RESUMO
We have identified a novel gene, Translin, encoding a protein which specifically binds to consensus sequences at breakpoint junctions of chromosomal translocations in many cases of lymphoid malignancies. The encoded protein, Translin, is a previously undescribed type with no significant similarity to known proteins. In the native form, Translin polypeptides form a multimeric structure which is responsible for its DNA binding activity. Nuclear localization of Translin is limited to lymphoid cell lines, raising the intriguing possibility that nuclear transport of Translin is regulated in a physiologically significant way such that active nuclear transport is associated with the lymphoid specific process known as Ig/TCR gene rearrangement.
Assuntos
Proteínas de Ligação a DNA/genética , Recombinação Genética , Translocação Genética , Sequência de Aminoácidos , Sequência de Bases , Transporte Biológico , Núcleo Celular/genética , Núcleo Celular/metabolismo , Clonagem Molecular , Sequência Consenso , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Rearranjo Gênico , Humanos , Zíper de Leucina , Leucemia-Linfoma de Células T do Adulto/genética , Leucemia-Linfoma de Células T do Adulto/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Células Tumorais CultivadasRESUMO
High purity phosphorus was recovered from municipal wastewater secondary effluent as phosphate, using a newly developed phosphorus adsorption and recovery system. A high-speed adsorbent having a unique porous structure was used in this system. The secondary effluent, showing total phosphorus (TP) of 0.1-2.1 mg P/L, was passed through an adsorbent packed column at high space velocity (SV) of 15 h(-1). The TP of the treated water was as low as 0.02-0.04 mg P/L, indicating that 97% of phosphorus in the secondary effluent was removed. The removed phosphorus was desorbed from the adsorbent by passing a sodium hydroxide aqueous solution through the column. Calcium hydroxide was added to this solution to precipitate the phosphorus as calcium phosphate. This precipitate was neutralized with hydrochloric acid aqueous solution, washed with water, and then solid-liquid separation was performed for the phosphorus recovery. The main constituent of the recovered phosphorus was apatite-type calcium phosphate, with 16% phosphorus content, which matched that of high-grade phosphorus ore. The hazardous elements content of the recovered phosphorus was exceedingly low. Therefore the recovered phosphorus can be applied to an alternative for phosphorus ore, or to a phosphate fertilizer.
Assuntos
Resíduos Industriais , Fósforo/isolamento & purificação , Poluentes Químicos da Água/isolamento & purificação , Adsorção , Difração de Raios XRESUMO
DNA helicase B is a major DNA helicase in mouse FM3A cells. A temperature-sensitive mutant defective in DNA replication, tsFT848, isolated from FM3A cells, has a heat-labile DNA helicase B. In this study, we purified DNA helicase B from mouse FM3A cells and determined partial amino acid sequences of the purified protein. By using a DNA probe synthesized according to one of the partial amino acid sequences, a cDNA was isolated, which encoded a 121.5 kDa protein containing seven conserved motifs for DNA/RNA helicase superfamily members. A database search revealed similarity between DNA helicase B and the alpha subunit of exodeoxyribonuclease V of a number of prokaryotes including Escherichia coli RecD protein, but no homologous protein was found in yeast. The cDNA encoding DNA helicase B from tsFT848 was sequenced and a mutation was found between DNA/RNA helicase motifs IV and V.
Assuntos
Adenosina Trifosfatases/genética , DNA Helicases/genética , Replicação do DNA/genética , DNA Complementar/genética , Proteínas de Escherichia coli , Adenosina Trifosfatases/isolamento & purificação , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Helicases/isolamento & purificação , DNA Helicases/metabolismo , DNA Complementar/química , Escherichia coli/enzimologia , Exodesoxirribonuclease V , Exodesoxirribonucleases/genética , Camundongos , Dados de Sequência Molecular , Mutação , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Temperatura , Células Tumorais CultivadasRESUMO
We have previously reported the construction of recombinant mouse salivary alpha-amylase secretion vector in Saccharomyces cerevisiae utilizing novel yeast secretion signal derived from killer 28 kDa precursor protein. Here, we have first purified recombinant mouse alpha-amylase to homogeneity from the culture medium of S. cerevisiae, and determined its NH2-terminal amino acid sequence. The sequencing data indicated that the 28 kDa killer secretion signal-alpha-amylase fusion protein was cleaved accurately at its native processing site, and that both the core-glycosylated and non-glycosylated alpha-amylases possessed the same NH2-terminal amino acid sequences.
Assuntos
Precursores Enzimáticos/genética , Micotoxinas/genética , Saccharomyces cerevisiae/enzimologia , alfa-Amilases/genética , Sequência de Aminoácidos , Animais , Cromatografia por Troca Iônica , Precursores Enzimáticos/isolamento & purificação , Proteínas Fúngicas/genética , Fatores Matadores de Levedura , Camundongos , Dados de Sequência Molecular , Peso Molecular , Plasmídeos , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Proteínas de Saccharomyces cerevisiae , Saliva/enzimologia , alfa-Amilases/isolamento & purificaçãoRESUMO
In order to better understand the cause of hereditary hearing impairment, we have performed a proteomic analysis of the inner ear proteins using two-dimensional gel electrophoresis. In the process of analysis, we have found very unique properties of the bovine homologue of the human COCH gene product. The COCH gene is responsible for one of the hereditary hearing impairments, DFNA9, and was recently suggested to be a possible genetic factor contributing to Ménière's disease. The Coch protein constitutes 70% of bovine inner ear proteins and is composed of 16 different protein spots, with charge and size heterogeneity. Heterogeneity of this protein suggests that the Coch gene is processed in several ways, at the transcriptional and/or posttranslational level. Much knowledge has accumulated about the hereditary hearing impairment genes; however, little research has been done regarding the protein products of those genes. This is the first report to characterize the Coch protein. Study of the Coch protein might provide more information on the mechanism of hearing and vestibular disorders.
Assuntos
Surdez/genética , Orelha Interna/metabolismo , Proteínas/genética , Sequência de Aminoácidos , Animais , Bovinos , Orelha Interna/química , Eletroforese em Gel Bidimensional , Endopeptidases , Dados de Sequência Molecular , Mapeamento de Peptídeos , Proteínas/química , Alinhamento de SequênciaRESUMO
Proteins having relations to hereditary dwarfism of the rdw rat (gene symbol: rdw) were searched for in various tissues of the rat with an improved two-dimensional gel electrophoresis technique followed by immunoblotting and microsequencing. Tissues inspected were cerebral cortex, cerebellum, brain trunk, hypothalamus, pituitary, thyroid gland, liver, testis, spleen, and thymus. Only pituitary and thyroid glands among those tissues showed abnormalities in protein contents. GH and PRL contents in the rdw pituitary were much less than in the normal one, which in the former were 1/15 and less than 1/30 times as much as in the latter, respectively, but the abnormalities in the rdw thyroid were far more serious than in the pituitary. At least 18 protein levels in the rdw thyroid were above, and 17 were below the normal. Those identified among the increased proteins were endoplasmin (GRP94), immunoglobulin heavy chain binding protein (BiP/GRP78), and heat shock protein 70 (hsp70), the contents of which respectively were 40 times, 10 times and more than 50 times as much in the rdw thyroid as in the normal tissue. Because BiP and endoplasmin are known to be ER resident proteins, and because all three belong to a chaperone protein family, accumulation of these proteins in the rdw thyroid suggests that protein folding and secreting disorders underlie the hypothyroidism of the rdw rat.
Assuntos
Nanismo/genética , Hipófise/patologia , Proteínas/análise , Animais , Northern Blotting , DNA Complementar/química , Feminino , Hipófise/química , RNA Mensageiro/análise , Ratos , Receptores da Tireotropina/química , Glândula Tireoide/químicaRESUMO
Hyperphosphorylated tau protein is known to be a major component of the paired helical filaments (PHFs) that accumulate in the brain of Alzheimer's patients. The kinase that phosphorylated Ser-208 and Ser-210 in PHF-tau had remained unknown. We used anti-pS208 and anti-pS210 antibodies and Western blots to confirm that the tau-tubulin kinase (TTK) phosphorylates tau at Ser-208 and at Ser-210. Using partial amino acid sequences of purified bovine brain TTK, a mouse cDNA of TTK was isolated and the sequence was determined. Its 963 bp coding region is composed of 320 amino acids and encodes a 36 kDa protein indistinguishable in size from authentic bovine brain TTK. Our immunoblot analysis demonstrated that TTK is ubiquitously distributed in the rat tissues, and that it is developmentally regulated in the rat brain.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Serina/metabolismo , Proteínas tau/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Especificidade de Anticorpos , Sequência de Bases , Western Blotting , Encéfalo/enzimologia , DNA Complementar/análise , Glutationa Transferase/genética , Quinase 3 da Glicogênio Sintase , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
We previously found a brain-specific glycoprotein in the rat brain. It postnatally increases and is rich in the mature brain. We cloned cDNA of this protein. It is composed of a signal peptide, a V-type immunoglobulin domain, two C1-type immunoglobulin domains, a transmembrane segment and a cytoplasmic region containing two tyrosine-based activation motifs (TAM) that are variants of the antigen receptor signaling motifs. The overall structure is similar to those of immune antigen receptors. This molecule, BIT (brain immunoglobulin-like molecule with TAMs), is a major endogenous substrates of brain tyrosine kinases in vitro. Cerebral cortical neurons could extend their neurites on BIT-coated substrate and anti-BIT monoclonal antibody specifically inhibited the effect. These findings and our recent study concerning BIT signal transduction mechanism suggest that BIT, an immune antigen receptor-like molecule of the brain, functions as a membrane signaling molecule that may participate in cell-cell interaction.
Assuntos
Antígenos de Diferenciação , Encéfalo/metabolismo , Glicoproteínas de Membrana , Proteínas do Tecido Nervoso/fisiologia , Molécula L1 de Adesão de Célula Nervosa , Receptores Imunológicos/fisiologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Sequência de Bases , Northern Blotting , Encéfalo/citologia , Encéfalo/imunologia , Células Cultivadas , Clonagem Molecular , Immunoblotting , Laminina/metabolismo , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Neuritos/ultraestrutura , Neurônios/citologia , Fosfotirosina/metabolismo , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/análise , Ratos , Receptores Imunológicos/química , Alinhamento de Sequência , Transdução de Sinais/fisiologiaRESUMO
During purification of tau protein kinase I and II from the bovine brain extract, a new tau protein kinase was detected and purified with phosphocellulose, gel filtration, S-Sepharose and AF-Heparin column chromatography. The molecular mass of the enzyme was determined to be 32 kDa by gel filtration and activity staining on SDS-PAGE. The enzyme is a Ser/Thr protein kinase phosphorylating tau, beta-tubulin, MAP2 and alpha-casein. Employing many synthetic peptides, the recognition site of this enzyme appears to be -SR-. The enzyme requires no second messenger and is inhibited with high concentration of heparin, but not by inhibitors of CKI. These results indicate that this enzyme, tau-tubulin kinase is novel and distinct from TPKI, II and CKI, II.
Assuntos
Encéfalo/enzimologia , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Serina-Treonina Quinases/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas tau/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Caseínas/metabolismo , Bovinos , Cromatografia , Eletroforese em Gel de Poliacrilamida , Heparina/farmacologia , Histonas/metabolismo , Cinética , Proteínas Associadas aos Microtúbulos/metabolismo , Dados de Sequência Molecular , Peso Molecular , Peptídeos/química , Peptídeos/metabolismo , Fosforilação , Cloreto de Potássio/farmacologia , Proteínas Serina-Treonina Quinases/química , Especificidade por SubstratoRESUMO
We previously reported that tau protein kinase II (TPKII) from bovine brain was composed of 30 kDa and 23 kDa subunits. The 30 kDa subunit of TPKII can be regarded as a catalytic subunit because of its ATP-binding activity. Antibodies directed against TPKII-phosphorylated tau also reacted with tau phosphorylated by cdc2 kinase obtained from starfish oocytes, indicating that TPKII and cdc2 kinase phosphorylate the same sites. We determined the amino acid sequence of the 30 kDa subunit and found it to be homologous with a cdc2-related kinase, PSSALRE/cdk5. Moreover, an antibody against PSSALRE/cdk5 reacted with the 30 kDa subunit. These results indicate that the 30 kDa subunit of TPKII is bovine homologue of PSSALRE/cdk5. Expression of the 30 kDa subunit mRNA was enhanced in juvenile rat brain. This result supports our previous hypothesis that the kinase works actively in juvenile brain.
Assuntos
Quinases Ciclina-Dependentes , Microtúbulos/enzimologia , Prolina/metabolismo , Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteína Quinase CDC2/química , Bovinos , Quinase 5 Dependente de Ciclina , DNA Complementar , Humanos , Dados de Sequência Molecular , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Homologia de Sequência de Aminoácidos , Proteínas tau/metabolismoRESUMO
We previously reported that tau protein kinase I (TPKI) induced normal tau protein into a state of paired helical filaments (PHF); this is further confirmed here by immunoblot analysis using several antibodies. We also present the amino acid sequence of TPKI, which is identical to glycogen synthase kinase 3 beta (GSK3 beta). Moreover, we found that TPKI activity was inseparable from GSK3 activity throughout the purification procedure. These results indicate that TPKI is identical to GSK3 beta.
Assuntos
Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas Quinases Dependentes de Cálcio-Calmodulina , Bovinos , DNA , Epitopos , Quinase 3 da Glicogênio Sintase , Quinases da Glicogênio Sintase , Humanos , Immunoblotting , Dados de Sequência Molecular , Proteínas Quinases/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Ratos , Especificidade por SubstratoRESUMO
In order to visualize the activity of the cAMP-dependent protein kinase (PKA) in living cells, we have constructed a new fluorescence PKA substrate by conjugating a fluorescence probe to a partial amino acid sequence of PKA regulatory domain II which contains a specific autophosphorylation site. The fluorescent peptide was cell-permeable and became phosphorylated when the intracellular cAMP concentration was increased, resulting in a decrease in its fluorescence intensity. In NG108-15 cells, PKA activity was localized to the cytosol around the nucleus. In cultured hippocampal neurons, addition of L-glutamate caused PKA activation associated with increase of the cellular cAMP.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/análise , Corantes Fluorescentes/metabolismo , Neurônios/enzimologia , Sequência de Aminoácidos , Animais , Permeabilidade da Membrana Celular , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citosol/enzimologia , Ativação Enzimática , Ácido Glutâmico/farmacologia , Hipocampo/citologia , Hipocampo/embriologia , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosforilação , Fosfosserina/metabolismo , Ratos , Células Tumorais CultivadasRESUMO
Tau protein kinase II (TPKII) was reported previously to be composed of a neuron-rich cdc2-related kinase (PSSALRE/cdk5) and 23 kDa subunit. Here we show that the 23 kDa subunit is a putative activator for the kinase activity. Amino acid sequence analysis revealed that the protein was novel and included a partial similarity of amino acids to a cyclin box important for the interaction with cdc2-related kinase. These results suggest that the 23 kDa subunit, but not cyclin, activates cdk5 in neuronal cells, which no longer exhibit cell cycling but are terminally differentiated cells.
Assuntos
Quinases Ciclina-Dependentes , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/metabolismo , Bovinos , Quinase 5 Dependente de Ciclina , Ciclinas/genética , Primers do DNA/genética , Sondas de DNA/genética , DNA Complementar/genética , Ativação Enzimática , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Conformação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Homologia de Sequência de AminoácidosRESUMO
A novel brain-specific 25 kDa protein (p25) was purified from a bovine brain extract. The protein was phosphorylated by Ser/Thr-Pro kinase (TPK II) in tau protein kinase fractions at the Ser residues of Ser-Pro sequences. Using immunoblot analysis, the protein was found only in brain extracts, and was most abundant in the brain regions such as cerebrum and hippocampus, but less abundant in cerebellum, medulla oblongata and olfactory bulb. The protein was detected in rat, bovine and human brain extracts, indicating that this protein specifically exists in mammalian brain tissues.
Assuntos
Encéfalo/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Bovinos , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Humanos , Dados de Sequência Molecular , Fosforilação , Ratos , Especificidade por Substrato , Proteínas tauRESUMO
Despite their high sequence homology, the peptide neurotoxins omega-conotoxin MVIIA and MVIIC selectively block N- and P/Q-type calcium channels, respectively. To study the recognition mechanism of calcium channel subtypes, two chimeric analogs of omega-conotoxin MVIIA and MVIIC were synthesized by exchanging their N- and C-terminal halves. Binding assay for both N- and P/Q-type calcium channels showed that amino acid residues restricted to the N-terminal half are important for the recognition of N-type channels, whereas essential residues for P/Q-type channel recognition are widely spread over the whole omega-conotoxin molecule.
Assuntos
Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/metabolismo , Canais de Cálcio/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptídeos/metabolismo , ômega-Conotoxinas , Sequência de Aminoácidos , Animais , Sítios de Ligação , Membrana Celular/metabolismo , Cerebelo/metabolismo , Dicroísmo Circular , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Peptídeos/química , Conformação Proteica , Dobramento de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Endoglycoceramidase (EGCase) cleaves the linkage between oligosaccharides and ceramides of various glycosphingolipids [Ito, M. & Yamagata, T. (1986) J. Biol. Chem. 261, 14278-14282]. A detergent was required for EGCase to express full activity, possibly due to its hydrophobic nature. Recently, activator proteins responsible for stimulating EGCase activity in the absence of detergents were isolated from the culture supernatant of Rhodococcus sp. [Ito, M., Ikegami, Y., & Yamagata, T. (1991) J. Biol. Chem. 266, 7919-7926]. The activity of activator II specific for EGCase II was heat-labile but insensitive to trypsin-treatment. This activator (69.2 kDa) was converted to the 27.9 kDa polypeptide via the 42 kDa intermediate by exhaustive trypsination, and the stimulatory activity of 27.9 kDa polypeptide on EGCase II was identical to that of the native form toward asialo GM1 and cell-surface GM3 of horse erythrocytes as substrates. This observation was successfully applied to obtain the purified activator without contamination with EGCase activity, which is abolished completely following treatment with trypsin.
Assuntos
Glicosídeo Hidrolases/metabolismo , Proteínas/metabolismo , Rhodococcus/enzimologia , Tripsina/metabolismo , Animais , Cromatografia de Afinidade/métodos , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Membrana Eritrocítica/química , Glicosídeo Hidrolases/isolamento & purificação , Glicoesfingolipídeos/sangue , Cavalos , Cinética , Peso Molecular , Fragmentos de Peptídeos/isolamento & purificação , Proteínas/isolamento & purificaçãoRESUMO
1. Four DNases were found in the dried liver extract of a top shell, Turbo cornutus. The major one was purified 120-fold by phosphocellulose column chromatography, sulfoethylcellulose column chromatography and gel-filtration on Sephadex G-150. The yield was 2.7%. 2. The enzyme activity was not affected by Mg2+ (10(-3)--10(-2)M), EDTA (10(-3)--10(-2)M), or NaCl (10(-1)M). It showed a pH optimum of 4.7--4.8. Ionic strength was found to be critical for the maximal activity. The isoelectric point was 8.5--9.0. On heating at 50 degrees C C for 5 min the enzymic activity fell to half the initial value. 3. The enzyme preparation degraded native as well as heat-denatured DNA, but not RNA. It degraded heat-denatured DNA endonucleolytically to give oligonucleotides with 3'-phosphates. 4. The 3'-phosphate and 5'-hydroxy termini of oligonucleotides were investigated. At both the 3'- and 5'-terminal positions, purine nucleotides were predominant.
Assuntos
Desoxirribonucleases/metabolismo , Moluscos/enzimologia , Desoxirribonucleases/isolamento & purificação , Ácido Edético/farmacologia , Temperatura Alta , Concentração de Íons de Hidrogênio , Fígado/enzimologia , Magnésio/farmacologia , Nucleotídeos/análise , Oligonucleotídeos/metabolismoRESUMO
1. Two main DNases were found in the dried liver extract of a snail, Achatina fulica. They were purified by the phosphocellulose batch method and by phosphocellulose column chromatography. The enzyme eluted earlier from the phosphocellulose column was designated as Achatina DNase-1 and the other as Achatina DNase-2. DNase-1 was purified further by QAE-Sephadex A-25 column chromatography (twice) just before use because of the instability of the purified enzyme. By these procedures, DNase-1 and 2 were purified 200- and 130-fold, respectively. 2. Divalent or monovalent cations had no marked effect on either enzyme. The showed pH optima of 4.8 (DNase-1) and 5.2 (DNase-2). Ionic strength was found to be critical for the maximal activity. The isoelectric points of DNase-1 and 2 were both 6.9. On heating at 70--75 degrees C for 5 min, each enzymic activity fell to half of the initial value. 3. The enzyme preparations degraded native DNA 1.5--2.5 times faster than heat-denatured DNA. They both degraded heat-denatured DNA endonucleolytically, to give oligonucleotides with 3'-phosphates. 4. The 3'-phosphoryl and 5'-hydroxy termini of the resulting oligonucleotides were analyzed. DNase-1 possessed marked specificity for dThd at 3'-termini and dAdo at 5'-termini in the early stages of degradation, but only for dAdo at 5'-termini in the later stages. DNase-2 showed some preference for purine nucleotides at both 3'- and 5'-termini in the later stages of degradation.
Assuntos
Desoxirribonucleases/isolamento & purificação , Fígado/enzimologia , Caramujos/enzimologia , Cromatografia/métodos , Cromatografia por Troca Iônica/métodos , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Desoxirribonucleases/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Desnaturação de Ácido Nucleico , Especificidade por SubstratoRESUMO
An inhibitory factor to mixed lymphocyte reaction (MLR) was purified from the supernatant of rat liver homogenate by procedures including chloroform treatment, ammonium sulfate precipitation, ion-exchange column chromatography, and gel filtration. The molecular weight of the purified inhibitor was 34,000 on SDS-PAGE. We determined the amino acid sequence of the N-terminal region of the purified inhibitor to be Glu-Glu-Pro-Trp-Met-Ser-Met-Ser-Ser-Lys-Pro-Lys-Pro-Ile-Glu-. This sequence shows a high homology to a rat arginase, the amino acid sequence of which was predicted from the nucleic acid sequence of cloned rat arginase cDNA. The amino acid sequence of the purified arginase is 6 amino acid residues longer than the predicted one. The purified MLR inhibitor showed a high arginase activity. The inhibition mechanism was studied and it was discovered that L-arginine was depleted in the culture medium, and that the supply of L-arginine to the cell culture caused recovery of the incorporation of tritium thymidine. Here we present evidence that the MLR inhibitor from rat liver homogenate is the liver arginase. It is noteworthy that immune response may be controlled by a liver factor.
Assuntos
Arginase/imunologia , Fígado/enzimologia , Teste de Cultura Mista de Linfócitos , Sequência de Aminoácidos , Sulfato de Amônio , Animais , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Imunoglobulina G/imunologia , Transplante de Fígado , Linfoma/metabolismo , Dados de Sequência Molecular , Peso Molecular , Ratos , Timidina/metabolismo , Células Tumorais Cultivadas/metabolismoRESUMO
The immunoaffinity-purified DNA polymerase alpha-primase complex from Drosophila melanogaster Kc cells contains three high molecular weight polypeptides besides the 180 kDa catalytic polypeptide. These polypeptides are immunologically cross-reactive with the 180 kDa polypeptide. When the immunoaffinity-purified complex was kept at 4 degrees C for about four weeks, the amounts of the three polypeptides increased, while the 180 kDa polypeptide completely disappeared. Sodium bisulfite inhibited the decrease in the 180 kDa polypeptide. The N-terminal amino acid sequences of all the polypeptides were all assigned to ones present in a portion close to the N-terminus of the 180 kDa polypeptide. The N-terminal residue of all the three polypeptides was Ser. The cleavage sites were Phe130-Ser131, Thr180-Ser181, and Phe237-Ser238. These results show that the three polypeptides are cleavage products of the 180 kDa catalytic polypeptide, the cleavage occurring at specific labile sites including a Ser residue. The amino acid residues at the sites are quite different from those (Lys-Lys) in the human 180 kDa catalytic polypeptide.