase activity that phosphorylates the epidermal growth factor (EGF) receptor at Thr669 has been described (Countaway, J. L., Northwood, I. C., and Davis, R. J. (1989) J. Biol. Chem. 264, 10828-10835). Anion-exchange chromatography demonstrated that this protein kinase activity was accounted for by two enzymes. The first peak of activity eluted from the column corresponded to the microtubule-associated protein 2 (MAP2) kinase. However, the second peak of activity was found to be a distinct enzyme. We present here the purification of this enzyme from human tumor KB cells by sequential ion-exchange chromatography. The isolated protein kinase was identified as a 46-kDa protein by polyacrylamide gel electrophoresis and silver staining. Gel filtration chromatography demonstrated that the enzyme was functional in a monomeric state. A kinetic analysis of the purified enzyme was performed at 22 degrees C using a synthetic peptide substrate based on the primary sequence of the EGF receptor (KREL VEPLT669PSGEAPNQALLR). The Km(app) for ATP was 40 +/- 5 microM (mean +/- S.D., n = 3). GTP was not found to be a substrate for the purified enzyme. The Km(app) for the synthetic peptide substrate was 260 +/- 40 microM (mean +/- S.D., n = 3). The Vmax(app) for the isolated protein kinase was determined to be 400-900 nmol/mg/min. The purified enzyme was designated EGF receptor Thr669 (ERT) kinase. It is likely that the MAP2 and ERT kinases account for the phosphorylation of the EGF receptor at Thr669 observed in cultured cells. The marked stimulation of protein kinase activity caused by growth factors indicates that these enzymes may have an important function during signal transduction.