RESUMO
cdk2.cyclin E and cdk5.p25 are two members of the cyclin-dependent kinase family that are potential therapeutic targets for oncology and Alzheimer's disease, respectively. In this study we have investigated the mechanism for these enzymes. Kinases catalyze the transfer of phosphate from ATP to a protein acceptor, thus utilizing two substrates, ATP and the target protein. For a two-substrate reaction, possible kinetic mechanisms include: ping-pong, sequential random, or sequential ordered. To determine the kinetic mechanism of cdk2.GST-cyclin E and cdk5.GST-p25, kinase activity was measured in experiments in which concentrations of peptide and ATP substrates were varied in the presence of dead-end inhibitors. A peptide identical to the peptide substrate, but with a substitution of valine for the phosphoacceptor threonine, competed with substrate with a K(i) value of 0.6 mm. An aminopyrimidine, PNU 112455A, was identified in a screen for inhibitors of cdk2. Nonlinear least squares and Lineweaver-Burk analyses demonstrated that the inhibitor PNU 112455A was competitive with ATP with a K(i) value of 2 microm. In addition, a co-crystal of PNU 112455A with cdk2 showed that the inhibitor binds in the ATP binding pocket of the enzyme. Analysis of the inhibitor data demonstrated that both kinases use a sequential random mechanism, in which either ATP or peptide may bind first to the enzyme active site. For both kinases, the binding of the second substrate was shown to be anticooperative, in that the binding of the first substrate decreases the affinity of the second substrate. For cdk2.GST-cyclin E the kinetic parameters were determined to be K(m, ATP) = 3.6 +/- 1.0 microm, K(m, peptide) = 4.6 +/- 1.4 microm, and the anticooperativity factor, alpha = 130 +/- 44. For cdk5.GST-p25, the K(m, ATP) = 3.2 +/- 0.7 microm, K(m, peptide) = 1.6 +/- 0.3 microm, and alpha = 7.2 +/- 1.8.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Quinases Ciclina-Dependentes/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Quinase 2 Dependente de Ciclina , Quinase 5 Dependente de Ciclina , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Cinética , Ligação Proteica , Conformação Proteica , Pirimidinas/química , Pirimidinas/metabolismo , Especificidade por Substrato , Sulfonamidas/química , Sulfonamidas/metabolismoRESUMO
Unregulated or overexpressed matrix metalloproteinases (MMPs), including stromelysin, collagenase, and gelatinase. have been implicated in several pathological conditions including arthritis and cancer. Small-molecule MMP inhibitors may have therapeutic value in the treatment of these diseases. In this regard, the solution structures of two stromelysin/ inhibitor complexes have been investigated using 1H, 13C, and 15N NMR spectroscopy. Both-inhibitors are members of a novel class of matrix metalloproteinase inhibitor that contain a thiadiazole group and that interact with stromelysin in a manner distinct from other classes of inhibitors. The inhibitors coordinate the catalytic zinc atom through their exocyclic sulfur atom, with the remainder of the ligand extending into the S1-S3 side of the active site. The binding of inhibitor containing a protonated or fluorinated aromatic ring was investigated using 1H and 19F NMR spectroscopy. The fluorinated ring was found to have a reduced ring-flip rate compared to the protonated version. A strong, coplanar interaction between the fluorinated ring of the inhibitor and the aromatic ring of Tyr155 is proposed to account for the reduced ring-flip rate and for the increase in binding affinity observed for the fluorinated inhibitor compared to the protonated inhibitor. Binding interactions observed for the thiadiazole class of ligands have implications for the design of matrix metalloproteinase inhibitors.
Assuntos
Inibidores Enzimáticos/química , Metaloproteinase 3 da Matriz/química , Inibidores de Metaloproteinases de Matriz , Tiadiazóis/química , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Soluções , Tiadiazóis/metabolismo , Ureia/análogos & derivados , Ureia/química , Ureia/metabolismo , Zinco/químicaRESUMO
A rapid and simple method for quantitating the reaction product of UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase (GalNAc-transferase) by scintillation proximity assay (SPA) was developed. The assay quantitates the radioactivity incorporated from 3H-labeled UDP-GalNAc into a biotin-labeled acceptor peptide, as measured after adsorption of the acceptor peptide to avidin-coated SPA beads. The acceptor peptide, PPASTSAPG (Elhammer et al. (1993) J. Biol. Chem. 268, 10029-10038) was conjugated to biotin using a di-beta-alanine spacer arm. The conjugated peptide reacted readily with the enzyme and it had an apparent Km comparable to that of the parent peptide. Using a reaction mixture consisting of 4 mg of SPA beads, 17 microM acceptor, 0.5 microM nucleotide sugar, and 7.5 U/ml enzyme, the time dependence of product formation obeyed Michaelis-Menten-type kinetics throughout the full course of the reaction-until exhaustion of the donor substrate-and the beginning portion of the reaction was sufficiently linear for calculating accurate initial rates. Analysis of the time dependency yielded an apparent Km of 0.38 +/- 0.12 microM for UDP-GalNAc. The assay is conveniently carried out in 96-well microtiter plates; it is ideally suited for assaying large numbers of samples and for screening large collections of chemicals for competitive inhibitors.
Assuntos
N-Acetilgalactosaminiltransferases/análise , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Cinética , Microesferas , Dados de Sequência Molecular , Peptídeos/metabolismo , Contagem de Cintilação , Estreptavidina , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
A heme-binding protein has been isolated and characterized from both the hemolymph and oocytes of the blood-sucking insect, Rhodnius prolixus. The protein from both sources is identical in most aspects studied. The Rhodnius heme-binding protein (RHBP) is composed of a single 15-kDa polypeptide chain coiled in a highly alpha-helical structure which binds non-covalently one heme/polypeptide chain. This RHBP is not produced by limited degradation of hemoglobin from the vertebrate host, since specific polyclonal antibodies against it do not cross-react with rabbit hemoglobin, and since it differs from hemoglobin in having a distinct amino-acid composition and NH2-terminal sequence. The spectrum of the dithionite-reduced protein has peaks at 426, 530, and 559 nm and resembles that of a b-type cytochrome. RHBP from hemolymph is not saturated with heme and promptly binds heme added to the solution. The oocyte protein, on the other hand, is fully saturated and is not capable of binding additional heme.
Assuntos
Proteínas de Transporte/isolamento & purificação , Hemeproteínas/isolamento & purificação , Hemolinfa/química , Rhodnius/química , Sequência de Aminoácidos , Aminoácidos/química , Animais , Western Blotting , Proteínas de Transporte/química , Feminino , Heme/metabolismo , Proteínas Ligantes de Grupo Heme , Hemeproteínas/química , Masculino , Dados de Sequência Molecular , Peso Molecular , Oócitos/química , Análise EspectralRESUMO
The specificity of UDP-GalNAc:polypeptide N-acetylgalactosaminytransferase (GalNAc-transferase) is consistent with the existence of an extended site composed of nine subsites, denoted by P4, P3, P2, P1, P0, P1', P2', P3', P4', where the acceptor at P0 is being either Ser or Thr. To predict whether a peptide will react with the enzyme to form a Ser- or Thr-conjugated glycopeptide, a vector projection method is proposed which uses a training set of amino acid sequences surrounding 90 Ser and 106 Thr O-glycosylation sites extracted from the National Biomedical Research Foundation Protein Database. The model postulates independent interactions of the 9 amino acid moieties with their respective binding sites. The high ratio of correct predictions vs. total predictions for the data in both the training and the testing sets indicates that the method is self-consistent and efficient. It provides a rapid means for predicting O-glycosylation and designing effective inhibitors of GalNAc-transferase.
Assuntos
N-Acetilgalactosaminiltransferases/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Glicosilação , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , N-Acetilgalactosaminiltransferases/química , N-Acetilgalactosaminiltransferases/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Serina/química , Treonina/químicaRESUMO
The tetracycline analogs minocycline and doxycycline are inhibitors of metalloproteinases (MMPs) and have been shown to inhibit angiogenesis in vivo. To further study the mechanism of action of these compounds we tested them in an in vitro model of angiogenesis: aortic sprouting in fibrin gels. Angiogenesis was quantitated in this system by a unique application of planar morphometry. Both compounds were found to potently inhibit angiogenesis in this model. To further characterize the activity of these compounds against MMPs, we determined the IC50S of both compounds against representatives of three classes of metalloproteinases: fibroblast collagenase, stromelysin, and gelatinase A. Doxycycline was found to inhibit collagenase, gelatinase A and stromelysin with IC50S of 452 microM, 56 microM and 32 microM, respectively. Minocycline was found to inhibit only stromelysin in the micromolar range with an IC50 of 290 microM. Since these results suggest that these compounds may not have been inhibiting in vitro angiogenesis by an MMP-dependent mechanism, we decided to test the effects of the potent MMP inhibitor BB-94. This compound failed to inhibit aortic sprouting in fibrin gels, thus strongly suggesting that both doxycycline and minocycline act by an MMP-independent mechanism. These results have implications for the mechanism of action of tetracycline analogs, particularly where they are being considered for the treatment of disorders of extracellular matrix degradation including periodontal disease, arthritis, and tumor angiogenesis.
Assuntos
Doxiciclina/farmacologia , Metaloendopeptidases/antagonistas & inibidores , Minociclina/farmacologia , Neovascularização Patológica/prevenção & controle , Animais , Aorta/efeitos dos fármacos , Gelatinases/antagonistas & inibidores , Masculino , Metaloproteinase 2 da Matriz , Metaloproteinase 3 da Matriz , Inibidores de Metaloproteinases de Matriz , Proteínas de Neoplasias/antagonistas & inibidores , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Ratos , Ratos Endogâmicos F344 , Tiofenos/farmacologiaRESUMO
FG glycoprotein is a recombinant chimeric protein consisting of the extracellular portions of human respiratory syncytial virus (RSV) F and G glycoproteins. In theory, highly purified FG glycoprotein may be effective as a RSV vaccine. Recombinant FG glycoprotein was expressed using the baculovirus/insect cell system. FG glycoprotein was isolated from cell culture supernatants using S Sepharose ion-exchange chromatography, Cu(2+)-immobilized metal affinity chromatography, preparative reversed-phase high-performance liquid chromatography, denaturation with 6 M guanidine hydrochloride, and protein refolding in Tween 80 detergent. The purified FG glycoprotein was concentrated on a S Sepharose column and exchanged into an appropriate buffer for vaccine formulation. Five batches of FG glycoprotein with protein purity of 92-99% were produced using this purification process. FG glycoprotein produced using reversed-phase chromatography and protein refolding was compared with nondenatured FG glycoprotein using a panel of 14 monoclonal antibodies directed against conformational and linear epitopes on RSV F and G glycoproteins. The results of these studies indicated that refolded FG glycoprotein had the same three-dimensional structure as nondenatured FG glycoprotein.
Assuntos
Proteína HN , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vírus Sincicial Respiratório Humano , Vacinas Sintéticas/isolamento & purificação , Proteínas Virais/isolamento & purificação , Vacinas Virais/isolamento & purificação , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Células Cultivadas , Cromatografia , Vetores Genéticos , Guanidina , Guanidinas , Camundongos , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes de Fusão/isolamento & purificação , Infecções por Vírus Respiratório Sincicial/imunologia , Spodoptera/citologia , Vacinas Sintéticas/genética , Proteínas do Envelope Viral , Proteínas Virais/genética , Vacinas Virais/genéticaRESUMO
The acceptor substrate specificity of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) was inferred from the amino acid sequences surrounding 196 O-glycosylation sites extracted from the National Biomedical Research Foundation Protein Database. When analyzed according to the cumulative enzyme specificity model (Poorman, R.A., Tomasselli, A.G., Heinrikson, R.L., and Kézdy, F.J. (1991) J. Biol. Chem. 266, 14554-14561) these data were found to be consistent with an enzymatic active site which interacts with an 8-amino-acid long segment of the substrate, spanning 3 amino acid residues preceding and 4 amino acid residues following the reactive serine or threonine. The model postulates independent interactions of the 8 amino acid moieties with their respective binding sites, designated as subsites P3 through P0 and P1' to P4'. High selectivity is expressed at all subsites toward serine, threonine, and proline. The inferred specificity was confirmed by in vitro bovine colostrum GalNAc-transferase-catalyzed glycosylation of unglycosylated proteins containing predicted sites for O-glycosylation and synthetic peptides designed to be GalNAc acceptors. In synthetic peptides the bovine colostrum GalNAc-transferase glycosylates threonine about 35 times faster than serine. Our results suggest that the specificity of the enzyme is not dependent on any particular secondary structure of the substrate but, rather, it is determined by the amino acids in the acceptor peptide segment as well as by the accessibility of this segment. It also appears likely that bovine colostrum GalNAc-transferase is able to catalyze in vivo the glycosylation of both threonine and serine residues.
Assuntos
Bases de Dados Factuais , N-Acetilgalactosaminiltransferases/metabolismo , Oligopeptídeos/metabolismo , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Colostro/enzimologia , Feminino , Glicosilação , Cinética , Dados de Sequência Molecular , N-Acetilgalactosaminiltransferases/isolamento & purificação , Oligopeptídeos/síntese química , Probabilidade , Relação Estrutura-Atividade , Especificidade por Substrato , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
We compared the receptor binding, antigenicity, biological activation, and cell-mediated proteolytic degradation properties of mouse nerve growth factor (mNGF) and human NGF (hNGF). The affinity of hNGF toward human NGF-receptor is greater than that of mNGF, but the affinity of mNGF toward rat NGF-receptor is greater than that of hNGF. Thus, the specificity of the interaction between NGF and its receptor resides both on the NGF and on its receptor. Using a group of anti-NGF monoclonal antibodies that competitively inhibit the binding of NGF to receptor, sites differing between mNGF and hNGF were detected. Together, these results indicate that the sites on hNGF and mNGF, responsible for binding to NGF-receptor, are similar but not identical. In comparing the relative abilities of mNGF and hNGF to stimulate a biological response in PC12 cells, we observed that mNGF was better at stimulating neurite outgrowth than was hNGF, consistent with the differences observed for receptor binding affinity. However, the ED50 for biological activation is approximately 100-fold lower than the Kd for receptor occupancy, and, thus, the dose-response curve is not consistent with a simple activation proportional to receptor occupancy. The data are consistent with a model requiring a low-level threshold occupancy of NGF-receptor (Kd = 10(-9) M) in order to stimulate full biological activity. Finally, we observed the degradation of NGF by PC12 cells. We found that the NGF molecule is significantly degraded via a receptor-mediated uptake mechanism. Together, the data provide insight into regions of the NGF molecule involved in contacts with the receptor leading to formation of the NGF:NGF-receptor complex. Additionally, they establish the link between occupancy of receptor and biological activation and the requirement for receptor-mediated uptake in order to degrade NGF proteolytically in cultured PC12 cells.
Assuntos
Fatores de Crescimento Neural/fisiologia , Animais , Anticorpos Monoclonais/imunologia , Sítios de Ligação , Ligação Competitiva , Humanos , Cinética , Camundongos , Microssomos/metabolismo , Fatores de Crescimento Neural/imunologia , Fatores de Crescimento Neural/metabolismo , Células PC12 , Receptores de Superfície Celular/metabolismo , Receptores de Fator de Crescimento Neural , Relação Estrutura-AtividadeRESUMO
Statistical analysis of an expanded data base of regions in viral polyproteins and in non-viral proteins that are sensitive to hydrolysis by the protease from human immunodeficiency virus (HIV) type 1 has generated a model which characterizes the substrate specificity of this retroviral enzyme. The model leads to an algorithm for predicting protease-susceptible sites from primary structure. Amino acids in each of the sites from P4 to P4' are tabulated for 40 protein substrates, and the frequency of occurrence for each residue is compared to the natural abundance of that amino acid in a selected data set of globular proteins. The results suggest that the highest stringency for particular amino acid residues is at the P2, P1, and P2' positions of the substrate. The broad specificity of the HIV-1 protease appears to be a consequence of its being able to bind productively substrates in which interactions with only a few Pi or Pi' side-chains need be optimized. The analysis, extended to 22 protein segments cleaved by the HIV-2 protease, delineates marked differences in specificity from that of the HIV-1 enzyme.
Assuntos
Protease de HIV/metabolismo , HIV-1/enzimologia , HIV-2/enzimologia , Actinas/metabolismo , Algoritmos , Sequência de Aminoácidos , Sítios de Ligação , Calmodulina/metabolismo , Sistemas de Informação , Modelos Biológicos , Dados de Sequência Molecular , Especificidade por SubstratoRESUMO
High affinity interactions were studied between the basement membrane form of heparan sulfate proteoglycan (HSPG) and the 695-, 751-, and 770-amino acid Alzheimer amyloid precursor (AAP) proteins. Based on quantitative analyses of binding data, we identified single binding sites for the HSPG on AAP-695 (Kd = 9 x 10(-10) M), AAP-751 (Kd = 10 x 10(-9) M), and AAP-770 (Kd = 9 x 10(-9) M). It is postulated that the "Kunitz" protease inhibitor domain which is present in AAP-751 and -770 reduces the affinity of AAPs for the HSPG through steric hindrance and/or conformational alteration. HSPG binding was inhibited by heparin and dextran sulfate, but not by dermatan or chondroitin sulfate. HSPG protein core, obtained by heparitinase digestion, also bound to the beta-amyloid precursor proteins with high affinity, indicating that the high affinity binding site is constituted by the polypeptide chain rather than the carbohydrate moiety. The effects of various cations on these interactions were also studied. Our results suggest that specific interactions between the AAP proteins and the extracellular matrix may be involved in the nucleation stages of Alzheimer's disease type amyloidogenesis.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Heparitina Sulfato/metabolismo , Inibidores de Proteases/metabolismo , Precursores de Proteínas/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide , Membrana Basal/metabolismo , Sítios de Ligação , Proteoglicanas de Sulfatos de Condroitina/isolamento & purificação , Fibronectinas/metabolismo , Proteoglicanas de Heparan Sulfato , Heparitina Sulfato/isolamento & purificação , Humanos , Cinética , Ligação Proteica , Precursores de Proteínas/genética , Proteínas Recombinantes/metabolismoRESUMO
Monoclonal antibodies were raised against a synthetic peptide (43 amino acid residues) that corresponds to the complete profragment of human prorenin. Seven monoclonal antibodies were chosen for further characterization. Two antibodies, 2-X-C1 and 4-X-E1, reacted with the middle region and C-terminus of the profragment and were isotyped IgG1. The affinity constants of these antibodies against the human profragment were 7.6 x 10(8) and 3.0 x 10(7) M-1, respectively. Immunoaffinity columns containing the antibodies 2-X-C1 and 4-X-E1, respectively, were used for the characterization of active prorenin in human plasma. This active prorenin strongly bound to the 4-X-E1 column and eluted as two separate peaks which corresponded to fully and partially active prorenin, respectively. The partially active prorenin had higher activity with a small substrate, tridecapeptide, than with a large one, angiotensinogen, although the fully active prorenin had the same renin activity irrespective of the size of the substrate. These data suggest that new forms of prorenin, active prorenin, exist in human plasma and that their active sites are completely or partially exposed to the substrates. Moreover, the active prorenin in plasma was found not only in human but also in all tested mammalians. Cross-reactivity among the profragments of mammalian plasma prorenins can be explained by conservation of the amino acid sequence (epitope) of the combining site.
Assuntos
Anticorpos Monoclonais/imunologia , Precursores Enzimáticos/imunologia , Renina/imunologia , Afinidade de Anticorpos , Sítios de Ligação de Anticorpos , Reações Cruzadas , Precursores Enzimáticos/sangue , Humanos , Immunoblotting , Mieloma Múltiplo/imunologia , Fragmentos de Peptídeos/imunologia , Renina/sangueRESUMO
The reactivity of the 16 thiol groups of rabbit skeletal muscle phosphofructokinase has been studied extensively over the past 20 years. Several of these thiols show high reactivity with a variety of reagents, display differential reactivity in the presence of allosteric ligands and substrates, and appear to be important to function because their modification changes activity and regulatory properties. In the present study, the location in the primary structure of several highly reactive thiol groups has been established by reaction with [14C]iodoacetate. In the course of these studies, 2 methionyl residues that are located at or near proposed ligand-binding sites are readily carboxymethylated by iodoacetate. In addition to confirming the presence of the most reactive thiol group at sequence position 88, a thiol protected from reaction by the presence of fructose-6-P and cyclic AMP has been found at position 169. Cysteine 169 is close to a residue important to the binding of fructose-6-P in the homologous structure from Bacillus stearothermophilis phosphofructokinase. The modification of Cys-169 brings about extensive, but not total, loss of activity. Another cysteine, at position 232, was found to be highly reactive also. Substrate provided partial protection against carboxymethylation at this position. Carboxymethylation of enzyme restricted to methionines 74 and 173 brought about no changes in the total activity or in the ATP inhibition profile of the enzyme. This is significant since position 74 was projected on the basis of the homologous procaryotic structure to be important in the binding of nucleotide to the allosteric site.
Assuntos
Cisteína , Metionina , Músculos/enzimologia , Fosfofrutoquinase-1/metabolismo , Sítio Alostérico , Sequência de Aminoácidos , Animais , Sítios de Ligação , Fenômenos Químicos , Química , AMP Cíclico/farmacologia , Escherichia coli/enzimologia , Frutosefosfatos/metabolismo , Geobacillus stearothermophilus/enzimologia , Iodoacetatos , Ácido Iodoacético , Fragmentos de Peptídeos/metabolismo , Coelhos , Relação Estrutura-Atividade , TripsinaRESUMO
Phosphorylation of rabbit skeletal muscle phosphofructokinase by the catalytic subunit of cyclic AMP-dependent protein kinase occurs with a Km of about 230 microM and Vmax approaching that seen with histone as a substrate. The rate of phosphorylation of phosphofructokinase by protein kinase is increased by allosteric activators of phosphofructokinase, whereas inhibitors of phosphofructokinase inhibit the phosphorylation. Inhibitors and activators change Vmax but not Km. The site of phosphorylation is a serine residue that is the sixth amino acid from the carboxyl terminus. Limited proteolysis by trypsin releases an octapeptide from the carboxyl terminus and a brief exposure to subtilisin releases a dodecapeptide from the carboxyl end. The sequence of the dodecapeptide is His-Ile-Ser-Arg-Lys-Arg-Ser(P)-Gly-Glu-Ala-Thr-Val. Phosphofructokinase isolated from a rabbit injected 18 h prior to killing with [32P]PO4 contained covalently bound radioactive phosphate. Approximately 80% of the phosphate was released in a trichloroacetic acid-soluble form following limited proteolysis by trypsin, under which conditions the enzyme remained with a monomer size of about 80,000 daltons. The position of elution from Sephadex G-25 of the phosphopeptide was identical with that found following limited trypsin proteolysis of in vitro labeled enzyme. Migration of the phosphopeptides on thin layer cellulose chromatography was also identical. We conclude that at least 80% of the radioactive phosphate introduced within 18 h of an intravenous injection of [32P]PO4 is found at the same site as that introduced by phosphorylation with the catalytic subunit of cyclic AMP-dependent protein kinase.