RESUMEN
Cancer of the cervix is the 8th most common cancer for women in Ireland.Cervical Check has organised and combated this clinical presentation nationwide and has shown encouraging figures since its launch in 2007. While working in Kerry General Hospital (KGH), the Southwest Specialist Training Scheme in General Practice carried out an audit of the colposcopy referrals being received from GPs in the southwest. Adherence to Cervical Check referral guidelines was the main focus of the audit. Very positive figures presented in round one of the audit cycle, with 51 (90%) of all GP referrals adhering to the guidelines. This was further improved by a GP information campaign, leading to 57 (93%) of referrals meeting the appropriate referral criteria. Overall, this paper highlights the excellent screening programme that is Cervical Check and the superb working relationship between primary and secondary care facilities.
Asunto(s)
Colposcopía , Auditoría Médica , Atención Primaria de Salud/normas , Derivación y Consulta , Neoplasias del Cuello Uterino , Colposcopía/normas , Femenino , Adhesión a Directriz/normas , Guías como Asunto , Humanos , Irlanda/epidemiología , Auditoría Médica/normas , Derivación y Consulta/normas , Derivación y Consulta/estadística & datos numéricos , Especialización , Neoplasias del Cuello Uterino/diagnóstico , Neoplasias del Cuello Uterino/epidemiologíaRESUMEN
The extreme acidothermophilic archaeon Sulfolobus solfataricus harbors a membrane-associated protein kinase activity. Its solubilization and stabilization required detergents, suggesting that this activity resides within an integral membrane protein. The archaeal protein kinase utilized purine nucleotides as phosphoryl donors in vitro. A noticeable preference for nucleotide triphosphates over nucleotide diphosphates and for adenyl nucleotides over the corresponding guanyl ones was observed. The molecular mass of the solubilized, partially purified enzyme was estimated to be approximately 125 kDa by gel filtration chromatography. Catalytic activity resided in a polypeptide with an apparent molecular mass of approximately 67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Challenges with several exogenous substrates revealed the protein kinase to be relatively selective. Only casein, histone H4, reduced carboxyamidomethylated and maleylated lysozyme, and a peptide modeled after myosin light chains (KKRAARATSNVFA) were phosphorylated to appreciable levels in vitro. All of the aforementioned substrates were phosphorylated on threonine residues, while histone H4 was phosphorylated on serine as well. Substitution of serine for the phosphoacceptor threonine in the myosin light chain peptide produced a noticeably inferior substrate. The protein kinase underwent autophosphorylation on threonine and was relatively insensitive to a set of known inhibitors of "eukaryotic" protein kinases.
Asunto(s)
Fosfotreonina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Sulfolobus/enzimología , Membrana Celular/enzimología , Inhibidores Enzimáticos/farmacología , Metales/metabolismo , Nucleótidos/metabolismo , Péptidos/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Proteínas/metabolismo , Solubilidad , Sulfolobus/crecimiento & desarrolloRESUMEN
Three dual-specific phosphatases [DSPs], IphP, VHR, and Cdc14, and three protein-tyrosine phosphatases [PTPs], PTP-1B, PTP-H1, and Tc-PTPa, were challenged with a set of low molecular weight phosphoesters to probe the factors underlying the distinct substrate specificities displayed by these two mechanistically homologous families of protein phosphatases. It was observed that beta-naphthyl phosphate represented an excellent general substrate for both PTPs and DSPs. While DSPs tended to hydrolyze alpha-naphthyl phosphate at rates comparable to that of the beta-isomer, the PTPs PTP-1B and Tc-PTPa did not. PTP-H1, however, displayed high alpha-naphthyl phosphatase activity. Intriguingly, PTP-H1 also displayed much higher protein-serine phosphatase activity in vitro, 0.2-0.3% that toward equivalent tyrosine phosphorylated proteins, than did PTP-1B or Tc-PTPa. The latter two PTPs discriminated between the serine- and tyrosine-phosphorylated forms of two test proteins by factors of >/=10(4)-10(6). While free phosphoserine represented an extremely poor substrate for all of the DSPs examined, the addition of a hydrophobic "handle" to form N-(cyclohexanecarboxyl)-O-phospho-l-serine produced a compound that was hydrolyzed by IphP with high efficiency, i.e., at a rate comparable to that of free phosphotyrosine or p-nitrophenyl phosphate. VHR also hydrolyzed N-(cyclohexanecarboxyl)-O-phospho-l-serine (1 mM) at a rate approximately one-tenth that of beta-naphthyl phosphate. None of the PTPs tested exhibited significant activity against this compound. However, N-(cyclohexanecarboxyl)-O-phospho-l-serine did not prove to be a universal substrate for DSPs as Cdc14 displayed little propensity to hydrolyze it.
Asunto(s)
Ciclohexanos/metabolismo , Complejos Multienzimáticos/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Serina/análogos & derivados , Sitios de Unión , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Cianobacterias/enzimología , Ciclohexanos/síntesis química , Ciclohexanos/química , Fosfatasa 3 de Especificidad Dual , Humanos , Hidrólisis , Isomerismo , Cinética , Peso Molecular , Muramidasa/metabolismo , Proteína Básica de Mielina/metabolismo , Naftalenos/química , Naftalenos/metabolismo , Compuestos Organofosforados/química , Compuestos Organofosforados/metabolismo , Fosfoserina/metabolismo , Fosfotirosina/metabolismo , Proteínas Tirosina Fosfatasas/química , Saccharomyces cerevisiae/enzimología , Serina/síntesis química , Serina/química , Serina/metabolismo , Especificidad por SustratoRESUMEN
A set of open reading frames (ORFs) potentially encoding signal transduction proteins are clustered around icfG, a gene implicated in the regulation of carbon metabolism, in the genome of Synechocystis sp. strain PCC 6803. slr1860 is the ORF for icfG, whose predicted product resembles the protein phosphatases SpoIIE, RsbU, and RsbX from Bacillus subtilis. Bracketing slr1860/icfG are (i) ORF slr1861, whose predicted product resembles the SpoIIAB, RsbT, and RsbW protein kinases from B. subtilis, and (ii) ORFs slr1856 and slr1859, whose predicted products resemble the respective phosphoprotein substrates for the B. subtilis protein kinases: SpoIIAA, RsbS, and RsbV. In order to determine whether the protein products encoded by these ORFs possessed the functional capabilities suggested by sequence comparisons, each was expressed in Escherichia coli as a histidine-tagged fusion protein and analyzed for its ability to participate in protein phosphorylation-dephosphorylation processes in vitro. It was observed that ORF slr1861 encoded an ATP-dependent protein kinase capable of phosphorylating Slr1856 and, albeit with noticeably lower efficiency, Slr1859. Site-directed mutagenesis suggests that Slr1861 phosphorylated these proteins on Ser-54 and Ser-57, respectively. Slr1860 exhibited divalent metal ion-dependent protein-serine phosphatase activity. It catalyzed the dephosphorylation of Slr1856, but not Slr1859, in vitro.
Asunto(s)
Proteínas Bacterianas/genética , Cianobacterias/genética , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas/genética , Monoéster Fosfórico Hidrolasas , Proteínas Quinasas/genética , Factor sigma , Factores de Transcripción , Adenosina Trifosfato/metabolismo , Cianobacterias/enzimología , Cartilla de ADN , Activación Enzimática/efectos de los fármacos , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Genoma Bacteriano , Hidrólisis , Magnesio/farmacología , Manganeso/farmacología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Sistemas de Lectura Abierta/genética , Fosfoproteínas Fosfatasas/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Quinasas/metabolismo , Homología de Secuencia de Aminoácido , Serina , Transducción de Señal/fisiología , Especificidad por SustratoRESUMEN
The structural gene for a putative PPP family protein-serine/threonine phosphatase from the microcystin-producing cyanobacterium Microcystis aeruginosa PCC 7820, pp1-cyano1, was cloned. The sequence of the predicted gene product, PP1-cyano1, was 98% identical to that of the predicted product of an open reading frame, pp1-cyano2, from a cyanobacterium that does not produce microcystins, M. aeruginosa UTEX 2063. By contrast, PP1-cyano1 displayed less than 20% identity with other PPP family protein phosphatases from eukaryotic, archaeal, or other bacterial organisms. PP1-cyano1 and PP1-cyano2 were expressed in Escherichia coli and purified to homogeneity. Both enzymes exhibited divalent metal dependent phosphohydrolase activity in vitro toward phosphoserine- and phosphotyrosine-containing proteins and 3-phosphohistidine- and phospholysine-containing amino acid homopolymers. This multifunctional potential also was apparent in samples of PP1-cyano1 and PP1-cyano2 isolated from M. aeruginosa. Catalytic activity was insensitive to okadaic acid or the cyanobacterially produced cyclic heptapeptide, microcystin-LR, both potent inhibitors of mammalian PP1 and PP2A. PP1-cyano1 and PP1-cyano2 displayed diadenosine tetraphosphatase activity in vitro. Diadenosine tetraphosphatases share conserved sequence features with PPP family protein phosphatases. The diadenosine tetraphosphatase activity of PP1-cyano1 and PP1-cyano2 confirms that these enzymes share a common catalytic mechanism.
Asunto(s)
Cianobacterias/genética , Genes Bacterianos , Péptidos Cíclicos/farmacología , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Nucleótidos de Adenina/metabolismo , Secuencia de Aminoácidos , Catálisis , Clonación Molecular , Escherichia coli , Concentración de Iones de Hidrógeno , Toxinas Marinas , Microcistinas , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/aislamiento & purificaciónRESUMEN
Prokaryotes contain at least five distinct families of protein O-phosphatases, including AceK, the chimeric isocitrate dehydrogenase kinase/phosphatase, and four protein phosphatase families first identified and characterized in Eukaryotes. The latter consist of the PPP and PPM families of protein-serine/threonine phosphatases, and the low molecular weight and conventional families of protein-tyrosine phosphatases. Prokaryotic protein O-phosphatases participate in the regulation of metabolic processes and the transduction of environmental signals. Certain pathogenic bacteria employ protein-tyrosine phosphatases as virulence factors, injecting them into host cells where they enzymatically perturb the phosphorylation state of proteins therein. While our understanding of protein O-phosphorylation events in Prokaryotes only now is emerging from its infancy, their phylogenetic diversity and malleability to genetic manipulation render these "simple'" organisms powerful vehicles for answering fundamental questions concerning the origins and evolution of this key biological regulatory mechanism.
Asunto(s)
Fosfoproteínas Fosfatasas/fisiología , Células Procariotas/fisiología , Fosforilación , Fosfotirosina/fisiología , Filogenia , Proteínas Quinasas/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Tirosina Fosfatasas/fisiología , Proteínas/metabolismoRESUMEN
Inspection of the genomes for the bacteria Bacillus subtilis 168, Borrelia burgdorferi B31, Escherichia coli K-12, Haemophilus influenzae KW20, Helicobacter pylori 26695, Mycoplasma genitalium G-37, and Synechocystis sp PCC 6803 and for the archaeons Archaeoglobus fulgidus VC-16 DSM4304, Methanobacterium thermoautotrophicum delta H, and Methanococcus jannaschii DSM2661 revealed that each contains at least one ORF whose predicted product displays sequence features characteristic of eukaryote-like protein-serine/threonine/tyrosine kinases and protein-serine/threonine/tyrosine phosphatases. Orthologs for all four major protein phosphatase families (PPP, PPM, conventional PTP, and low molecular weight PTP) were present in the bacteria surveyed, but not all strains contained all types. The three archaeons surveyed lacked recognizable homologs of the PPM family of eukaryotic protein-serine/threonine phosphatases; and only two prokaryotes were found to contain ORFs for potential phosphatases from all four major families. Intriguingly, our searches revealed a potential ancestral link between the catalytic subunits of microbial arsenate reductases and the protein-tyrosine phosphatases; they share similar ligands (arsenate versus phosphate) and features of their catalytic mechanism (formation of arseno-versus phospho-cysteinyl intermediates). It appears that all prokaryotic organisms, at one time, contained the genetic information necessary to construct protein phosphorylation-dephosphorylation networks that target serine, threonine, and/or tyrosine residues on proteins. However, the potential for functional redundancy among the four protein phosphatase families has led many prokaryotic organisms to discard one, two, or three of the four.
Asunto(s)
Proteínas Arqueales/genética , Proteínas Bacterianas/genética , Sistemas de Lectura Abierta/genética , Fosfoproteínas Fosfatasas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Secuencia de Aminoácidos , Archaeoglobus fulgidus/enzimología , Archaeoglobus fulgidus/genética , Bacillus subtilis/enzimología , Bacillus subtilis/genética , Grupo Borrelia Burgdorferi/enzimología , Grupo Borrelia Burgdorferi/genética , Cianobacterias/enzimología , Cianobacterias/genética , Escherichia coli/enzimología , Escherichia coli/genética , Haemophilus influenzae/enzimología , Haemophilus influenzae/genética , Helicobacter pylori/enzimología , Helicobacter pylori/genética , Methanobacterium/enzimología , Methanobacterium/genética , Methanococcus/enzimología , Methanococcus/genética , Datos de Secuencia Molecular , Mycoplasma/enzimología , Mycoplasma/genética , Alineación de SecuenciaAsunto(s)
Fosfoproteínas Fosfatasas/aislamiento & purificación , Células Procariotas/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Methanosarcina/enzimología , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/clasificación , Fosfoproteínas Fosfatasas/genética , Filogenia , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , Sulfolobus/enzimologíaRESUMEN
More than a hundred different enzymes impinging on aspects of cell function ranging from carbohydrate and lipid metabolism to signal transduction and gene expression to biomolecule degradation have been detected by the assay of their enzymatic activities following SDS-PAGE. The strategies by which this has been accomplished are as varied as the enzymes themselves and offer testimony to the creativeness and ingenuity of life scientists. Assay of enzyme activity following SDS-PAGE is well adapted to identifying the source of catalytic activity in a heterogeneous protein mixture or a heterooligomeric protein (20), or determining if multiple catalytic activities reside in a single polypeptide (60). The alliance of versatile enzyme assay techniques with the molecular resolution of SDS-PAGE offers a powerful means for meeting the increasing demand for the high-throughput screening arising from protein engineering, combinatorial chemistry, and functional genomics.
Asunto(s)
Enzimas/análisis , Animales , Electroforesis en Gel de Poliacrilamida/métodos , Enzimas/química , Enzimas/metabolismo , Humanos , Desnaturalización Proteica , Dodecil Sulfato de SodioRESUMEN
When soluble extracts from the extreme acidophilic archaeon Sulfolobus solfataricus were incubated with [gamma-32P]ATP, several radiolabeled polypeptides were observed following SDS-PAGE. The most prominent of these migrated with apparent molecular masses of 14, 18, 35, 42, 46, 50, and 79 kDa. Phosphoamino acid analysis revealed that all of the proteins contained phosphoserine, with the exception of the 35-kDa one, whose protein-phosphate linkage proved labile to strong acid. The observed pattern of phosphorylation was influenced by the identity of the divalent metal ion cofactor used, Mg2+ versus Mn2+, and the choice of incubation temperature. The 35- and 50-kDa phosphoproteins were purified and their amino-terminal sequences determined. The former polypeptide's amino-terminal sequence closely matched a conserved portion of the alpha-subunit of succinyl-CoA synthetase, which forms an acid-labile phosphohistidyl enzyme intermediate during its catalytic cycle. This identification was confirmed by the ability of succinate or ADP to specifically remove the radiolabel. The 50-kDa polypeptide's sequence contained a heptapeptide motif, Phe/Pro-Gly-Thr-Asp/Ser-Gly-Val/Leu-Arg, found in a similar position in several hexosephosphate mutases. The catalytic mechanism of these mutases involves formation of a phosphoseryl enzyme intermediate. The identity of p50 as a hexosephosphate mutase was confirmed by (1) the ability of sugars and sugar phosphates to induce removal of the labeled phosphoryl group from the protein, and (2) the ability of [32P]glucose 6-phosphate to donate its phosphoryl group to the protein.
Asunto(s)
Fosfoglucomutasa/química , Fosfoproteínas/química , Sulfolobus/enzimología , Adenosina Difosfato/metabolismo , Secuencia de Aminoácidos , Proteínas Arqueales/química , Histidina/análogos & derivados , Histidina/análisis , Datos de Secuencia Molecular , Fosforilación , Fosfoserina/análisis , Análisis de Secuencia , Homología de Secuencia de Aminoácido , Succinato-CoA Ligasas/química , Ácido Succínico/metabolismo , Fosfatos de Azúcar/metabolismoRESUMEN
With oligonucleotides modelled after conserved regions within the protein-serine/threonine phosphatases (PPs) of the PP1/2A/2B superfamily, the gene for the archaeal protein phosphatase PP1-arch2 was identified, cloned, and sequenced from the methanogenic archaeon Methanosarcina thermophila TM-1. The DNA-derived amino acid sequence of PP1-arch2 exhibited a high degree of sequence identity, 27 to 31%, with members of the PP1/2A/2B superfamily such as PP1-arch1 from Sulfolobus solfataricus, PP1alpha from rats, PP2A from Saccharomyces cerevisiae, and PP2B from humans. The activity of the recombinant PP1-arch2 was sensitive to several naturally occurring microbial toxins known to potently inhibit eucaryal PP1 and PP2A, including microcystin-LR, okadaic acid, tautomycin, and calyculin A.
Asunto(s)
Methanosarcina/genética , Fosfoproteínas Fosfatasas/genética , Acetatos/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Expresión Génica , Genes Bacterianos , Methanosarcina/enzimología , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/metabolismo , Fosfoserina/metabolismo , Fosfotirosina/metabolismo , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Public health nurses are in a strategic position to maintain, promote, and protect the health of populations both now and in the future. In the changing health care environment of the 1990s, defining the expanding and evolving role of public health nursing assists in the effective utilization of public health nurses. During a time when the health care environment is emphasizing the protection and promotion of health, access to health services, and prevention of illness, it is necessary for public health nurses to be in the forefront in the changing focus to primary prevention. Therefore, a model for public health nursing is proposed to provide a framework for defining public health nursing roles and practice. A flowering tree is one symbol of public health nursing. The tree consists of seven parts symbolizing nine public health nursing concepts. This model is proposed to articulate clearly the capacity of public health nursing within today's ever-changing environment and to provide a framework for public health nursing theory, public health nursing practice, policy development, and public health research.
Asunto(s)
Modelos de Enfermería , Enfermería en Salud Pública , Enfermería en Salud Comunitaria , Participación de la Comunidad , Educación en Enfermería , Promoción de la Salud , Humanos , Investigación en Enfermería , Poder PsicológicoRESUMEN
Components of a protein tyrosine phosphorylation/dephosphorylation network were identified in the cyanobacterium Anabaena sp. strain PCC 7120. Three phosphotyrosine (P-Tyr) proteins of 27, 36, and 52 kDa were identified through their conspicuous immunoreactions with RC20H monoclonal antibodies specific for P-Tyr. These immunoreactions were outcompeted completely by free P-Tyr (5 mM) but not by phosphoserine or phosphothreonine. The P-Tyr content of the three major P-Tyr proteins and several minor proteins increased with their time of incubation in the presence of Mg-ATP and the protein phosphatase inhibitors sodium orthovanadate and sodium fluoride. Incubation of the same extracts with [gamma-32P]ATP but not [alpha-32P]ATP led to the phosphorylation of five polypeptides with molecular masses of 20, 27, 52, 85, and 100 kDa. Human placental protein tyrosine phosphatase 1B, with absolute specificity for P-Tyr, liberated significant quantities of 32Pi from four of the polypeptides, confirming that a portion of the protein-bound phosphate was present as 32P-Tyr. Alkaline phosphatase and the dual-specificity protein phosphatase IphP from the cyanobacterium Nostoc commune UTEX 584 also dephosphorylated these proteins and did so with greater apparent efficiency. Two of the polypeptides were partially purified, and phosphoamino analysis identified 32P-Tyr, [32P]phosphoserine, and [32P]phosphothreonine. Anabaena sp. strain PCC 7120 cell extracts contained a protein tyrosine phosphatase activity that was abolished in the presence of sodium orthovanadate and inhibited significantly by the sulfhydryl-modifying agents p-hydroxymercuriphenylsulfonic acid and p-hydroxymercuribenzoate as well as by heparin. In Anabaena sp. strain PCC 7120 the presence and/or phosphorylation status of P-Tyr proteins was influenced by incident photon flux density.
Asunto(s)
Anabaena/metabolismo , Proteínas Bacterianas/metabolismo , Fosfoproteínas/metabolismo , Fosfotirosina/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Anabaena/enzimología , Peso Molecular , Fosforilación , Proteínas Tirosina Fosfatasas/metabolismoRESUMEN
Sulfolobus sulfataricus ATCC 35091, Haloferax volcanii, and Methanosarcina thermophila TM-1, representing the Euryarchaeota and Crenarchaeota subdomains of the Archaea, contain proteins which are phosphorylated on tyrosine. These data raise fundamental questions as to the origin and evolution of tyrosine phosphorylation, a protein modification that is of pivotal importance in the regulation of the physiology of eukaryotic cells.
Asunto(s)
Archaea/metabolismo , Proteínas Bacterianas/metabolismo , Methanosarcina/metabolismo , Fosfoproteínas/metabolismo , Fosfotirosina/metabolismo , Sulfolobus/metabolismo , Peso MolecularRESUMEN
Bacteria play host to a wide range of protein phosphorylation-dephosphorylation systems (Fig. 1). As little as five years ago the known systems were thought to be late-emerging and absolutely prokaryote specific. Today we know that most protein kinases and protein phosphatases are descended from a set of common, and possibly quite ancient, prototypes. Prokaryote- and eukaryote-specific protein kinases and protein phosphatases are rare and represent exceptions, not the rule as previously thought. Commonality suggests that a dynamic and versatile regulatory mechanism was first adapted to the modulation of protein function as early if not earlier than more "basic" mechanisms such as allosterism, etc. The existence of common molecular themes confirms that the microbial world offers a unique, largely untapped library and a powerful set of tools for the understanding of a regulatory mechanism which is crucial to all organisms, tools whose diversity and experimental malleability will provide new avenues for exploring and understanding key modes of cellular regulation.
Asunto(s)
Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Células Procariotas/metabolismo , Animales , Bacterias/clasificación , Bacterias/genética , Cianobacterias/genética , Cianobacterias/metabolismo , Histidina Quinasa , Modelos Biológicos , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Filogenia , Proteínas Quinasas/metabolismoRESUMEN
The substrate specificity of the cyanobacterial dual-specificity protein phosphatase, IphP, was explored using a variety of potential substrates. The enzyme displayed phosphomonoesterase activity toward a broad range of peptide, protein, and low molecular weight organophosphate compounds. It displayed little or no hydrolase activity toward phosphodiesters, phosphoramides, carboxyl esters, or sulfoesters. However, it did display measurable pyrophosphatase activity, especially toward ADP and ATP. Among the low molecular weight phosphomonoesters, the presence of an aromatic ring either as part of the leaving group alcohol or immediately adjacent thereto, as in 5'-AMP, was a strong positive determinant for hydrolysis. Among peptide and protein substrates, a rough, but imperfect, correlation between charge character and hydrolysis was noted in which proteins and phosphorylation sites of an acidic nature seemed favored. Heparin affected IphP activity in a substrate-dependent manner. Toward small organophosphates, heparin had no significant effect, but it was inhibitory toward most protein and peptide substrates. However, toward phosphoseryl casein and MAP kinase, it enhanced activity as much as 10-fold. This enhancement was attributed to the ability of heparin to bind to these substrate proteins, as well as IphP, and recruit them to the same microenvironment.
Asunto(s)
Cianobacterias/enzimología , Proteínas Tirosina Fosfatasas/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Proteínas Quinasas Dependientes de Calcio-Calmodulina/aislamiento & purificación , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Escherichia coli , Cinética , Datos de Secuencia Molecular , Organofosfatos/metabolismo , Proteínas Tirosina Fosfatasas/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Abstract A gene, CYP119, for a potential cytochrome P450 has been isolated and sequenced from the extreme acidothermophilic archaeon Sulfolobus solfataricus. The gene predicts a polypeptide of 368 amino acids containing the consensus heme-binding sequence Phe-Gly-Xaa-Gly-Xaa-His-Xaa-Cys-Xaa-Gly- Xaa3-Ala-Arg-Xaa-Glu. It most closely resembles the cytochrome P450s found in the bacterium Bacillus subtilis, with which it shares 129 identical amino acid residues (35%). This first sequence of a potential archaeal cytochrome P450 represents an important step in tracing the complex evolutionary history of this biologically important enzyme family.