RESUMEN
The erythroid membrane cytoskeletal protein 4.1 is the prototypical member of a genetically and topologically complex family that is generated by combinatorial alternative splicing pathways and is localized at diverse intracellular sites including the nucleus. To explore the molecular determinants for nuclear localization, we transfected COS-7 cells with epitope-tagged versions of natural red cell protein 4.1 (4.1R) isoforms as well as mutagenized and truncated derivatives. Two distant topological sorting signals were required for efficient nuclear import of the 4.1R80 isoform: a basic peptide, KKKRER, encoded by alternative exon 16 and acting as a weak core nuclear localization signal (4.1R NLS), and an acidic peptide, EED, encoded by alternative exon 5. 4.1R80 isoforms lacking either of these two exons showed decreased nuclear import. Fusion of various 4.1R80 constructs to the cytoplasmic reporter protein pyruvate kinase confirmed a requirement for both motifs for full NLS function. 4.1R80 was efficiently imported in the nuclei of digitonin-permeabilized COS-7 cells in the presence of recombinant Rch1 (human importin alpha2), importin beta, and GTPase Ran. Quantitative analysis of protein-protein interactions using a resonant mirror detection technique showed that 4.1R80 bound to Rch1 in vitro with high affinity (KD = 30 nM). The affinity decreased at least 7- and 20-fold, respectively, if the EED motif in exon 5 or if 4.1R NLS in exon 16 was lacking or mutated, confirming that both motifs were required for efficient importin-mediated nuclear import of 4.1R80.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Membrana Eritrocítica/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Neuropéptidos , Células 3T3/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Células COS/metabolismo , Proteínas Portadoras/metabolismo , Citoplasma/metabolismo , Exones , Genes Reporteros , Humanos , Ratones , Mutación , Proteínas Nucleares/metabolismo , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Mapeo Peptídico , Isoformas de Proteínas , Piruvato Quinasa/metabolismo , Fracciones Subcelulares , Transfección , alfa CarioferinasRESUMEN
Erythrocyte membrane skeletal protein 4.1 isoforms have been identified in a variety of non-erythroid cells. However, interactions between protein 4.1 and its binding partners in non-erythroid cell membranes are poorly understood. In the erythrocyte membrane, protein 4.1 binds to the cytoplasmic domain of band 3 and, through this interaction, modulates ankyrin binding to band 3. The sequences LRRRY or IRRRY in band 3 mediate the interaction between band 3 and protein 4.1. The cytoplasmic domain of CD44, a transmembrane glycoprotein found in erythroid as well as non-erythroid cells, has internal sequences SRRRC and QKKKL. We wanted to determine if protein 4.1 binds to CD44 in a fashion analogous to its binding to band 3 and through this interaction modulates ankyrin binding to CD44. We report here that protein 4.1 binds to the cytoplasmic domain of CD44 with a dissociation constant on the order of 10(-7) M and that Ca2+ and calmodulin reduce the affinity of this interaction. Furthermore, although independent binding of both protein 4.1 and ankyrin to CD44 could be documented, binding of protein 4.1 prevented subsequent ankyrin binding. These studies have enabled us to identify a potentially important functional role for protein 4.1 in modulating ankyrin binding to CD44.
Asunto(s)
Calcio/metabolismo , Calmodulina/metabolismo , Proteínas del Citoesqueleto , Receptores de Hialuranos/metabolismo , Proteínas de la Membrana/metabolismo , Neuropéptidos , Ancirinas/metabolismo , Membrana Eritrocítica/ultraestructura , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Queratinocitos/metabolismo , Cinética , Sustancias Macromoleculares , Péptidos/metabolismo , Unión Proteica , Proteínas RecombinantesRESUMEN
DNA synthesis and postreplication mismatch repair were measured in vitro using cell-free extracts from cultured human SY5Y neuroblastoma and WI38 fibroblast cells in different growth states. All extracts, including differentiated SY5Y and quiescent WI38 fibroblasts, catalyzed SV40 origin-dependent DNA synthesis, totally dependent on SV40 T-antigen. Thus, although differentiated neuroblastoma and quiescent fibroblasts cells were essentially nondividing, their extracts were competent for DNA replication using DNA polymerases delta, alpha, and possibly epsilon, with proliferating cell nuclear antigen. Nonreplicative DNA synthesis and lesion bypass by either alpha- or beta-polymerases were detected independently in extracts using primed or gapped single-stranded DNA templates. Long-patch postreplication mismatch repair was measured for the first time in neuroblastoma cell-free extracts. Extracts from subconfluent and high-density SY5Y cells catalyzed postreplication mismatch repair with efficiencies comparable to those of HeLa cell extracts. No significant differences were observed in repair between SY5Y differentiated and undifferentiated cell extracts. Mismatch repair efficiencies were threefold lower in extracts from subconfluent WI38 cells, and repair in WI38 quiescent cells was fourfold less than in subconfluent cells, suggesting that mismatch repair may be regulated. The spectrum of mismatch repair in SY5Y extracts closely resembled the mismatch removal specificities of HeLa extracts: T . G and G . G mismatches were repaired most efficiently; C . A, A . A, A . G and a five-base loop were repaired with intermediate efficiency; repair of G . A, C . C, and T . T mismatches was extremely inefficient.
Asunto(s)
Reparación del ADN/fisiología , Replicación del ADN/fisiología , Neuroblastoma , Antineoplásicos/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Extractos Celulares/genética , ADN Ligasas/fisiología , ADN Polimerasa I/metabolismo , ADN Polimerasa beta/metabolismo , ADN Viral/genética , Fibroblastos/citología , Fibroblastos/enzimología , Células HeLa , Humanos , Origen de Réplica/genética , Virus 40 de los Simios/genética , Tretinoina/farmacologíaRESUMEN
Yeast splicing factor Prp43, a DEAH box protein of the putative RNA helicase/RNA-dependent NTPase family, is a splicing factor that functions late in the pre-mRNA splicing pathway to facilitate spliceosome disassembly. In this paper we report cDNA cloning and characterization of mDEAH9, an apparent mammalian homologue of Prp43. Amino acid sequence comparison revealed that the two proteins are approximately 65% identical over a 500-aa region spanning the central helicase domain and the C-terminal region. Expression of mDEAH9 in S. cerevisiae bearing a temperature-sensitive mutation in prp43 was sufficient to restore growth at the nonpermissive temperature. This functional complementation was specific, as mouse mDEAH9 failed to complement mutations in related splicing factor genes prp16 or prp22. Finally, double label immunofluorescence experiments performed with mammalian cells revealed colocalization of mDEAH9 and splicing factor SC35 in punctate nuclear speckles. Thus, the hypothesis that mDEAH9 represents the mammalian homologue of yeast Prp43 is supported by its high sequence homology, functional complementation, and colocalization with a known splicing factor in the nucleus. Our results provide additional support for the hypothesis that the spliceosomal machinery that mediates regulated, dynamic changes in conformation of pre-mRNA and snRNP RNAs has been highly conserved through evolution.
Asunto(s)
Proteínas Nucleares/genética , ARN Helicasas , ARN Nucleotidiltransferasas/genética , Empalme del ARN , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Animales , Compartimento Celular , Clonación Molecular , Secuencia Conservada , ARN Helicasas DEAD-box , Prueba de Complementación Genética , Ratones , Datos de Secuencia Molecular , ARN Mensajero/aislamiento & purificación , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
Structural protein 4.1 was first characterized as an important 80-kDa protein in the mature red cell membrane skeleton. It is now known to be a member of a family of protein isoforms detected at diverse intracellular sites in many nucleated mammalian cells. We recently reported that protein 4.1 isoforms are present at interphase in nuclear matrix and are rearranged during the cell cycle. Here we report that protein 4.1 epitopes are present in centrosomes of human and murine cells and are detected by using affinity-purified antibodies specific for 80-kDa red cell 4.1 and for 4.1 peptides. Immunofluorescence, by both conventional and confocal microscopy, showed that protein 4.1 epitopes localized in the pericentriolar region. Protein 4.1 epitopes remained in centrosomes after extraction of cells with detergent, salt, and DNase. Higher resolution electron microscopy of detergent-extracted cell whole mounts showed centrosomal protein 4.1 epitopes distributed along centriolar cylinders and on pericentriolar fibers, at least some of which constitute the filamentous network surrounding each centriole. Double-label electron microscopy showed that protein 4.1 epitopes were predominately localized in regions also occupied by epitopes for centrosome-specific autoimmune serum 5051 but were not found on microtubules. Our results suggest that protein 4.1 is an integral component of centrosome structure, in which it may play an important role in centrosome function during cell division and organization of cellular architecture.
Asunto(s)
Centrosoma/metabolismo , Proteínas del Citoesqueleto , Proteínas de la Membrana/análisis , Neuropéptidos , Animales , Línea Celular , Centrosoma/ultraestructura , Humanos , Mamíferos , Proteínas de la Membrana/ultraestructura , Microscopía ElectrónicaRESUMEN
Structural protein 4.1, first identified as a crucial 80-kD protein in the mature red cell membrane skeleton, is now known to be a diverse family of protein isoforms generated by complex alternative mRNA splicing, variable usage of translation initiation sites, and posttranslational modification. Protein 4.1 epitopes are detected at multiple intracellular sites in nucleated mammalian cells. We report here investigations of protein 4.1 in the nucleus. Reconstructions of optical sections of human diploid fibroblast nuclei using antibodies specific for 80-kD red cell 4.1 and for 4.1 peptides showed 4.1 immunofluorescent signals were intranuclear and distributed throughout the volume of the nucleus. After sequential extractions of cells in situ, 4.1 epitopes were detected in nuclear matrix both by immunofluorescence light microscopy and resinless section immunoelectron microscopy. Western blot analysis of fibroblast nuclear matrix protein fractions, isolated under identical extraction conditions as those for microscopy, revealed several polypeptide bands reactive to multiple 4.1 antibodies against different domains. Epitope-tagged protein 4.1 was detected in fibroblast nuclei after transient transfections using a construct encoding red cell 80-kD 4.1 fused to an epitope tag. Endogenous protein 4.1 epitopes were detected throughout the cell cycle but underwent dynamic spatial rearrangements during cell division. Protein 4.1 was observed in nucleoplasm and centrosomes at interphase, in the mitotic spindle during mitosis, in perichromatin during telophase, as well as in the midbody during cytokinesis. These results suggest that multiple protein 4.1 isoforms may contribute significantly to nuclear architecture and ultimately to nuclear function.
Asunto(s)
Núcleo Celular/química , Proteínas del Citoesqueleto , Fibroblastos/química , Proteínas de la Membrana/análisis , Neuropéptidos , Ribonucleoproteínas , Células 3T3 , Secuencia de Aminoácidos , Animales , Carcinoma de Células Escamosas , División Celular , Línea Celular , Replicación del ADN , Diploidia , Epítopos/análisis , Membrana Eritrocítica/química , Femenino , Fibroblastos/citología , Humanos , Ratones , Datos de Secuencia Molecular , Matriz Nuclear , Proteínas Nucleares/análisis , Péptidos , Antígeno Nuclear de Célula en Proliferación/análisis , Empalme del ARN , Factores de Empalme Serina-Arginina , Huso Acromático/química , Células Tumorales Cultivadas , Neoplasias del Cuello UterinoRESUMEN
Ethanol appears to modulate the function of selective mammalian receptors and transporters by interacting with highly specific membrane protein sites. Of the multiple types of nucleoside transporters known to be present in mammalian cells, we observed that ethanol inhibits only one class of facilitative nucleoside transporters, that inhibited by nitrobenzylmercaptopurine riboside. Because there are biochemical similarities between facilitative glucose transporters and nitrobenzylmercaptopurine riboside-sensitive nucleoside transporters, we tested whether ethanol might selectively inhibit a unique class of facilitative glucose transporters. We report here that ethanol inhibits hexose uptake in human lymphocytes and several cell lines expressing the ubiquitous facilitative type 1 glucose transporter (GLUT1). Ethanol inhibition of hexose uptake by GLUT1 is independent of ethanol inhibition of facilitative nucleoside transport. We also determined the ethanol sensitivity of various cloned human facilitative glucose transporters expressed in Chinese hamster ovary cells and we found that ethanol inhibits hexose uptake by GLUT1 but not uptake by GLUT3 or GLUT4 transporters. Our results suggest that a protein motif or motifs present in the GLUT1 amino acid sequence but absent in GLUT3 or GLUT4 proteins may confer ethanol sensitivity.
Asunto(s)
Etanol/farmacología , Proteínas de Transporte de Monosacáridos/antagonistas & inhibidores , Proteínas Musculares , Proteínas del Tejido Nervioso , Animales , Transporte Biológico , Células CHO , Células Cultivadas , Cricetinae , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 3 , Transportador de Glucosa de Tipo 4 , Hexosas/metabolismo , Humanos , Proteínas de Transporte de Monosacáridos/efectos de los fármacos , Proteínas de Transporte de Monosacáridos/metabolismo , Nucleósidos/antagonistas & inhibidores , Nucleósidos/metabolismoRESUMEN
Adenosine uptake via nucleoside transporters is inhibited when S49 and NG108-15 cell lines cells are exposed to ethanol. This inhibition leads to an accumulation of extracellular adenosine that binds to adenosine A2 receptors and increases cAMP production. Subsequently, there is a heterologous desensitization of receptors coupled to adenylyl cyclase for which adenosine also is required. There are multiple classes of facilitative and concentrative nucleoside transporters that could be inhibited by ethanol to initiate this cascade of events. In this paper, we establish that adenosine uptake by only one type of nucleoside transporter, an NBMPR-sensitive facilitative transporter, is inhibited by ethanol. There is no effect on other classes of nucleoside transporters even when present in the same cell. Thus, ethanol-induced extracellular accumulation of adenosine results specifically from inhibition of NBMPR-sensitive facilitative nucleoside transporters. We also find that human lymphocytes express only facilitative nucleoside transporters and that the NBMPR-sensitive type is predominant. Thus, inhibition of this type of transporter by ethanol may be related to the desensitization of cAMP signal transduction that we have reported in lymphocytes from alcoholics.
Asunto(s)
Adenosina/sangre , Proteínas Sanguíneas/efectos de los fármacos , Proteínas Portadoras/efectos de los fármacos , Etanol/farmacología , Proteínas de la Membrana/efectos de los fármacos , Adulto , Transporte Biológico/efectos de los fármacos , Proteínas Sanguíneas/metabolismo , Proteínas Portadoras/sangre , Formicinas/sangre , Glutamatos/sangre , Ácido Glutámico , Humanos , Técnicas In Vitro , Isoleucina/sangre , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Masculino , Proteínas de la Membrana/sangre , Persona de Mediana Edad , Proteínas de Transporte de Nucleósidos , Sodio/sangre , Tioinosina/análogos & derivados , Tioinosina/farmacologíaAsunto(s)
Adenosina/metabolismo , Alcoholismo/metabolismo , Etanol/farmacología , Linfocitos/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Transporte Biológico/efectos de los fármacos , Línea Celular , AMP Cíclico/fisiología , Humanos , Cinética , Linfoma , Ratones , Valores de ReferenciaRESUMEN
Protein kinase C (Ca2+/phospholipid-dependent protein kinase) purified from rat brain or endogenous to cell-free extracts from HeLa cells stimulates, by a factor of 2-3, HeLa DNA polymerase alpha but not beta or gamma. Monoclonal antibody to the kinase prevents the stimulation, and monoclonal antibody to human DNA polymerase alpha neutralizes the enhanced activity. Reduced DNA polymerase alpha activity is obtained from noncycling HeLa cells and this activity has lower fidelity when copying synthetic primer-templates than that obtained from log phase cultures. After exposure to the kinase, the fidelities and activities of the polymerase from both sources increase by 2- to 3-fold. This improved accuracy is not accompanied by the appearance of triphosphatase or DNase activities. Exposure to the protein kinase reduces the Km for activated DNA and for poly(dA-dT) but not for dNTPs. Moreover, the Vmax for activated DNA but not for poly(dA-dT) is increased approximately 2- to 3-fold. These alterations suggest a role for protein phosphorylation in modulating DNA polymerase alpha.
Asunto(s)
ADN Polimerasa II/metabolismo , Proteína Quinasa C/metabolismo , Animales , Encéfalo/enzimología , ADN Polimerasa II/aislamiento & purificación , Activación Enzimática , Células HeLa/enzimología , Humanos , Cinética , Fosfolípidos/farmacología , RatasRESUMEN
DNA polymerase activities from HeLa cells and from cultured diploid human fibroblasts in various growth states were compared. alpha-Polymerase activities from log phase fibroblasts treated with sodium butyrate and from stationary phase HeLa cells had DEAE-cellulose elution patterns that differed from those of polymerases from dividing cells. Moreover, alpha- and beta-polymerases from nondividing cells replicated synthetic polymers less faithfully. Although similar changes were observed previously for polymerases from late-passage and postconfluent early passage fibroblasts, amounts of alpha-polymerase activity recovered from nondividing cells in this study did not dramatically decline as they had in the former cases. The alpha-polymerase activities from HeLa cells and fibroblasts in various growth states sedimented near 7.5S in 0.4 M KCI and could be inhibited by a monoclonal IgG fraction prepared against KB cell alpha-polymerase. By several criteria, there was no significant differences in levels of UV-stimulated repair synthesis observed in early or late-passage postconfluent fibroblasts or in log phase fibroblasts treated with sodium butyrate. In summary, levels of alpha-polymerase do not necessarily correlate either with replicative activity or with apparent levels of repair synthesis. However, cells with decreased replicative activity always yielded enzyme with decreased fidelity in vitro and altered chromatographic behavior. It appears, therefore, that the alterations observed for alpha-polymerase from late-passage cells may be attributed more generally to the nondividing nature of these cells.
Asunto(s)
ADN Polimerasa II/análisis , ADN Polimerasa I/análisis , Replicación del ADN , Línea Celular , Supervivencia Celular , Cromatografía DEAE-Celulosa , ADN/biosíntesis , Reparación del ADN/efectos de la radiación , Fibroblastos/enzimología , Células HeLa , Humanos , InterfaseRESUMEN
DNA polymerase I* is a form of the DNA polymerase I isolated from Escherichia coli which are expressing recA/lexA (SOS) functions. Induction of recA or polA1 cells by nalidixic acid does not result in the appearance of pol I*, but lexA or recA mutants that are constitutive for SOS functions constitutively express pol I* and mutants which lack functional recA protein produce pol I* when they carry a lexA mutation which renders the lexA repressor inoperative. Pol I* has been induced by nalidixic acid in dinA, dinD, dinF, and umuC mutants. Polymerase I* has a lower affinity for single-stranded DNA-agarose than polymerase I and it sediments through sucrose gradients in a dispersed manner between 6.6-10.5 S, whereas polymerase I sediments at 5 S. Whereas pol I* migrates significantly faster than pol I in nondenaturing polyacrylamide gels, the active polypeptide of both forms migrates at the same rate in denaturing polyacrylamide gels. Compared with polymerase I, polymerase I* has an enhanced capacity to incorporate the adenine analog, 2-amino-purine, into activated salmon sperm DNA and a relatively low fidelity in replicating synthetic polydeoxyribonucleotides. Both the 3'----5' (proofreading) and 5'----3' (nick-translational) exonuclease activities of pol I* and pol I are indistinguishable. Estimates of processivity give a value of approximately 6 for both forms of the enzyme.
Asunto(s)
ADN Polimerasa I/metabolismo , Reparación del ADN , Escherichia coli/enzimología , Centrifugación por Gradiente de Densidad , Cromatografía de Afinidad , ADN Bacteriano/biosíntesis , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Mutación , Poli dA-dT/metabolismoRESUMEN
DNA polymerases from IMR-90 human diploid fibroblasts at various passage levels and from HeLa cells were purified and fractionated into alpha 1, alpha 2, alpha 3, beta, and gamma species and subspecies, and than the accuracy with which each one copied synthetic template-primers was measured in the presence of Mn2+ or Mg2+. All activities from fibroblasts of later population doubling levels incorporated noncomplementary triphosphates more frequently than did the same polymerase type from earlier population doubling levels. HeLa polymerase activities copied several different templates in the presence of Mn2+ with greater fidelity than enzymes from fibroblasts of population doubling level 27 or greater. The total DNA polymerase activity extracted from IMR-90 cells decreased with increasing population doubling levels. The alpha-polymerase activity generally declined with increasing population doubling levels, while beta-polymerase activity remained relatively constant, except at the very end of the cellular replicative life span. In addition, the amounts of alpha 2 and alpha 3 became progressively lower relative to alpha 1, and a new alpha-type polymerase activity, alpha 0, appeared upon diethylaminoethylcellulose chromatography. HeLa cells also contained three alpha species, though two of them eluted from diethylaminoethylcellulose at higher phosphate concentrations than alpha species from fibroblasts. Postconfluent IMR-90 cells of population doubling level 21 had a decreased level of alpha-polymerase relative to that recovered from rapidly growing cells. This polymerase activity had some chromatographic properties similar to enzyme from late-passage cells. In addition, the alpha-, beta-, and gamma-polymerases from these cells had decreased fidelities relative to those isolated from subconfluent cells.
Asunto(s)
Supervivencia Celular , ADN Polimerasa Dirigida por ADN/metabolismo , División Celular , Inhibición de Contacto , ADN Polimerasa Dirigida por ADN/aislamiento & purificación , Humanos , Especificidad por SustratoRESUMEN
A novel form of DNA polymerase I (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, DNA nucleotidyltransferase, EC 2.7.7.7) activity has been isolated from Escherichia coli cells that had been activated for expression of the DNA damage-inducible genes. Induction was by treatment of normal cells or cells carrying the spr-51 and tif-1 mutations with nalidixic acid. This activity, DNA polymerase I, seems to be a form of DNA polymerase I because it is insensitive to N-ethylmaleimide, is inhibited by antibody to DNA polymerase I, and does not appear in a polA1 strain. DNA polymerase I activity sediments through sucrose gradients as a broad peak with s20.w = 6.6--10.5, compared with an s20,w = 4.8--5.5 for DNA polymerase I. The fidelity during polymerization reactions of DNA polymerase I is relatively low with a variety of synthetic templates and deoxynucleoside triphosphates, although the enzyme appears to have a normal level of 3' greater than 5' exonuclease. Polymerase I has properties that might implicate it in some form of mutagenic DNA repair.
Asunto(s)
ADN Polimerasa I/genética , Reparación del ADN , ADN Polimerasa Dirigida por ADN/genética , Escherichia coli/genética , Escherichia coli/enzimología , Exonucleasas/metabolismo , Mutación , Recombinación Genética , Especificidad por SustratoAsunto(s)
Reparación del ADN , Endodesoxirribonucleasas/metabolismo , Endonucleasas/metabolismo , Proteínas de Escherichia coli , Exonucleasas/metabolismo , ADN Ligasas/metabolismo , ADN Polimerasa I/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Escherichia coli/enzimología , Especificidad por Sustrato , Fagos T/enzimologíaAsunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Placenta/enzimología , ADN Polimerasa I/metabolismo , ADN Polimerasa II/metabolismo , ADN Polimerasa III/metabolismo , ADN Polimerasa Dirigida por ADN/aislamiento & purificación , Exonucleasas/aislamiento & purificación , Exonucleasas/metabolismo , Femenino , Humanos , Isoenzimas/metabolismo , Cinética , Magnesio/farmacología , Manganeso/farmacología , Embarazo , Especificidad por Sustrato , Moldes GenéticosAsunto(s)
Hipoxantina Fosforribosiltransferasa , Aminoácidos/análisis , Bromuro de Cianógeno , Electroforesis en Gel de Poliacrilamida/métodos , Eritrocitos/enzimología , Células HeLa/enzimología , Humanos , Hipoxantina Fosforribosiltransferasa/sangre , Radioisótopos de Yodo , Microquímica , Peso Molecular , Fragmentos de Péptidos/análisis , Radioisótopos de Azufre , TritioRESUMEN
Hypoxanthine phosphoribosyltransferase (HPRT, IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) can be purified 5-to 10,000-fold from extracts of HeLa (human) cells by a three-step procedure consisting of high-speed centrifugation, adsorption to Sepharose-conjugated HPRT antibody, and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Purified enzyme labeled in vivo with radioactive lysine, arginine, or methionine was digested with trypsin and the tryptic peptides were separated by column chromatography on Bio-Rad cation exchanger Aminex A-5. Less than 50 ng (2 pmol) of HPRT is required to produce a tryptic peptide pattern. A methionine-labeled peptide was identified as the COOH-terminus because it was not labeled with either lysine or arginine. We have compared the tryptic peptide patterns of normal HeLaHPRT and a crossreacting HPRT protein lacking enzyme activity from HeLa mutant H23 [Milman et al. (1976) Proc. Natl. Acad. Sci. USA 73, 4589--4593]. The mutant protein has a new lysine-labeled peptide, but the chromatography patterns of arginine- or methionine-labeled peptides appear identical to those of the normal protein. The appearance in the H23 mutant HPRT protein of a new tryptic peptide provides strong evidence for a mutation in the HPRT structural gene. The tryptic peptide patterns were used to determine the total number of residues of labeled amino acid in the protein, and the values are reasonably consistent with those determined by conventional amino acid analysis pf erythrocyte HPRT.
Asunto(s)
Hipoxantina Fosforribosiltransferasa/análisis , Mutación , Células HeLa , Hipoxantina Fosforribosiltransferasa/aislamiento & purificación , Péptidos/análisis , TripsinaRESUMEN
In an effort to understand the regulation of expression of the factors required for protein biosynthesis, we have measured the turnover of elongation factor G (EF-G) and initiation factor 2 (IF-2). Using a quantitative assay which involves immunoprecipitation and gel electrophoresis, it was found that both of these factors are very stable cellular proteins, but that their rates of accumulation are quite different, indicating a differential rate of synthesis. The effects of amino acid starvation and the relaxed response were also examined. The results showed that under these conditions EF-G and IF-2 are neither synthesized nor degraded.
Asunto(s)
Proteínas Bacterianas/metabolismo , Escherichia coli/metabolismo , Factores de Elongación de Péptidos , Factores de Iniciación de Péptidos , Proteínas Bacterianas/inmunología , Marcaje Isotópico , Cinética , Leucina/metabolismo , Radioisótopos de Azufre , Factores de Tiempo , Tritio , Valina/metabolismoRESUMEN
These studies are directed toward determining whether the structural genes for protein biosynthetic factors comprise an operon subject to coordinate regulation in Escherichia coli. To assess coordinate expression of these genes, an immunoassay was devided to enable accurate quantitation of initiation and elongation factors in crude bacterial extracts. The antibodies made against highly purified initiation factor 2 (IF-2A and IF-2B) and elongation factor G (EF-G) are shown to inhibit the appropriate in vitro reactions and precipitate proteins co-migrating with appropriate factors on polyacrylamide gels. Immunoprecipitation in combination with gel electrophoresis was employed to make quantitative measurements of the amounts of IF-2 (A and B) relative to EF-G present in cells at different growth rates. The results show that the ratio of EF-G to IF-2 varies in a consistent way with the generation time of the cell. IF-2 levels remain constant as cells double more rapidly, WHEREAS THE EF-G content increases with more rapid cell growth.