RESUMEN
The transcription factor T-bet (Tbox protein expressed in T cells) is one of the master regulators of both the innate and adaptive immune responses. It plays a central role in T-cell lineage commitment, where it controls the TH1 response, and in gene regulation in plasma B-cells and dendritic cells. T-bet is a member of the Tbox family of transcription factors; however, T-bet coordinately regulates the expression of many more genes than other Tbox proteins. A central unresolved question is how T-bet is able to simultaneously recognize distant Tbox binding sites, which may be located thousands of base pairs away. We have determined the crystal structure of the Tbox DNA binding domain (DBD) of T-bet in complex with a palindromic DNA. The structure shows a quaternary structure in which the T-bet dimer has its DNA binding regions splayed far apart, making it impossible for a single dimer to bind both sites of the DNA palindrome. In contrast to most other Tbox proteins, a single T-bet DBD dimer binds simultaneously to identical half-sites on two independent DNA. A fluorescence-based assay confirms that T-bet dimers are able to bring two independent DNA molecules into close juxtaposition. Furthermore, chromosome conformation capture assays confirm that T-bet functions in the direct formation of chromatin loops in vitro and in vivo. The data are consistent with a looping/synapsing model for transcriptional regulation by T-bet in which a single dimer of the transcription factor can recognize and coalesce distinct genetic elements, either a promoter plus a distant regulatory element, or promoters on two different genes.
Asunto(s)
Cromatina/química , ADN/química , Genoma , Proteínas de Dominio T Box/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cromatina/metabolismo , Cristalografía por Rayos X , ADN/genética , ADN/metabolismo , Elementos de Facilitación Genéticos , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Secuencias Invertidas Repetidas , Ratones , Modelos Moleculares , Regiones Promotoras Genéticas , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Xenopus laevisRESUMEN
The FIKK family of kinases is unique to parasites of the Apicomplexan order, which includes all malaria parasites. Plasmodium falciparum, the most virulent form of human malaria, has a family of 19 FIKK kinases, most of which are exported into the host red blood cell during malaria infection. Here, we confirm that FIKK 8 is a non-exported member of the FIKK kinase family. Through expression and purification of the recombinant kinase domain, we establish that emodin is a relatively high-affinity (IC50=2µM) inhibitor of PfFk8. Closely related anthraquinones do not inhibit PfFk8, suggesting that the particular substitution pattern of emodin is critical to the inhibitory pharmacophore. This first report of a P. falciparum FIKK kinase inhibitor lays the groundwork for developing specific inhibitors of the various members of the FIKK kinase family in order to probe their physiological function.
Asunto(s)
Emodina/química , Emodina/farmacología , Plasmodium falciparum/enzimología , Proteínas Quinasas/química , Proteínas Quinasas/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Secuencia de Aminoácidos , Antraquinonas/química , Emodina/metabolismo , Activación Enzimática/efectos de los fármacos , Eritrocitos/metabolismo , Eritrocitos/parasitología , Humanos , Concentración 50 Inhibidora , Microscopía Fluorescente , Plasmodium falciparum/efectos de los fármacos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismoRESUMEN
Mycobacterium tuberculosis (Mtb) employs multiple strategies to evade host immune responses and persist within macrophages. We have previously shown that the cell envelope-associated Mtb serine hydrolase, Hip1, prevents robust macrophage activation and dampens host pro-inflammatory responses, allowing Mtb to delay immune detection and accelerate disease progression. We now provide key mechanistic insights into the molecular and biochemical basis of Hip1 function. We establish that Hip1 is a serine protease with activity against protein and peptide substrates. Further, we show that the Mtb GroEL2 protein is a direct substrate of Hip1 protease activity. Cleavage of GroEL2 is specifically inhibited by serine protease inhibitors. We mapped the cleavage site within the N-terminus of GroEL2 and confirmed that this site is required for proteolysis of GroEL2 during Mtb growth. Interestingly, we discovered that Hip1-mediated cleavage of GroEL2 converts the protein from a multimeric to a monomeric form. Moreover, ectopic expression of cleaved GroEL2 monomers into the hip1 mutant complemented the hyperinflammatory phenotype of the hip1 mutant and restored wild type levels of cytokine responses in infected macrophages. Our studies point to Hip1-dependent proteolysis as a novel regulatory mechanism that helps Mtb respond rapidly to changing host immune environments during infection. These findings position Hip1 as an attractive target for inhibition for developing immunomodulatory therapeutics against Mtb.
Asunto(s)
Proteínas Bacterianas/fisiología , Chaperonina 60/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Mycobacterium tuberculosis/enzimología , Serina Endopeptidasas/fisiología , Serina Proteasas/fisiología , Animales , Proteínas Bacterianas/metabolismo , Células Cultivadas , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Activación de Macrófagos , Ratones , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/inmunología , Unión Proteica , Multimerización de Proteína , Proteolisis , Serina Endopeptidasas/metabolismo , Serina Proteasas/metabolismoRESUMEN
Colleges and universities are learning to provide relevant virtual lab experiences for students due to the COVID-19 pandemic. Even schools attempting in-person instruction often need to utilize virtual experiences for students absent due to quarantine or illness. Much of biochemistry is amenable to molecular visualization and/or computational study; however, many faculty face learning how to utilize new computational and molecular visualization software. We present a set of virtual lab exercises with detailed instructions to engage students in the discovery of novel antiviral compounds against the SARS-CoV-2 main protease.
Asunto(s)
Bioquímica/economía , COVID-19 , Biología Computacional/educación , Diseño de Fármacos , Educación a Distancia , Pandemias , SARS-CoV-2 , HumanosRESUMEN
Corals are vulnerable to increasing ocean temperatures. It is known that elevated temperatures lead to the breakdown of an essential mutualistic relationship with photosynthetic algae. The molecular mechanisms of this temperature-dependent loss of symbiosis are less well understood. Here, the thermal stability of a critical metabolic enzyme, glyceraldehyde-3-phosphate dehydrogenase, from the stony coral Acropora millepora was found to increase significantly in the presence of its cofactor NAD+. Determination of the structure of the cofactor-enzyme complex (PDB ID 6PX2) revealed variable NAD+ occupancy across the four monomers of the tetrameric enzyme. The structure of the fully occupied monomers was compared to those with partial cofactor occupancy, identifying regions of difference that may account for the increased thermal stability.
RESUMEN
Benzaldehyde lyase (BAL) from Pseudomonas putida is a thiamin diphosphate (ThDP)-dependent enzyme that catalyzes the breakdown of (R)-benzoin. Here we report that a point mutant, BAL A28S, not only catalyzes the decarboxylation of benzoylformate but, like benzoylformate decarboxylase (BFDC), is also inactivated by the benzoylformate analogues methyl benzoylphosphonate (MBP) and benzoylphosphonate (BP). The latter has no effect on wild-type BAL, and the inactivation of the A28S variant is shown to result from phosphorylation of the newly introduced serine residue. This lends support to the proposal that an appropriately placed nucleophile facilitates the expulsion of carbon dioxide from the active site in many ThDP-dependent decarboxylases.
Asunto(s)
Aldehído-Liasas/metabolismo , Mutación Puntual , Ingeniería de Proteínas , Aldehído-Liasas/antagonistas & inhibidores , Aldehído-Liasas/química , Dominio Catalítico , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Modelos Moleculares , Organofosfonatos/química , Organofosfonatos/farmacología , Relación Estructura-ActividadRESUMEN
The mechanism of the enzyme benzoylformate decarboxylase (BFDC), which carries out a typical thiamin diphosphate (ThDP)-dependent nonoxidative decarboxylation reaction, was studied with the chromophoric alternate substrate (E)-2-oxo-4(pyridin-3-yl)-3-butenoic acid (3-PKB). Addition of 3-PKB resulted in the appearance of two transient intermediates formed consecutively, the first one to be formed a predecarboxylation ThDP-bound intermediate with lambda(max) at 477 nm, and the second one corresponding to the first postdecarboxylation intermediate the enamine with lambda(max) at 437 nm. The time course of formation/depletion of the PKB-ThDP covalent complex and of the enamine showed that decarboxylation was slower than formation of the PKB-ThDP covalent adduct. When the product of decarboxylation 3-(pyridin-3-yl)acrylaldehyde (PAA) was added to BFDC, again an absorbance with lambda(max) at 473 nm was formed, corresponding to the tetrahedral adduct of PAA with ThDP. Addition of well-formed crystals of BFDC to a solution of PAA resulted in a high resolution (1.34 A) structure of the BFDC-bound adduct of ThDP with PAA confirming the tetrahedral nature at the C2alpha atom, rather than of the enamine, and supporting the assignment of the lambda(max) at 473 nm to the PAA-ThDP adduct. The structure of the PAA-ThDP covalent complex is the first example of a product-ThDP adduct on BFDC. Similar studies with 3-PKB indicated that decarboxylation had taken place. Evidence was also obtained for the slow formation of the enamine intermediate when BFDC was incubated with benzaldehyde, the product of the decarboxylation reaction thus confirming its presence on the reaction pathway.
Asunto(s)
Butiratos/química , Carboxiliasas/química , Pseudomonas putida/enzimología , Piridinas/química , Tiamina Pirofosfato/química , Sitios de Unión , Butiratos/metabolismo , Carboxiliasas/aislamiento & purificación , Carboxiliasas/metabolismo , Catálisis , Cristalografía por Rayos X , Descarboxilación , Colorantes Fluorescentes/química , Cinética , Unión Proteica , Piridinas/metabolismo , Especificidad por Sustrato , Tiamina Pirofosfato/metabolismo , Factores de TiempoRESUMEN
Benzoylformate decarboxylase (BFDC) is a thiamin diphosphate- (ThDP-) dependent enzyme acting on aromatic substrates. In addition to its metabolic role in the mandelate pathway, BFDC shows broad substrate specificity coupled with tight stereo control in the carbon-carbon bond-forming reverse reaction, making it a useful biocatalyst for the production of chiral alpha-hydroxy ketones. The reaction of methyl benzoylphosphonate (MBP), an analogue of the natural substrate benzoylformate, with BFDC results in the formation of a stable analogue (C2alpha-phosphonomandelyl-ThDP) of the covalent ThDP-substrate adduct C2alpha-mandelyl-ThDP. Formation of the stable adduct is confirmed both by formation of a circular dichroism band characteristic of the 1',4'-iminopyrimidine tautomeric form of ThDP (commonly observed when ThDP forms tetrahedral complexes with its substrates) and by high-resolution mass spectrometry of the reaction mixture. In addition, the structure of BFDC with the MBP inhibitor was solved by X-ray crystallography to a spatial resolution of 1.37 A (PDB ID 3FSJ). The electron density clearly shows formation of a tetrahedral adduct between the C2 atom of ThDP and the carbonyl carbon atom of the MBP. This adduct resembles the intermediate from the penultimate step of the carboligation reaction between benzaldehyde and acetaldehyde. The combination of real-time kinetic information via stopped-flow circular dichroism with steady-state data from equilibrium circular dichroism measurements and X-ray crystallography reveals details of the first step of the reaction catalyzed by BFDC. The MBP-ThDP adduct on BFDC is compared to the recently solved structure of the same adduct on benzaldehyde lyase, another ThDP-dependent enzyme capable of catalyzing aldehyde condensation with high stereospecificity.
Asunto(s)
Carboxiliasas/antagonistas & inhibidores , Carboxiliasas/química , Inhibidores Enzimáticos/química , Organofosfonatos/química , Aldehído-Liasas/química , Aldehído-Liasas/metabolismo , Unión Competitiva , Biocatálisis , Carboxiliasas/metabolismo , Dicroismo Circular , Cristalografía por Rayos X , Inhibidores Enzimáticos/metabolismo , Cinética , Organofosfonatos/metabolismo , Estereoisomerismo , Especificidad por SustratoRESUMEN
The increasing rate of autoimmune disorders and cancer in recent years has been a controversial issue in all aspects of prevention, diagnosis, prognosis and treatment. Among dietary factors, flavonoids have specific immunomodulatory effects that might be of importance to several cancers. Over different types of immune cells, T lymphocytes play a critical role in protecting the immune system as well as in the pathogenesis of specific autoimmune diseases. One of the important mediators of metabolism and immune system is mTOR, especially in T lymphocytes. In the current review, we assessed the effects of flavonoids on the immune system and then their impact on the mTOR pathway. Flavonoids can suppress mTOR activity and are consequently able to induce the T regulatory subset.
Asunto(s)
Flavonoides/farmacología , Factores Inmunológicos/farmacología , Inmunomodulación/efectos de los fármacos , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Metabolismo Energético/efectos de los fármacos , Humanos , Fenómenos del Sistema Inmunológico/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Benzaldehyde lyase (BAL) catalyzes the reversible cleavage of ( R)-benzoin to benzaldehyde utilizing thiamin diphosphate and Mg (2+) as cofactors. The enzyme is important for the chemoenzymatic synthesis of a wide range of compounds via its carboligation reaction mechanism. In addition to its principal functions, BAL can slowly decarboxylate aromatic amino acids such as benzoylformic acid. It is also intriguing mechanistically due to the paucity of acid-base residues at the active center that can participate in proton transfer steps thought to be necessary for these types of reactions. Here methyl benzoylphosphonate, an excellent electrostatic analogue of benzoylformic acid, is used to probe the mechanism of benzaldehyde lyase. The structure of benzaldehyde lyase in its covalent complex with methyl benzoylphosphonate was determined to 2.49 A (Protein Data Bank entry 3D7K ) and represents the first structure of this enzyme with a compound bound in the active site. No large structural reorganization was detected compared to the complex of the enzyme with thiamin diphosphate. The configuration of the predecarboxylation thiamin-bound intermediate was clarified by the structure. Both spectroscopic and X-ray structural studies are consistent with inhibition resulting from the binding of MBP to the thiamin diphosphate in the active centers. We also delineated the role of His29 (the sole potential acid-base catalyst in the active site other than the highly conserved Glu50) and Trp163 in cofactor activation and catalysis by benzaldehyde lyase.
Asunto(s)
Aldehído-Liasas/química , Aldehído-Liasas/metabolismo , Benzaldehídos/química , Benzaldehídos/metabolismo , Benzoína/química , Benzoína/metabolismo , Sitios de Unión , Dicroismo Circular , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Especificidad por Sustrato , Tiamina Pirofosfato/química , Tiamina Pirofosfato/metabolismoRESUMEN
The development of antimalarial drugs remains a public health priority, and the orotidine 5'-monophosphate decarboxylase from Plasmodium falciparum (PfOMPDC) has great potential as a drug target. The crystallization of PfOMPDC with substrate bound represents an important advance for structure-based drug-design efforts [Tokuoka et al. (2008), J. Biochem. 143, 69-78]. The complex of the enzyme bound to the substrate OMP (PDB entry 2za1) would be of particular utility in this regard. However, re-refinement of this structure of the Michaelis complex shows that the bound ligand is the product rather than the substrate. Here, the re-refinement of a set of three structures, the apo enzyme and two versions of the product-bound form (PDB entries 2za1, 2za2 and 2za3), is reported. The improved geometry and fit of these structures to the observed electron density will enhance their utility in antimalarial drug design.
Asunto(s)
Orotidina-5'-Fosfato Descarboxilasa/química , Plasmodium falciparum/química , Proteínas Protozoarias/química , Uridina Monofosfato/análogos & derivados , Uridina Monofosfato/química , Antimaláricos/química , Sitios de Unión , Ligandos , Modelos Moleculares , Orotidina-5'-Fosfato Descarboxilasa/metabolismo , Plasmodium falciparum/enzimología , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Protozoarias/metabolismo , Especificidad por Sustrato , Uridina Monofosfato/metabolismoRESUMEN
A relatively high-affinity inhibitor of FIKK kinase from the malaria parasite Plasmodium vivax was identified by in vitro assay of recombinant kinase. The FIKK kinase family is unique to parasitic organisms of the Apicomplexan order and has been shown to be critical in malaria parasites. The recombinant kinase domain was expressed and screened against a small molecule library, revealing a number of tyrosine kinase inhibitors that block FIKK kinase activity. A family of tyrphostins was further investigated, to begin exploring the FIKK kinase pharmacophore. Finally, emodin was identified as a relatively high-affinity FIKK kinase inhibitor, identifying this family of anthraquinones as potential lead compounds for the development of antimalarials targeting the FIKK kinase.
RESUMEN
The caging of specific residues of proteins is a powerful tool. This discussion attempts to alert the reader to the considerations that must be made in preparing and analyzing a caged protein through nonsense suppression. Although the suppression methodology is conceptually straightforward, it not possible to provide a failsafe "cook book" method for using caged unnaturals. We have emphasized the preparation of caged receptors expressed in Xenopus oocytes, but these approaches can clearly be adapted to many other systems.
Asunto(s)
Aminoácidos/química , Aminoácidos/genética , Animales , Codón de Terminación , Mutagénesis , ARN Mensajero/genética , Transcripción Genética , XenopusRESUMEN
P. falciparum causes the most deadly form of malaria, resulting from the adherence of infected red blood cells to blood vessels. During the blood stage of infection, the parasite secretes a large number of proteins into the host erythrocyte. The secretion of a 20-member family of protein kinases known as FIKK kinases, after a conserved Phe-Ile-Lys-Lys sequence motif, is unique to P. falciparum. Identification of physiological substrates of these kinases may provide perspective on the importance of FIKK kinase activity to P. falciparum virulence. We demonstrate, for the first time, the heterologous expression and purification of a FIKK kinase (PfFk4.1, PFD1165w). The recombinant kinase is active against general substrates and phosphorylates itself. Having demonstrated kinase activity, we incubated recombinant Fk4.1 with parasite and human erythrocyte lysates. No parasite-derived substrates were identified. However, treatment of erythrocyte ghosts shows that the FIKK kinase Fk4.1 phosphorylates dematin, a cytoskeletal protein found at the red blood cell spectrin-actin junction.
Asunto(s)
Eritrocitos/parasitología , Proteínas de Microfilamentos/metabolismo , Fosfotransferasas/metabolismo , Plasmodium falciparum/enzimología , Plasmodium falciparum/metabolismo , Procesamiento Proteico-Postraduccional , Clonación Molecular , Expresión Génica , Humanos , Fosforilación , Fosfotransferasas/genética , Fosfotransferasas/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Accumulation of misfolded protein in the endoplasmic reticulum (ER) triggers an adaptive stress response-termed the unfolded protein response (UPR)-mediated by the ER transmembrane protein kinase and endoribonuclease inositol-requiring enzyme-1alpha (IRE1alpha). We investigated UPR signaling events in mice in the absence of the proapoptotic BCL-2 family members BAX and BAK [double knockout (DKO)]. DKO mice responded abnormally to tunicamycin-induced ER stress in the liver, with extensive tissue damage and decreased expression of the IRE1 substrate X-box-binding protein 1 and its target genes. ER-stressed DKO cells showed deficient IRE1alpha signaling. BAX and BAK formed a protein complex with the cytosolic domain of IRE1alpha that was essential for IRE1alpha activation. Thus, BAX and BAK function at the ER membrane to activate IRE1alpha signaling and to provide a physical link between members of the core apoptotic pathway and the UPR.
Asunto(s)
Apoptosis , Retículo Endoplásmico/metabolismo , Endorribonucleasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Animales , Proteínas de Unión al ADN/metabolismo , Retículo Endoplásmico/ultraestructura , Chaperón BiP del Retículo Endoplásmico , Regulación de la Expresión Génica , Proteínas de Choque Térmico/metabolismo , Humanos , Riñón/citología , Riñón/efectos de los fármacos , Riñón/metabolismo , Hígado/citología , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Fosforilación , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Recombinantes/metabolismo , Factores de Transcripción del Factor Regulador X , Transducción de Señal , Factor de Transcripción CHOP/metabolismo , Factores de Transcripción , Tunicamicina/farmacología , Proteína Destructora del Antagonista Homólogo bcl-2/química , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína X Asociada a bcl-2/genética , eIF-2 Quinasa/metabolismoRESUMEN
We present the chemical and biological synthesis of caged phosphoproteins using the in vitro nonsense codon suppression methodology. Specifically, phosphoamino acid analogues of serine, threonine, and tyrosine with a single photocleavable o-nitrophenylethyl caging group were synthesized as the amino acyl tRNA adducts for insertion into full-length proteins. For this purpose, a novel phosphitylating agent was developed. The successful incorporation of these bulky and charged amino acids into the alpha-subunit of the nicotinic acetyl choline receptor (nAChR) and the vasodilator-stimulated phosphoprotein (VASP) using an in vitro translation system is reported.
Asunto(s)
Fosfoproteínas/biosíntesis , Fosfoproteínas/síntesis química , Serina/análogos & derivados , Treonina/análogos & derivados , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfoproteínas/química , ARN de Transferencia Aminoácido-Específico/química , ARN de Transferencia Aminoácido-Específico/metabolismo , Serina/metabolismo , Treonina/metabolismoRESUMEN
A series of tryptophan analogues has been introduced into the binding site regions of two ion channels, the ligand-gated nicotinic acetylcholine and serotonin 5-HT(3A) receptors, using unnatural amino acid mutagenesis and heterologous expression in Xenopus oocytes. A cation-pi interaction between serotonin and Trp183 of the serotonin channel 5-HT(3A)R is identified for the first time, precisely locating the ligand-binding site of this receptor. The energetic contribution of the observed cation-pi interaction between a tryptophan and the primary ammonium ion of serotonin is estimated to be approximately 4 kcal/mol, while the comparable interaction with the quaternary ammonium of acetylcholine is approximately 2 kcal/mol. The binding mode of nicotine to the nicotinic receptor of mouse muscle is examined by the same technique and found to differ significantly from that of the natural agonist, acetylcholine.