RESUMEN
Phospholipase activity is elevated in dividing cells. In response to growth factor stimulation, phospholipase C-gamma (PLC-gamma) binds to activated tyrosine kinase receptors via SH2 binding domains, resulting in phosphorylation of PLC-gamma and activation of its enzyme activity. These observations suggest that PLC-gamma participates in the signal transduction pathway employed by growth factors to promote mitogenesis. Consistent with this hypothesis, microinjection of purified bovine PLC-gamma into quiescent fibroblasts has been previously reported to initiate a mitogenic response [Smith et al. (1989) Proc. Natl. Acad. Sci. 86, 3659]. We have reproduced this result using recombinant rat PLC-gamma protein. Surprisingly, however, a catalytically inactive mutant of PLC-gamma, H335Q, also elicited a full mitogenic response. The capacity to induce mitogenesis by microinjection of PLC-gamma was mapped to the 'Z' domain of the protein, which contains PLC-gamma's SH2 and SH3 motifs. Inactivation of the phosphorylated tyrosine binding properties of both SH2 domains had no effect on the mitogenic activity of the Z-domain peptide. However, deletion of the SH3 domain resulted in a complete loss of activity. These results suggest that PLC-gamma's mitogenic properties do not require the enzyme's phospholipase activity, but are instead mediated by a novel pathway for mitogenic stimulation which is dependent upon an intact SH3 domain.
Asunto(s)
Isoenzimas/farmacología , Mitógenos/farmacología , Fosfolipasas de Tipo C/farmacología , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Catálisis , Cartilla de ADN , Humanos , Isoenzimas/química , Ratones , Microinyecciones , Mitógenos/química , Datos de Secuencia Molecular , Fosfolipasa C gamma , Fosforilación , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Fosfolipasas de Tipo C/químicaRESUMEN
E2F is a mammalian transcription factor involved in cell cycle regulation. The retinoblastoma gene product, pRB, binds to E2F in a cell cycle-dependent manner and appears to turn E2F from a transcriptional activator into a repressor. We show here that in vitro binding of pRB has three major effects on the DNA binding properties of E2F affinity-purified from HeLa cells; pRB binding increases the half-life of E2F.DNA complexes 10-15-fold, it reduces E2F specific DNA binding in the presence of nonspecific DNA by sequestering E2F, and it partially reverses the DNA bending induced by E2F. Upon specific DNA binding, E2F induces a DNA bend with a flexure angle of 125 degrees. Both full-length pRB105 and the N-terminally truncated pRB60 bind to the E2F.DNA complex with a Kd,app of 150 pM and reduce the apparent DNA bending to less than 80 degrees. DNA footprinting analysis indicates that the nonspecific DNA binding activity of pRB is not involved in this effect. Our biochemical data suggest that transcriptional activation by E2F may involve DNA bending and that the reversal of bending upon binding of pRB may turn E2F into a repressor.
Asunto(s)
Proteínas Portadoras , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , ADN/química , ADN/metabolismo , Oligodesoxirribonucleótidos/metabolismo , Proteína de Retinoblastoma/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Bases , Sitios de Unión , Cromatografía de Afinidad , Desoxirribonucleasa I , Factores de Transcripción E2F , Células HeLa , Humanos , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Represoras/metabolismo , Proteína 1 de Unión a Retinoblastoma , Factor de Transcripción DP1 , Factores de Transcripción/aislamiento & purificaciónRESUMEN
E2F is a mammalian transcription factor that appears to play an important role in cell cycle regulation. While at least two proteins (E2F-1 and DP-1) with E2F-like activity have been cloned, studies from several laboratories suggest that additional homologs may exist. A novel protein with E2F-like properties, designated E2F-2, was cloned by screening a HeLa cDNA library with a DNA probe derived from the DNA binding domain of E2F-1 (K. Helin, J. A. Lees, M. Vidal, N. Dyson, E. Harlow, and A. Fattaey, Cell 70:337-350, 1992). E2F-2 exhibits overall 46% amino acid identity to E2F-1. Both the sequence and the function of the DNA and retinoblastoma gene product binding domains of E2F-1 are conserved in E2F-2. The DNA binding activity of E2F-2 is dramatically enhanced by complementation with particular sodium dodecyl sulfate-polyacrylamide gel electrophoresis-purified components of HeLa cell E2F, and anti-E2F-2 antibodies cross-react with components of purified HeLa cell E2F. These observations are consistent with a model in which E2F binds DNA as a heterodimer of two distinct proteins, and E2F-2 is functionally and immunologically related to one of these proteins.
Asunto(s)
Proteínas Portadoras , Proteínas de Ciclo Celular , Factores de Transcripción/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión/genética , Clonación Molecular , Secuencia Conservada , Cartilla de ADN/genética , ADN Complementario/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Factor de Transcripción E2F2 , Glutatión Transferasa/genética , Células HeLa , Humanos , Datos de Secuencia Molecular , Conformación Proteica , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/genética , Proteína 1 de Unión a Retinoblastoma , Factor de Transcripción DP1 , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
To assess the effect of oral candidiasis (OC) associated with human immunodeficiency virus (HIV) at initial hospital admission on both survival and hospital use, a retrospective analysis was performed in 1,172 hospitalized patients identified by an HIV surveillance program at an inner city public hospital in East Harlem, New York. Survival times were compared using three different HIV staging schemes placing patients with OC into either a common stage with adenopathy patients (Scheme IHS-URV), a common stage with acquired immune deficiency syndrome patients (AIDS; Scheme WRCDC), or an intermediate stage between AIDS patients and all others (Scheme ORAL). Patients without AIDS demonstrated a significantly increased risk of dying (relative risk, 2.61; 95% confidence interval [CI], 1.69, 4.03) if they were initially admitted with OC. Survival times for different stages of disease showed the best between-stage distinction for a Scheme ORAL, with the OC stage having a median survival of 643 days. Mean days of hospitalization also showed best distinctions for Scheme ORAL. Other staging schemes did not distinguish patients as well in terms of both survival times or mean hospitalization days. HIV-infected patients admitted with OC but without AIDS had a discrete survival prognosis and hospitalization course. Therefore, presence of OC even without other immunologic data has implications for institutional resource allocation and planning. These data support, in this context, a separate clinical designation for OC patients.
Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/mortalidad , Síndrome de Inmunodeficiencia Adquirida/mortalidad , Candidiasis Bucal/mortalidad , Adolescente , Adulto , Anciano , Femenino , Humanos , Tiempo de Internación , Masculino , Persona de Mediana Edad , Admisión del Paciente , Pronóstico , Sistema de Registros , Estudios Retrospectivos , Riesgo , Tasa de SupervivenciaRESUMEN
E2F is a mammalian transcription factor that appears to play an important role in cell cycle control. DNA affinity column-purified E2F from HeLa cells reproducibly exhibits multiple protein bands when analyzed by SDS/PAGE. After electrophoretic purification, electroelution, and refolding of the individual protein components, the E2F DNA binding activity of the individual proteins was poor. However, upon mixing the individual components together, a dramatic (100- to 1000-fold) increase in specific DNA binding activity was observed. The five protein bands isolated can be separated into two groups based on apparent molecular mass. Optimal reconstitution of activity requires one of the two proteins found in the group of larger molecular mass (approximately 60 kDa) and one of the three proteins in the smaller-sized group (approximately 50 kDa). The reconstituted heterodimer is identical to authentic affinity-purified E2F by three criteria: DNA-binding specificity, DNA pattern, and binding to the retinoblastoma gene product. A recently cloned protein with E2F-like activity, RBP3/E2F-1, is related to the protein components of the group of larger molecular mass, as determined by Western blot analysis and reconstitution experiments. These data suggest that E2F, like many other transcription factors, binds DNA as an oligomeric complex composed of at least two distinct proteins.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Factores de Transcripción/metabolismo , Adenovirus Humanos/genética , Secuencia de Bases , Sitios de Unión , Western Blotting , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Cromatografía de Afinidad , ADN Viral/genética , ADN Viral/metabolismo , Proteínas de Unión al ADN/aislamiento & purificación , Factores de Transcripción E2F , Glutatión Transferasa/genética , Glutatión Transferasa/aislamiento & purificación , Glutatión Transferasa/metabolismo , Células HeLa , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/metabolismo , Regiones Promotoras Genéticas , Desnaturalización Proteica , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Proteína de Retinoblastoma/metabolismo , Proteína 1 de Unión a Retinoblastoma , Especificidad por Sustrato , Factor de Transcripción DP1 , Factores de Transcripción/aislamiento & purificaciónRESUMEN
Human papillomaviruses (HPVs) are the etiological agents for genital warts and contribute to the development of cervical cancer in humans. The HPV E7 gene product is expressed in these diseases, and the E7 genes from HPV types 16 and 18 contribute to transformation in mammalian cells. Mutation and deletion analysis of this gene suggests that the transforming activity of the protein product resides in the same domain as that which is directly involved in complex formation with the retinoblastoma gene product (pRB). This domain is one of two conserved regions (designated CRI and CRII) shared by E7 and other viral oncoproteins which bind pRB, including adenovirus E1A protein. Binding of HPV type 16 E7 protein to pRB has previously been shown to affect pRB's ability to bind DNA and to form complexes with other cellular proteins. In the current study, we map the functional interaction between E7 protein and pRB by monitoring the association between a 60-kDa version of the pRB, pRB60, and the cellular transcription factor E2F. We observe that CRII of E7 (amino acids 20 to 29), which completely blocks binding of full-length E7 protein, is necessary but not sufficient to inhibit E2F/pRB60 complex formation. While CRI of E1A (amino acids 37 to 55) appears to be sufficient to compete with E2F for binding to pRB60, the equivalent region of E7 is neither necessary nor sufficient. Only E7 fragments that contained both CRII and at least a portion of the zinc-binding domain (amino acids 60 to 98) inhibited E2F/pRB60 complex formation. These results suggest that pRB60 associates with E7 and E2F through overlapping but distinct domains.
Asunto(s)
Proteínas Portadoras , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Proteínas Oncogénicas Virales/metabolismo , Proteína de Retinoblastoma/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Unión Competitiva , ADN/metabolismo , Factores de Transcripción E2F , Epítopos , Células HeLa , Humanos , Datos de Secuencia Molecular , Proteínas Oncogénicas Virales/química , Proteínas E7 de Papillomavirus , Fragmentos de Péptidos/metabolismo , Proteína de Retinoblastoma/química , Proteína 1 de Unión a Retinoblastoma , Factor de Transcripción DP1 , Factores de Transcripción/químicaRESUMEN
The retinoblastoma susceptibility gene (RB) encodes a 928-amino acid protein (pRB) that is hypothesized to function in a pathway that restricts cell proliferation. The immortalizing proteins from three distinct DNA tumor viruses (SV40 large T antigen, adenovirus E1a, and human papilloma virus Type 16 E7) have been shown to interact with RB protein through two noncontiguous regions comprised of amino acids 393-572 (domain A) and 646-772 (domain B). We constructed a truncated form of RB (RB p60) that retains these two domains but eliminates the N-terminal 386 amino acids of RB. RB p60 was expressed in Escherichia coli in inclusion bodies. After solubilization, it was refolded in the presence of magnesium chloride, and the active protein was isolated with an E7 peptide affinity column. The protein that elutes from this column is functionally homogenous in its ability to bind immobilized E7 protein. Thermal denaturation studies provide additional evidence for the conformational homogeneity of the isolated protein. This purification scheme allows the isolation of significant amounts of RB p60 protein that is suitable for structural and functional studies.
Asunto(s)
Proteína de Retinoblastoma/aislamiento & purificación , Cromatografía de Afinidad , Dicroismo Circular , ADN/genética , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Expresión Génica , Genes Bacterianos , Calor , Humanos , Proteínas Oncogénicas Virales/metabolismo , Proteínas E7 de Papillomavirus , Plásmidos , Conformación Proteica , Desnaturalización Proteica , Proteína de Retinoblastoma/metabolismoRESUMEN
The recent increase of tuberculosis nationally and in New York City has been attributed in part to the progression of the acquired immunodeficiency syndrome (AIDS)/human immunodeficiency syndrome (HIV) epidemic. The East Harlem/South Bronx/Bushwick sections of New York City have an especially high incidence of tuberculosis. An HIV/AIDS registry (1986-1990) consisting of 1,312 patients from a community hospital serving East Harlem was examined for population characteristics associated with documented tuberculosis in the registrants. Tuberculosis affected males more commonly than females and was observed in comparable frequency in patients with AIDS-related complex (ARC) (12.9%) and AIDS (15.0%). The proportion of cases in blacks (18.3%) was significantly greater than that in Hispanics (10.4%, chi 2 = 15.196, p = 0.0003) or whites (8.7%, chi 2 = 5.62, p = 0.0171). Among intravenous (IV) drug users, the proportion of tuberculosis cases was also significantly higher in blacks than in Hispanics. These data could be consistent with a difference in exposure to tuberculosis and/or the purported racial susceptibility of blacks to tuberculosis infection. A review of new tuberculosis cases in East Harlem also suggests that blacks are at a greater risk for developing tuberculosis than Hispanics.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/complicaciones , Infecciones por VIH/complicaciones , Tuberculosis/epidemiología , Complejo Relacionado con el SIDA/complicaciones , Complejo Relacionado con el SIDA/etnología , Síndrome de Inmunodeficiencia Adquirida/etnología , Adulto , Negro o Afroamericano , Femenino , Infecciones por VIH/etnología , Hispánicos o Latinos , Humanos , Masculino , Persona de Mediana Edad , Ciudad de Nueva York/epidemiología , Sistema de Registros , Factores de Riesgo , Tuberculosis/complicaciones , Tuberculosis/etnología , Población BlancaRESUMEN
Human interleukin-1 alpha, cloned and expressed in E. coli, has been purified and structurally characterized by various physiochemical methods, including mass spectrometry. The recombinant protein has been crystallized by the hanging drop vapor diffusion method using dimethyl sulfoxide as the precipitating agent. The space group is P2(1)2(1)2(1). Unit cell dimensions are a = 44.1, b = 57.1, and c = 61.7 A and alpha = beta = gamma = 90 degrees. The crystals diffract to beyond 1.7 A and are suitable for high resolution data collection. Native diffraction data were collected. Screens for heavy atom derivatives have been initiated.
Asunto(s)
Interleucina-1/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Difracción de Rayos X , Secuencia de Aminoácidos , Cromatografía en Agarosa , Cromatografía DEAE-Celulosa , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Cromatografía de Gases y Espectrometría de Masas , Humanos , Focalización Isoeléctrica , Datos de Secuencia MolecularRESUMEN
The stereochemical configurations of the Mn(II) complexes with the resolved epimers of adenosine 5'-O-(1-thiodiphosphate) (ADP alpha S), bound at the active site of creatine kinase, have been determined in order to assess the relative strengths of enzymic stereoselectivity versus Lewis acid/base preferences in metal-ligand binding. Electron paramagnetic resonance (EPR) data have been obtained for Mn(II) in anion-stabilized, dead-end (transition-state analogue) complexes, in ternary enzyme-MnIIADP alpha S complexes, and in the central complexes of the equilibrium mixture. The modes of coordination of Mn(II) at P alpha in the nitrate-stabilized, dead-end complexes with each epimer of ADP alpha S were ascertained by EPR measurements with (Rp)-[alpha-17O]ADP alpha S and (Sp)-[alpha-17O]ADP alpha S. The EPR spectrum for the complex with (Rp)-[alpha-17O]ADP alpha S showed inhomogeneous broadening due to unresolved superhyperfine coupling from coordinated 17O at P alpha. By contrast, the EPR spectrum for Mn(II) in complex with (Sp)-[alpha-17O]ADP alpha S is indistinguishable from that obtained for a matched sample with unlabeled (Sp)-ADP alpha S. A reduction in the magnitude of the 55Mn hyperfine coupling constant in the spectrum for the complex containing (Sp)-ADP alpha S is indicative of Mn(II)-thio coordination at P alpha.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Adenosina Difosfato/análogos & derivados , Creatina Quinasa/metabolismo , Magnesio/metabolismo , Manganeso/metabolismo , Tionucleótidos/metabolismo , Adenosina Difosfato/metabolismo , Animales , Sitios de Unión , Espectroscopía de Resonancia por Spin del Electrón , Cinética , Conformación Molecular , Músculos/enzimología , Conejos , Especificidad por SustratoRESUMEN
Recently, p-cresol has been shown to be a mechanism-based inhibitor of dopamine beta-hydroxylase (DBH; EC 1.14.17.1) [Goodhart, P. J., DeWolf, W. E., Jr., & Kruse, L. I. (1987) Biochemistry 26, 2576-2583]. This inactivation was suggested to result from alkylation of an active site residue by an aberrant 4-hydroxybenzyl radical intermediate. In support of this hypothesis, we report here the isolation and characterization of two modified tryptic peptides from DBH inactivated by p-cresol. Using a combination of automated Edman sequencing, mass spectroscopy (MS), and tandem MS, we have determined the sequence of the putative active site peptides, identified the site of attachment of p-cresol, and defined the chemical nature of the adduct formed. Both modified peptides are the same primary sequence: Ala-Pro-Asp-Val-Leu-Ile-Pro-Gly-Gln-Gln-Thr-Thr-Tyc-Trp-Cys-Tyr-Va l-Thr-Glu- Leu-Pro-Asp-Gly-Phe-Pro-Arg, where Tyc is an amino acid residue with the in-chain mass of a cresol-Tyr adduct (106 + 163 Da). Gas-phase deuterium exchange studies (employing N2H3-DCI MS) of the isolated phenylthiohydantoin (Pth) derivatives of modified residue 13 demonstrate that p-cresol forms two chemically distinct covalent adducts and support the hypothesis that a (4-hydroxyphenyl)methyl radical is generated during catalysis. Rearrangement to a (4-methylphenyl)oxy radical may also occur prior to inactivation.
Asunto(s)
Cresoles/farmacología , Dopamina beta-Hidroxilasa/antagonistas & inhibidores , Alquilación , Secuencia de Aminoácidos , Sitios de Unión , Cromatografía Líquida de Alta Presión , Dopamina beta-Hidroxilasa/análisis , Activación Enzimática/efectos de los fármacos , Espectrometría de Masas , Modelos Químicos , Datos de Secuencia Molecular , Fragmentos de Péptidos/aislamiento & purificación , Mapeo PeptídicoRESUMEN
The mechanism-based inhibition of dopamine beta-hydroxylase (DBH; EC 1.14.17.1) by p-cresol (4-methylphenol) and other simple structural analogues of dopamine, which lack a basic side-chain nitrogen, is reported. p-Cresol binds DBH by a mechanism that is kinetically indistinguishable from normal dopamine substrate binding [DeWolf, W. E., Jr., & Kruse, L. I. (1985) Biochemistry 24, 3379]. Under conditions (pH 6.6) of random oxygen and phenethylamine substrate addition [Ahn, N., & Klinman, J. P. (1983) Biochemistry 22, 3096] p-cresol adds randomly, whereas at pH 4.5 or in the presence of fumarate "activator" addition of p-cresol precedes oxygen binding as is observed with phenethylamine substrate. p-Cresol is shown to be a rapid (kinact = 2.0 min-1, pH 5.0) mechanism-based inactivator of DBH. This inactivation exhibits pseudo-first-order kinetics, is irreversible, is prevented by tyramine substrate or competitive inhibitor, and is dependent upon oxygen and ascorbic acid cosubstrates. Inhibition occurs with partial covalent incorporation of p-cresol into DBH. A plot of -log kinact vs. pH shows maximal inactivation occurs at pH 5.0 with dependence upon enzymatic groups with apparent pK values of 4.51 +/- 0.06 and 5.12 +/- 0.06. p-Cresol and related alkylphenols, unlike other mechanism-based inhibitors of DBH, lack a latent electrophile. These inhibitors are postulated to covalently modify DBH by a direct insertion of an aberrant substrate-derived benzylic radical into an active site residue.
Asunto(s)
Médula Suprarrenal/enzimología , Cresoles/farmacología , Dopamina beta-Hidroxilasa/antagonistas & inhibidores , Fenoles/farmacología , Animales , Bovinos , Gránulos Cromafines/enzimología , Hidroxilación , Cinética , Relación Estructura-Actividad , Tritio , Tiramina/farmacologíaRESUMEN
The synthesis and kinetics characterization of a new class of dopamine beta-hydroxylase (DBH; EC 1.14.17.1) inhibitor, 1-(4-hydroxybenzyl)imidazole-2-thiol, is reported. These inhibitors, which incorporate a phenethylamine substrate mimic and an oxygen mimic into a single molecule, exhibit both the kinetic properties and the potency (Kis approximately 10(-9) M) expected for a multisubstrate inhibitor and are therefore classified as such. Steady-state kinetic experiments with these multisubstrate inhibitors and their substructural analogues support the recently proposed pH-dependent changes in substrate binding order [Ahn, N., & Klinman, J. P. (1983) Biochemistry 22, 3106] and a mechanism whereby the inhibitor binds specifically to the reduced Cu+ form of enzyme at both the phenethylamine substrate site and the active-site copper atom(s). A Yonetani-Theorell double-inhibition experiments indicates mutually exclusive binding of the inhibitor substructures p-cresol and 1-methylimidazole-2-thiol to suggest an extremely short intersite distance between the phenethylamine binding site and the active-site copper atom(s).
Asunto(s)
Dopamina beta-Hidroxilasa/antagonistas & inhibidores , Imidazoles/síntesis química , Médula Renal/enzimología , Animales , Bovinos , Gránulos Cromafines/enzimología , Imidazoles/farmacología , Indicadores y Reactivos , Cinética , Matemática , Relación Estructura-ActividadRESUMEN
Coordination of Mn(II) to the phosphate groups of the substrates and products in the central complexes of the creatine kinase reaction mixture has been investigated by electron paramagnetic resonance (EPR) spectroscopy with regiospecifically 17O-labeled substrates. The EPR pattern for the equilibrium mixture is a superposition of spectra for the two central complexes, and this pattern differs from those observed for the ternary enzyme-Mn(II)-nucleotide complexes and from that for the dead-end complex enzyme-Mn(II)ADP-creatine. In order to identify those signals that are associated with each of the central complexes of the equilibrium mixture, spectra were obtained for a complex of enzyme, Mn(II)ATP, and a nonreactive analogue of creatine, 1-(carboxymethyl)-2-iminoimidazolidin-4-one, which is a newly synthesized competitive inhibitor. This inhibitor permits an unobstructed view of the EPR spectrum for Mn(II)ATP in the closed conformation of the active site. The EPR spectrum for this nonreactive complex with Mn(II)ATP matches one subset of signals in the spectrum for the equilibrium mixture, i.e., those due to the enzyme-Mn(II)-ATP-creatine complex. Chemical quenching of the samples followed by chromatographic assays for both ATP and ADP indicates that the enzyme-Mn(II)ADP-phosphocreatine and the enzyme-Mn(II)ATP-creatine complexes are present in a ratio of approximately 0.7 to 1. A similar value for the equilibrium constant for enzyme-bound substrates is obtained directly from the EPR spectrum for the equilibrium mixture.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Adenosina Difosfato , Adenosina Trifosfato , Creatina Quinasa , Fosfocreatina , Animales , Sitios de Unión , Creatina Quinasa/antagonistas & inhibidores , Espectroscopía de Resonancia por Spin del Electrón , Cinética , Manganeso , Músculos/enzimología , Isótopos de Oxígeno , Unión Proteica , ConejosRESUMEN
Pyruvate kinase from rabbit muscle catalyzes an ATP-dependent phosphorylation of glycolate to yield 2-phosphoglycolate (F. J. Kayne (1974) Biochem. Biophys. Res. Commun. 59, 8-13). An investigation of anologous reactions with other alpha-substituted carboxylic acids reveals several new substrates for such a phosphorylation reaction. Thus the alpha-hydroxy carboxylic acids L-lactate, D-lactate, DL-alpha-hydroxybutyrate, DL-alpha-hydroxyvalerate, L-glycerate, D-glycerate, DL-nitrolactate, and DL-beta-chlorolactate are phosphorylated on the alpha-hydroxy group to give the corresponding phosphoesters. Thioglycolate is also a slow substrate for phosphorylation of the thiol group to give the phosphothioglycolate, and DL-thiolactate is phosphorylated in a very slow reaction to give phosphothiolactate. beta-Hydroxypyruvate is a substrate; but, unlike the reaction with pyruvate, with beta-hydroxypyruvate the equilibrium for the reaction lies in favor of ADP and the phosphorylated product which appears from 31P NMR data to be tartronate-semialdehyde-2-phosphate. 31P NMR spectroscopy has been used to verify the identity of the products for all of the reactions. Steady-state kinetic constants have been obtained for some of the more rapid reactions. The reactions with glycolate, L-glycerate, and beta-hydroxypyruvate have kcat values that are close to that for phosphorylation of pyruvate in the reverse of the physiological reaction.
Asunto(s)
Adenosina Trifosfato/farmacología , Ácidos Carboxílicos/metabolismo , Piruvato Quinasa/metabolismo , Animales , Glicolatos/metabolismo , Cinética , Espectroscopía de Resonancia Magnética , Fosforilación , Piruvatos/metabolismo , Conejos , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The presence of highly resistant gram-negative microogranisms is an area of major concern in the treatment of burned patients. Criteria for the germicide used for hydrotherapy must include effectiveness against the organism, absence of gross side effects, and conservation of human effort and materials. In the program described, these criteria seem to be successfully met. Microbial samplings from numerous patients and repeated examinations of residue taken from equipment demonstrate the elimination of pseudomonas and other gram-negative organisms.