RESUMEN
A novel human plasma protein has been identified as a universal component of complement deposits, when complement is detected immunohistochemically in vivo. The protein is homologous to complement factor H and related proteins and has been designated factor H-related protein 5 (FHR-5). FHR-5 was identified by a monoclonal antibody raised using pathologic human glomerular preparations as the immunogen. FHR-5 was purified by affinity chromatography from complement-lysed erythrocytes, and the peptide sequence was obtained. The cDNA was cloned from a human liver library, and FHR-5 was deduced to be a protein containing 551 amino acids organized into nine short consensus repeat motifs. The short consensus repeats of FHR-5 show homology to Factor H and to other Factor H-related proteins, with some unique features demonstrated. Recombinant FHR-5, expressed in insect cells, was shown to bind C3b in vitro. The strong association of FHR-5 with tissue complement deposits in vivo suggests that this additional member of the Factor H family of proteins has a function in complement regulation.
Asunto(s)
Proteínas Sanguíneas/análisis , Factor H de Complemento , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Apolipoproteínas/análisis , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Northern Blotting , Western Blotting , Clonación Molecular , Factor H de Complemento/metabolismo , Datos de Secuencia Molecular , Conejos , Proteínas Recombinantes/análisisRESUMEN
BACKGROUND: Heart failure is a common cause of hospitalization and death across the industrialized world. Improving the diagnosis and care of patients with heart failure is therefore likely to have a major impact on morbidity, mortality, and health care costs. METHODS AND RESULTS: To determine the relation between cardiac function and plasma levels of amino-terminal brain natriuretic peptide precursor (NT-proBNP), plasma NT-proBNP levels and ventricular function (by radionucleotide ventriculography) were measured in healthy patients, patients with renal failure, patients with recent myocardial infarction, and patients investigated for cardiorespiratory symptoms. Plasma NT-proBNP levels were greater in healthy women (median, 1.5 fmol/mL; range, 1.0 to 13.8 fmol/mL; n = 34) than healthy men (median, 1.0 fmol/mL; range, 1.0 to 3.3 fmol/mL; n = 33; P = .012). NT-proBNP levels were elevated in subjects with renal failure (geometric mean, 314 fmol/mL; range, 18 to 5,800 fmol/mL) and were related to left ventricular ejection fraction (LVEF) (r = -0.86; P < .0001; n = 19). NT-proBNP levels were also related to LVEF in patients with recent myocardial infarction (r = -0.62; P = .0003; n = 29) and those investigated for cardiorespiratory symptoms (r = -0.56; P < .0001; n = 129). Applying an upper limit of normal of 5 fmol/mL for men and 15 fmol/mL for women (specificity, 100%), elevated plasma NT-proBNP levels had 100% sensitivity for the detection of LVEF less than 45% after myocardial infarction and 97% sensitivity for the detection of LVEF less than 45% in patients investigated for cardiorespiratory symptoms. NT-proBNP levels were also elevated in 87% of the patients with normal systolic function (LVEF > or = 45%) after myocardial infarction and in 87% of the patients investigated for cardiorespiratory symptoms with heart failure and normal systolic function (LVEF > or = 45%). CONCLUSIONS: Plasma NT-proBNP level is a sensitive indicator of cardiac dysfunction, both in the presence and absence of systolic dysfunction, and may prove to be a useful tool for the identification and management of cardiac dysfunction in the general community.
Asunto(s)
Insuficiencia Cardíaca/diagnóstico , Infarto del Miocardio/diagnóstico , Proteínas del Tejido Nervioso/sangre , Fragmentos de Péptidos/sangre , Disfunción Ventricular Izquierda/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Femenino , Insuficiencia Cardíaca/sangre , Insuficiencia Cardíaca/fisiopatología , Humanos , Fallo Renal Crónico/sangre , Fallo Renal Crónico/complicaciones , Masculino , Persona de Mediana Edad , Infarto del Miocardio/sangre , Infarto del Miocardio/fisiopatología , Péptido Natriurético Encefálico , Pronóstico , Radioinmunoensayo , Ventriculografía con Radionúclidos , Sensibilidad y Especificidad , Volumen Sistólico , Disfunción Ventricular Izquierda/sangre , Disfunción Ventricular Izquierda/etiología , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
Dun1p and Rad53p of the budding yeast Saccharomyces cerevisiae are members of a conserved family of cell cycle checkpoint protein kinases that contain forkhead-associated (FHA) domains. Here, we demonstrate that these FHA domains contain 130-140 residues, and are thus considerably larger than previously predicted by sequence comparisons (55-75 residues). In vivo, expression of the proteolytically defined Dun1p FHA domain, but not a fragment containing only the predicted domain boundaries, inhibited the transcriptional induction of repair genes following replication blocks. This indicates that the non-catalytic FHA domain plays an important role in the transcriptional function of the Dun1p protein kinase.
Asunto(s)
Dominio Catalítico , Proteínas de Ciclo Celular , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Dominio Catalítico/genética , Dominio Catalítico/fisiología , Quinasa de Punto de Control 2 , Quimotripsina/metabolismo , Replicación del ADN/efectos de los fármacos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Genes Fúngicos/genética , Hidroxiurea/farmacología , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Proteínas Quinasas/genética , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Alineación de Secuencia , Serina Endopeptidasas/metabolismo , Transcripción Genética/genéticaRESUMEN
A heterotrimeric member of the AMP-activated protein kinase (AMPK) isoenzyme family was purified from rat skeletal muscle by immunoaffinity chromatography, consisting of an alpha2 catalytic and two non-catalytic subunits, beta2 and gamma1. The AMPK beta2 cDNA (271 amino acids (aa), molecular weight (MW)=30¿ omitted¿307, pI 6. 3) was cloned from skeletal muscle and found to share an overall identity of 70% with beta1 (270 aa, MW=30¿ omitted¿475, pI 6.0). In the liver AMPK beta1 subunit, Ser-182 is constitutively phosphorylated whereas in skeletal muscle beta2 isoform, we find that Ser-182 is only partially phosphorylated. In addition, the autophosphorylation sites Ser-24, Ser-25 found in the beta1 are replaced by Ala-Glu in the beta2 isoform. beta2 contains seven more Ser and one less Thr residues than beta1, raising the possibility of differential post-translational regulation. Immunoblot analysis further revealed that soleus muscle (slow twitch) contains exclusively beta1 associated with alpha2, whereas extensor digitorum longus muscle alpha2 (EDL, fast twitch) associates with beta2 as well as beta1. Sequence analysis revealed that glycogen synthase, a known AMPK substrate, co-immunoprecipitated with the AMPK alpha2beta2gamma1 complex.
Asunto(s)
Músculo Esquelético/enzimología , Proteínas Quinasas/metabolismo , Proteínas Quinasas Activadas por AMP , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/metabolismo , Immunoblotting , Isoenzimas , Hígado/enzimología , Masculino , Datos de Secuencia Molecular , Complejos Multienzimáticos , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , Ratas , Ratas Sprague-Dawley , Homología de Secuencia de AminoácidoRESUMEN
The mammalian S100A1 protein can activate the invertebrate myosin-associated giant protein kinase twitchin in a Ca(2+)-dependent manner by more than 1000-fold in vitro; however, no mammalian S100-dependent protein kinases are known. In an attempt to identify novel mammalian Ca(2+)/S100A1-regulated protein kinases, brain extracts were subjected to combined Ca(2+)-dependent affinity chromatography with S100A1 and an ATP analogue. This resulted in the purification to near-homogeneity of the four major synapsin isoforms Ia, Ib, IIa and IIb. All four synapsins were specifically affinity-labelled with the ATP analogue 5'-p-fluorosulphonylbenzoyladenosine. S100A1 bound to immobilized synapsin IIa in BIAcore experiments in a Ca(2+)-dependent and Zn(2+)-enhanced manner with submicromolar affinity; this interaction could be competed for with synthetic peptides of the proposed S100A1-binding sites of synapsin. Double-labelling confocal immunofluorescence microscopy demonstrated that synapsins and S100A1 are both present in the soma and neurites of PC12 cells, indicating their potential to interact in neurons in vivo.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Neuronas/química , Sinapsinas/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Química Encefálica , Compartimento Celular , Cromatografía de Afinidad , Datos de Secuencia Molecular , Neuritas/química , Neuronas/metabolismo , Células PC12 , Unión Proteica , Isoformas de Proteínas/metabolismo , Ratas , Proteínas S100 , Análisis de Secuencia de Proteína , Sinapsinas/aislamiento & purificaciónRESUMEN
Endothelial nitric oxide synthase (eNOS) is an important modulator of angiogenesis and vascular tone [1]. It is stimulated by treatment of endothelial cells in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent fashion by insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) [2] [3] and is activated by phosphorylation at Ser1177 in the sequence RIRTQS(1177)F (in the single-letter amino acid code) [4]. The protein kinase Akt is an important downstream target of PI 3-kinase [5] [6], regulating VEGF-stimulated endothelial cell survival [7]. Akt phosphorylates substrates within a defined motif [8], which is present in the sequence surrounding Ser1177 in eNOS. Both Akt [5] [6] and eNOS [9] are localized to, and activated at, the plasma membrane. We found that purified Akt phosphorylated cardiac eNOS at Ser1177, resulting in activation of eNOS. Phosphorylation at this site was stimulated by treatment of bovine aortic endothelial cells (BAECs) with VEGF or IGF-1, and Akt was activated in parallel. Preincubation with wortmannin, an inhibitor of Akt signalling, reduced VEGF- or IGF-1-induced Akt activity and eNOS phosphorylation. Akt was detected in immunoprecipitates of eNOS from BAECs, and eNOS in immunoprecipitates of Akt, indicating that the two enzymes associate in vivo. It is thus apparent that Akt directly activates eNOS in endothelial cells. These results strongly suggest that Akt has an important role in the regulation of normal angiogenesis and raise the possibility that the enhanced activity of this kinase that occurs in carcinomas may contribute to tumor vascularization and survival.
Asunto(s)
Óxido Nítrico Sintasa/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Línea Celular , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Linfocinas/farmacología , Datos de Secuencia Molecular , Óxido Nítrico Sintasa/química , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo III , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Transfección , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Parathyroid hormone-related protein (PTHrP) is expressed by a wide variety of cells and is considered to act as a secreted factor; however, evidence is accumulating for it to act in an intracrine manner. We have determined that PTHrP localizes to the nucleus at the G1 phase of the cell cycle and is transported to the cytoplasm when cells divide. PTHrP contains a putative nuclear localization sequence (NLS) (residues 61-94) similar to that of SV40 T-antigen, which may be implicated in the nuclear import of the molecule. We identified that Thr85 immediately prior to the NLS of PTHrP was phosphorylated by CDC2-CDK2 and phosphorylation was cell cycle-dependent. Mutation of Thr85 to Ala85 resulted in nuclear accumulation of PTHrP, while mutation to Glu85 to mimic a phosphorylated residue resulted in localization of PTHrP to the cytoplasm. Combined, the data demonstrate that the intracellular localization of PTHrP is phosphorylation- and cell cycle-dependent, and such control further supports a potential intracellular role (10,34,35) for PTHrP.
Asunto(s)
Núcleo Celular/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Proteínas de Neoplasias/metabolismo , Hormona Paratiroidea/metabolismo , Proteínas/metabolismo , Treonina/metabolismo , Transporte Biológico , Línea Celular , Humanos , Inmunohistoquímica , Proteína Relacionada con la Hormona Paratiroidea , Fosforilación , Fase S , Espectrometría de FluorescenciaRESUMEN
The AMP-activated protein kinase (AMPK) is a member of a metabolite-sensing protein kinase family that is found in all eukaryotes. AMPK activity is regulated by vigorous exercise, nutrient starvation and ischemia/hypoxia, and modulates many aspects of mammalian cell metabolism. The AMPK yeast homolog, Snf1p, plays a major role in adaption to glucose deprivation. In mammals, AMPK also has diverse roles that extend from energy metabolism through to transcriptional control.
Asunto(s)
Complejos Multienzimáticos/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Quinasas Activadas por AMP , Colesterol/biosíntesis , Creatina Quinasa/metabolismo , Metabolismo Energético , Ácidos Grasos/metabolismo , Glucosa/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Complejos Multienzimáticos/química , Conformación Proteica , Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
The AMP-activated protein kinase (AMPK) in rat skeletal and cardiac muscle is activated by vigorous exercise and ischaemic stress. Under these conditions AMPK phosphorylates and inhibits acetyl-coenzyme A carboxylase causing increased oxidation of fatty acids. Here we show that AMPK co-immunoprecipitates with cardiac endothelial NO synthase (eNOS) and phosphorylates Ser-1177 in the presence of Ca2+-calmodulin (CaM) to activate eNOS both in vitro and during ischaemia in rat hearts. In the absence of Ca2+-calmodulin, AMPK also phosphorylates eNOS at Thr-495 in the CaM-binding sequence, resulting in inhibition of eNOS activity but Thr-495 phosphorylation is unchanged during ischaemia. Phosphorylation of eNOS by the AMPK in endothelial cells and myocytes provides a further regulatory link between metabolic stress and cardiovascular function.
Asunto(s)
Endotelio Vascular/enzimología , Complejos Multienzimáticos/metabolismo , Óxido Nítrico Sintasa/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Quinasas Activadas por AMP , Secuencia de Aminoácidos , Animales , Aorta , Calmodulina/metabolismo , Bovinos , Endotelio Vascular/citología , Activación Enzimática , Cinética , Hígado/enzimología , Datos de Secuencia Molecular , Isquemia Miocárdica/enzimología , Miocardio/enzimología , Óxido Nítrico Sintasa de Tipo III , Fosforilación , Pruebas de Precipitina , Unión Proteica , Ratas , Proteínas Recombinantes/metabolismo , Serina/metabolismo , Treonina/metabolismoRESUMEN
Accumulating evidence supports the existence of nonthyroidal calcitonin (CT)-like peptides, more similar to fish CTs, which may act as endogenous regulators of CT receptors in brain and other tissues. In this study, we have carried out large-scale extractions from Sprague-Dawley rat brain diencephalon and pituitary, and purified a novel, biologically active, CT-like peptide from pituitary. Monitoring of the calcitonin-like activity of the peptides from rat brain and pituitary required different detection systems. While the brain CT cross-reacted with C-terminally directed salmon CT-specific antisera, the pituitary CT did not. However, the pituitary CT was biologically active, exhibiting specific interaction with CT receptors to activate adenylate cyclase. Conventional chromatographic techniques were employed to purify the CT-like peptides. Although the brain CT was not purified to homogeneity, size exclusion chromatography revealed the presence of multiple molecular weight forms of immunoreactive CT. Of these, only the lowest molecular weight form was biologically active. Purification from the pituitary resulted in the isolation of a biologically active peptide with a mass of 3267 Da. This mass differs from the mass of both salmon and thyroid-derived rat CT. Initial amino acid sequencing of the pituitary CT indicated that it was N-terminally blocked. Following aminopeptidase digestion, a unique six amino acid sequence, EKSQSP, was identified. Elucidation of the amino acid composition provided supporting evidence that the peptide was novel and was consistent with a full length peptide of approximately 30 amino acids. These data support the existence of novel, nonthyroidal, CTs which are potential regulators of CT receptor-mediated functions.
Asunto(s)
Calcitonina/aislamiento & purificación , Diencéfalo/química , Hipófisis/química , Secuencia de Aminoácidos , Animales , Calcitonina/química , Células Cultivadas , Humanos , Datos de Secuencia Molecular , Ratas , Ratas Sprague-Dawley , Receptores de Calcitonina/análisisRESUMEN
The AMP-activated protein kinase (AMPK) consists of catalytic alpha and noncatalytic beta and gamma subunits and is responsible for acting as a metabolic sensor for AMP levels. There are multiple genes for each subunit and the rat liver AMPK alpha1 and alpha2 catalytic subunits are associated with beta1 and gamma1 noncatalytic subunits. We find that the isolated gamma1 subunit is N-terminally acetylated with no other posttranslational modification. The isolated beta1 subunit is N-terminally myristoylated. Transfection of COS cells with AMPK subunit cDNAs containing a nonmyristoylatable beta1 reduces, but does not eliminate, membrane binding of AMPK heterotrimer. The isolated beta1 subunit is partially phosphorylated at three sites, Ser24/25, Ser182, and Ser108. The Ser24/25 and Ser108 sites are substoichiometrically phosphorylated and can be autophosphorylated in vitro. The Ser-Pro site in the sequence LSSS182PPGP is stoichiometrically phosphorylated, and no additional phosphate is incorporated into this site with autophosphorylation. Based on labeling studies in transfected cells, we conclude that alpha1 Thr172 is a major, although not exclusive, site of both basal and stimulated alpha1 phosphorylation by an upstream AMPK kinase.
Asunto(s)
Complejos Multienzimáticos/metabolismo , Proteínas Quinasas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas , Proteínas Quinasas Activadas por AMP , Secuencia de Aminoácidos , Animales , Catálisis , Hígado/enzimología , Espectrometría de Masas , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Ácido Mirístico , Ácidos Mirísticos/metabolismo , Mapeo Peptídico , Fosforilación , Proteínas Quinasas/química , Ratas , Fracciones Subcelulares/enzimologíaRESUMEN
The AMP-activated protein kinase (AMPK) consists of catalytic alpha and non-catalytic, beta and gamma (38 kDa) subunits and is responsible for acting as a metabolic sensor for AMP levels. There are multiple genes for each subunit and we find that rat liver AMPK-alpha2 isoform catalytic subunit is associated with beta1 and gamma1 and not with beta2 or gamma2 subunit isoforms. The beta1 and gamma1 isoforms are also subunits of the alpha1 isoform. The sequence of cloned human AMPK-beta1 is 95% identical in amino acid sequence with rat beta1. Human chromosomal localizations were determined for AMPK-alpha1 (5p11-p14), AMPK-beta1 (12q24.1-24.3) and AMPK-gamma1 (12q12-q14), respectively.
Asunto(s)
Mapeo Cromosómico , Isoenzimas/química , Isoenzimas/genética , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , Proteínas Quinasas Activadas por AMP , Secuencia de Aminoácidos , Animales , Cromosomas Humanos Par 12 , Cromosomas Humanos Par 5 , Cromosomas Humanos Par 7 , Humanos , Hígado/enzimología , Masculino , Metafase/genética , Datos de Secuencia Molecular , Familia de Multigenes , RatasRESUMEN
The SH3 domains of src and other nonreceptor tyrosine kinases have been shown to associate with the motif PXXP, where P and X stand for proline and an unspecified amino acid, but a motif that binds to the SH3 domain of myosin has thus far not been characterized. We previously showed that the SH3 domain of Acanthamoeba myosin-IC interacts with the protein Acan125. We now report that the Acan125 protein sequence contains two tandem consensus PXXP motifs near the C terminus. To test for binding, we expressed a polypeptide, AD3p, which includes 344 residues of native C-terminal sequence and a mutant polypeptide, AD3delta977-994p, which lacks the sequence RPKPVPPPRGAKPAPPPR containing both PXXP motifs. The SH3 domain of Acanthamoeba myosin-IC bound AD3p and not AD3delta977-994p, showing that the PXXP motifs are required for SH3 binding. The sequence of Acan125 is related overall to a protein of unknown function coded by Caenorhabditis elegans gene K07G5.1. The K07G5.1 gene product contains a proline-rich segment similar to the SH3 binding motif found in Acan125. The aligned sequences show considerable conservation of leucines and other hydrophobic residues, including the spacing of these residues, which matches a motif for leucine-rich repeats (LRRs). LRR domains have been demonstrated to be sites for ligand binding. Having an LRR domain and an SH3-binding domain, Acan125 and the C. elegans homologue define a novel family of bifunctional binding proteins.
Asunto(s)
Proteínas Portadoras/metabolismo , Leucina/genética , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Proteínas Protozoarias/metabolismo , Acanthamoeba , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Datos de Secuencia Molecular , Proteínas Protozoarias/genética , Secuencias Repetitivas de Ácidos Nucleicos , Alineación de SecuenciaRESUMEN
The 5'-AMP-activated protein kinase (AMPK) mediates several cellular responses to metabolic stress. Rat liver contains at least two isoforms of this enzyme, either alpha1 or alpha2 catalytic subunits together with beta and gamma noncatalytic subunits in a trimeric complex. The alpha1 isoform is purified using a peptide substrate affinity chromatography column with ADR1 (222-234)P229 (LKKLTRRPSFSAQ), corresponding to the cAMP-dependent protein kinase phosphorylation site in the yeast transcriptional activator of the ADH2 gene, ADR1. This peptide is phosphorylated at Ser230 by AMPK alpha1 with a Km of 3.8 microM and a Vmax of 4.8 micromol/min/mg compared to the commonly used rat acetyl-CoA carboxylase (73-87)A77R86-87 peptide substrate, HMRSAMSGLHLVKRR, with a Km of 33.3 microM and a Vmax of 8.1 micromol/min/mg. Thus, the AMPK exhibits some overlapping specificity with the cAMP-dependent protein kinase. The rat liver AMPK alpha1 isoform has a Kcat approximately 250-fold higher than the AMPK alpha2 isoform isolated from rat liver. The AMPK alpha1 isoform readily phosphorylates peptides corresponding to the reported AMPK phosphorylation sites in rat, chicken, and yeast acetyl-CoA carboxylase and rat hydroxymethylglutaryl-CoA reductase but not phosphorylase kinase. Based on previous peptide substrate specificity studies (Dale, S., Wilson, W. A., Edelman, A. M., and Hardie, G. (1995) FEBS Lett. 361, 191-195) using partially purified enzyme and variants of the peptide AMARAASAAALARRR, it was proposed that the AMPK preferred the phosphorylation site motif Phi(X, beta)XXS/TXXXPhi (Phi, hydrophobic; beta, basic). In good AMPK alpha1 peptide substrates, a hydrophobic residue at the P-5 position is conserved but not at the P+4 position. Oxidation of the Met residues in the rat acetyl-CoA carboxylase (73-87)A77R86-87 peptide increased the Km 6-fold and reduced the Vmax to 4% of the reduced peptide.
Asunto(s)
Isoenzimas/aislamiento & purificación , Complejos Multienzimáticos/aislamiento & purificación , Proteínas Quinasas/aislamiento & purificación , Proteínas Serina-Treonina Quinasas , Proteínas de Saccharomyces cerevisiae , Proteínas Quinasas Activadas por AMP , Acetil-CoA Carboxilasa/metabolismo , Animales , Proteínas de Unión al ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Isoenzimas/metabolismo , Cinética , Complejos Multienzimáticos/metabolismo , Fosforilación , Proteínas Quinasas/metabolismo , Ratas , Especificidad por Sustrato , Porcinos , Factores de Transcripción/metabolismo , Dedos de ZincRESUMEN
We have purified to near homogeneity from rat brain two Ca(2+)-calmodulin-dependent protein kinase I (CaM kinase I) activating kinases, termed here CaM kinase I kinase-alpha and CaM kinase I kinase-beta (CaMKIK alpha and CaMKIK beta, respectively). Both CaMKIK alpha and CaMKIK beta are also capable of activating CaM kinase IV. Activation of CaM kinase I and CaM kinase IV occurs via phosphorylation of an equivalent Thr residue within the "activation loop" region of both kinases, Thr-177 and Thr-196, respectively. The activities of CaMKIK alpha and CaMKIK beta are themselves strongly stimulated by the presence of Ca(2+)-CaM, and both appear to be capable of Ca(2+)-CaM-dependent autophosphorylation. Automated microsequence analysis of the purified enzymes established that CaMKIK alpha and -beta are the products of distinct genes. In addition to rat, homologous nucleic acids corresponding to these CaM kinase kinases are present in humans and the nematode, Caenorhabditis elegans. CaMKIK alpha and CaMKIK beta are thus representatives of a family of enzymes, which may function as key intermediaries in Ca(2+)-CaM-driven signal transduction cascades in a wide variety of eukaryotic organisms.
Asunto(s)
Encéfalo/enzimología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Calcio/metabolismo , Calmodulina/metabolismo , Isoenzimas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/aislamiento & purificación , Cromatografía Liquida , Activación Enzimática , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Datos de Secuencia Molecular , Ratas , Ratas Sprague-Dawley , Homología de Secuencia de AminoácidoRESUMEN
The mammalian 5'-AMP-activated protein kinase (AMPK) is related to a growing family of protein kinases in yeast and plants that are regulated by nutritional stress. We find the most prominent expressed form of the hepatic AMPK catalytic subunit (alpha 1) is distinct from the previously cloned kinase subunit (alpha 2). The alpha 1 (548 residues) and alpha 2 (552 residues) isoforms have 90% amino acid sequence identity within the catalytic core but only 61% identity elsewhere. The tissue distribution of the AMPK activity most closely parallels the low abundance 6-kilobase alpha 1 mRNA distribution and the alpha 1 immunoreactivity rather than alpha 2, with substantial amounts in kidney, liver, lung, heart, and brain. Both alpha 1 and alpha 2 isoforms are stimulated by AMP and contain noncatalytic beta and gamma subunits. The liver alpha 1 isoform accounts for approximately 94% of the enzyme activity measured using the SAMS peptide substrate. The tissue distribution of the alpha 2 immunoreactivity parallels the alpha 2 8.5-kilobase mRNA and is most prominent in skeletal muscle, heart, and liver. Isoforms of the beta and gamma subunits present in the human genome sequence reveal that the AMPK consists of a family of isoenzymes.
Asunto(s)
Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/análisis , Secuencia de Aminoácidos , Animales , Humanos , Datos de Secuencia Molecular , Especificidad de Órganos , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Alineación de Secuencia , Análisis de Secuencia , Especificidad de la Especie , PorcinosRESUMEN
The rapid whole blood test, developed for the detection of circulating antibodies to human immunodeficiency virus type 1 (HIV-1), is based on agglutination of autologous red blood cells using an anti-human glycophorin antibody conjugated to the HIV-1 immunodominant epitope of gp41 (579-613). A simplified procedure for preparing antibody-peptide conjugates for use in the autologous red cell agglutination test is described. F(ab')2 fragments of the anti-glycophorin antibody were prepared by pepsin digestion and reduced to F(ab') fragments with the use of tri-n-butylphosphine (TBP). This permitted the simultaneous reduction of the F(ab') fragments and coupling of a bromoacetyl derivative of the synthetic immunodominant peptide gp41 (579-613) [Cys-Acm 598, Lys-BrAc 604] containing epsilon-bromoacetyl-lysine at residue 604 to the resultant F(ab') fragment. Conjugation to the F(ab') fragment resulted in a stable thio-ether linkage between the peptide Lys-604 and the inter heavy chain cysteines of the F(ab'). The resultant F(ab')-peptide conjugate was comparable to the previously described disulfide coupled conjugate when used in the autologous red cell agglutination test. This simplified conjugation chemistry may also be useful for the development of reagents for FACS analysis as well as targetted vaccines.
Asunto(s)
Anticuerpos Anti-VIH/sangre , VIH-1/inmunología , Pruebas de Hemaglutinación/métodos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos de Péptidos/inmunología , Secuencia de Aminoácidos , Reactivos de Enlaces Cruzados , Electroforesis en Gel de Poliacrilamida , Eritrocitos/inmunología , Anticuerpos Anti-VIH/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/químicaRESUMEN
The T-lymphotropic lentivirus, feline immunodeficiency virus (FIV) is now recognised as a major viral pathogen affecting domestic cat populations worldwide. A rapid, autologous red cell agglutination test for antibodies to FIV has been developed. A synthetic peptide analog corresponding to the immunodominant epitope within the FIV transmembrane glycoprotein gp40 residues (680-715) KVEAMEKFLYTAFAMQELGC (Acm)NQNQFFK(BrAc)KIPLELWTR was conjugated to an anti-feline erythrocyte antibody using a thio-ether linkage. Within 3 min of adding this reagent to 20 microliters of whole blood, circulating antibody to the peptide epitope caused agglutination of the red blood cells. The performance of this simple test is comparable with the two commercially available enzyme immunoassay (EIA) kits and an EIA based on this peptide. A variant of the gp40 (680-715) peptide corresponding to the FIV, PPR strain gp40 (678-716) sequence was also synthesised and no difference in reactivity was observed in an EIA on 211 seropositive samples, indicating that the peptide-based test may be applicable to other known strains of the virus.
Asunto(s)
Anticuerpos Antivirales/análisis , Eritrocitos/inmunología , Virus de la Inmunodeficiencia Felina/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Gatos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Síndrome de Inmunodeficiencia Adquirida del Felino/diagnóstico , Hemaglutinación/inmunología , Pruebas de Hemaglutinación/veterinaria , Epítopos Inmunodominantes/inmunología , Datos de Secuencia Molecular , Proteínas del Envoltorio Viral/síntesis química , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
The AMP-activated protein kinase is responsible for the regulation of fatty acid synthesis by phosphorylation of acetyl-CoA carboxylase. It may also regulate cholesterol synthesis via phosphorylation and inactivation of hormone-sensitive lipase and hydroxymethylglutaryl-CoA reductase. We have purified the AMP-activated protein kinase 14,000-fold from porcine liver. The 63-kDa catalytic subunit co-purifies with two proteins of 40 and 38 kDa that may function as subunits. Partial amino acid sequence of the 63-kDa subunit revealed a striking homology with the catalytic domain of the yeast protein kinase transcriptional regulator Snf1 and its plant homologs. The Snf1 (72 kDa) and Snf4 (36 kDa) complex was also purified and found to phosphorylate the AMP-activated protein kinase peptide substrate, HMRSAMSGLHLVKRR-amide, but was not activated by AMP. Both Snf1/4 and the AMP-activated protein kinase phosphorylate and inactivate yeast acetyl-CoA carboxylase in vitro. These results indicate that during evolution the catalytic domain sequences of the Snf1 protein kinase subfamily have been exploited in the control of mammalian lipid metabolism and raise the possibilities that the AMP-activated protein kinase may have other substrates involved in regulating gene expression pathways, as well as Snf1 homologs participating in the control of lipid metabolism in many eukaryotic organisms.
Asunto(s)
Hígado/enzimología , Complejos Multienzimáticos/química , Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/química , Saccharomyces cerevisiae/enzimología , Proteínas Quinasas Activadas por AMP , Acetil-CoA Carboxilasa/metabolismo , Secuencia de Aminoácidos , Animales , Electroforesis en Gel de Poliacrilamida , Proteínas Fúngicas/química , Sustancias Macromoleculares , Datos de Secuencia Molecular , Peso Molecular , Complejos Multienzimáticos/aislamiento & purificación , Complejos Multienzimáticos/metabolismo , Fosforilación , Proteínas Quinasas/aislamiento & purificación , Proteínas Quinasas/metabolismo , Ratas , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The reported cDNA structure of chicken smooth muscle myosin light chain kinase (smMLCK) encodes a protein of 972 residues (Olson et al. Proc. Natl. Acad. Sci USA, 87:2284-2288, 1990). The calculated M(r) is 107,534 whereas the estimate by SDS-PAGE is approximately 130,000. Gibson and Higgins (DNA Sequence (in press)) have recently reported the possibility of errors in the cDNA sequence for non-muscle MLCK and that the NH2-terminus of both it and smMLCK may extend beyond the reported coding region. The native smMLCK is NH2-terminally blocked. A CNBr peptide derived from smMLCK contains the NH2-terminal sequence Asp-Phe-Arg-Ala corresponding to residues 2 to 4 in the smMLCK sequence indicating that Met-1 is present. Using a limited thermolysin digest we isolated an NH2-terminally blocked peptide by reversed-phase HPLC. This thermolytic peptide had a mass of approximately 797 by time of flight mass spectrometry. Amino acid analysis and Edman sequencing of a CNBr-subfragment of the thermolytic peptide indicated that it had the composition and sequence, (Met)-Asp-Phe-Arg-Ala-Asn, with a calculated mass of 753. The difference in mass corresponds to the NH2-terminal Met being blocked by acetylation. The results demonstrate that the NH2-terminal sequence of smMLCK inferred from the reported cDNA sequence is correct and that the proposed initiating Met is not removed, but modified by alpha-NH2 acetylation of the translation product.