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1.
J Lipid Res ; 50(7): 1497-504, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19318686

RESUMO

The expression of recombinant apolipoproteins provides experimental avenues that are not possible with plasma purified protein. The ability to specifically mutate residues or delete entire regions has proven to be a valuable tool for understanding the structure and function of apolipoproteins. A common feature of many recombinant systems is an affinity tag that allows for straightforward and high-yield purification of the target protein. A specific protease can then cleave the tag and yield the native recombinant protein. However, the application of this strategy to apolipoproteins has proven somewhat problematic because of the tendency for these highly flexible proteins to be nonspecifically cleaved at undesired sites within the native protein. Although systems have been developed using a variety of proteases, many suffer from low yield and, especially, the high cost of the enzyme.We developed a method that utilizes the tobacco etch virus protease to cleave a histidine-tag from apolipoproteins A-I and A-IV expressed in Escherichia coli. This protease can be easily and inexpensively expressed within most laboratories. We found that the protease efficiently cleaved the affinity tags from both apolipoproteins without nonspecific cleavage. All structural and functional measurements showed that the proteins were equivalent to native or previously characterized protein preparations. In addition to cost-effectiveness, advantages of the tobacco etch virus protease include a short cleavage time, low reaction temperature, and easy removal using the protease's own histidine-tag.


Assuntos
Apolipoproteína A-I/isolamento & purificação , Apolipoproteínas A/isolamento & purificação , Endopeptidases/metabolismo , Proteínas Recombinantes/isolamento & purificação , Sequência de Aminoácidos , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Apolipoproteínas A/genética , Apolipoproteínas A/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
2.
Zhonghua Yan Ke Za Zhi ; 43(2): 151-7, 2007 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-17459247

RESUMO

OBJECTIVE: To identify the active anti-angiogenic region in the amino acid sequence of human apolipoprotein (a) [apo (a)] kringle V (KV), and to evaluate the role of this synthetic peptide on VEGF-induced angiogenesis of mouse cornea in vivo. METHODS: The characterization of the structure and biological activity of the amino acid sequence of apo (a) KV was analyzed using the bioinformatic methods which included sequence alignment, analysis of antigenicity, surface accessibility and hydrophilicity, and then a peptides was selected. The peptide was synthesized with a high efficiency solid-phase method. Corneal neovascularization was induced with a pellet containing 160 ng vascular endothelial growth factor (VEGF) in a mouse corneal micropocket model. 40 C57BL/6 mice (40 eyes) were divided randomly into 4 groups (10 eyes per group). Four kinds of pellets were made containing 160 ng VEGF plus the dose range of 0.0, 0.5, 1.0 and 1.5 microg synthetic peptide for control group, group A, group B and group C, respectively. Neovascularization was observed biomicroscopically on day 7 after the operation, and the corneas were then examined histologically. RESULTS: The result of bioinformatic analysis showed that the peptide contained a majority of conservative residues and possessed fine properties of antigenicity, surface accessibility and hydrophilicity. The synthetic peptide at the doses of 1.0 microg and 1.5 microg showed significant inhibition of mouse corneal neovascularization induced by VEGF in the parameters of vessel length, clock hours and area compared with the control group on day 7 after the operation (P < 0.01). There was no difference in the two doses (1.0 microg and 1.5 microg peptide) in the inhibition of the neovascularization. The dose of 0.5 microg peptide did not show any significant inhibition of the neovascularization compared with the control group (P > 0.05). CONCLUSIONS: The peptide, selected from the amino acid sequence of apo (a) KV by bioinformatics, appears to inhibit VEGF-induced angiogenesis in a mouse corneal micropocket assay in vivo, therefore, the study suggest that this amino acid sequence may locate at the active anti-angiogenic region of apo (a) KV.


Assuntos
Apolipoproteínas A/genética , Biologia Computacional , Neovascularização da Córnea , Peptídeos/genética , Sequência de Aminoácidos , Animais , Apolipoproteínas A/isolamento & purificação , Apolipoproteínas A/uso terapêutico , Neovascularização da Córnea/tratamento farmacológico , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/isolamento & purificação , Peptídeos/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/metabolismo
3.
Biosci Biotechnol Biochem ; 70(4): 916-25, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16636459

RESUMO

In this study, the Kringle V domain (Glu4225-Ser4310) of human apolipoprotein A, an antiangiogenic polypeptide, was expressed as a secreted form in Pichia pastoris, and was purified via a process consisting of three chromatographic steps. The chromatographically purified kringle V domain contained a C-terminal serine-deleted form and several high-molecular-weight forms, which were suspected to represent glycosylated derivatives. In order to remove these derivatives, we employed a crystallization process. The crystallization of kringle V resulted in an 85% recovery yield, and also resulted in the complete removal of the aforementioned high-molecular-weight forms. However, we were still able to detect a trace of the C-terminal serine-deleted form. The prepared Kringle V crystals were stable within a pH range of 7.0 to 8.0, and were completely dissolved by dilution, which is a crucial factor in the preparation of a highly concentrated formulation. The chromatogram of the crystallized kringle V on reversed-phase HPLC analysis was identical to that observed without crystallization. Also, we noted that the original anti-wound migration activities of the molecule toward human umbilical vein endothelial cells were completely retained.


Assuntos
Inibidores da Angiogênese/química , Inibidores da Angiogênese/metabolismo , Apolipoproteínas A/química , Apolipoproteínas A/metabolismo , Inibidores da Angiogênese/isolamento & purificação , Inibidores da Angiogênese/farmacologia , Apolipoproteínas A/isolamento & purificação , Apolipoproteínas A/farmacologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Cromatografia Líquida , Cristalização , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Kringles , Espectrometria de Massas , Peso Molecular , Solubilidade , Cordão Umbilical/citologia , Cordão Umbilical/efeitos dos fármacos
4.
Protein Expr Purif ; 45(1): 216-25, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16260151

RESUMO

A kringle fragment (type IV (9)-IV (10)-V) from human apolipoprotein (a) (called LK68) was expressed in an inclusion body in Escherichia coli. The LK68 in this inclusion body was rendered soluble with urea, and efficiently refolded via oxidation in the presence of re-dox couple. The refolded LK68 was then purified via two steps of ion exchange chromatography, concentrated via preparative reversed-phase chromatography, and freeze-dried, at a final yield of approximately 30%. The purified LK68 exhibited profound affinity for lysine and fibrinogen, which suggests the proper folding of the kringle fragment, and also indicates that the native characteristics of apolipoprotein (a) were preserved. The purified LK68 was determined to be highly homogeneous upon reversed-phase HPLC analysis and size-exclusion HPLC analysis, in the presence of 20% (v/v) acetonitrile. However, on size-exclusion HPLC analysis without acetonitrile, it was determined to be somewhat heterogeneous, and this was corroborated by native analyses, including native PAGE and IEF.


Assuntos
Inibidores da Angiogênese/química , Apolipoproteínas A/química , Escherichia coli/química , Corpos de Inclusão/química , Kringles/genética , Proteínas Recombinantes/química , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/isolamento & purificação , Apolipoproteínas A/genética , Apolipoproteínas A/isolamento & purificação , Cromatografia de Afinidade/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Fibrinogênio/química , Fibrinogênio/isolamento & purificação , Regulação da Expressão Gênica , Engenharia Genética , Humanos , Técnicas In Vitro , Corpos de Inclusão/genética , Corpos de Inclusão/metabolismo , Lisina/química , Peso Molecular , Dobramento de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Fatores de Tempo
5.
Proteomics ; 3(5): 666-74, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12748946

RESUMO

Hepatitis B virus (HBV), a serious infectious and widespread human pathogen, represents a major health problem worldwide. Chronic HBV infection has a very high risk of evolving into hepatocellular carcinoma. Although considerable progress was made during the recent past, the pathogenesis of HBV infection is still elusive and a definite diagnosis of HBV infected liver information still relies on biopsy histological test. In this report, we used proteomics technology to globally examine HBV infected serum samples aiming at searching for disease-associated proteins that can be used as serological biomarkers for diagnosis and/or target proteins for pathogenetic study. By comparing with normal and HBV negative serum samples, we found that at least seven proteins were significantly changed in HBV infected sera. These greatly altered proteins were identified to be haptoglobin beta and alpha2 chain, apolipoprotein A-I and A-IV, alpha1-antitrypsin, transthyretin and DNA topoisomerase IIbeta. The alteration of these proteins is displayed not only in quantity but also in patterns (or specificity), which can be correlated with necroinflammatory scores. In particular, apolipoprotein A-I presents heterogeneous change in expression level with different isoforms and alpha1-antitrypsin produces evidently different fragments implying diverse cleavage pathways. These unique phenomena appear specific to HBV infection. A combination simultaneously considering the quantities and isoforms of these proteins could be a useful serum biomarker (or index) for HBV diagnosis and therapy.


Assuntos
Hepatite B Crônica/sangue , Proteômica/métodos , Adolescente , Adulto , Apolipoproteínas A/sangue , Apolipoproteínas A/isolamento & purificação , Biomarcadores/sangue , Proteínas Sanguíneas/isolamento & purificação , DNA Topoisomerases Tipo II/sangue , DNA Topoisomerases Tipo II/isolamento & purificação , Proteínas de Ligação a DNA , Eletroforese em Gel Bidimensional , Feminino , Haptoglobinas/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Pré-Albumina/isolamento & purificação , Prata , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Coloração e Rotulagem , alfa 1-Antitripsina/isolamento & purificação
6.
Biochem Biophys Res Commun ; 285(4): 903-8, 2001 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-11467836

RESUMO

Protein material was extracted from amyloid-rich sections of formalin-fixed and paraffin-embedded heart tissue from an individual with senile systemic amyloidosis, known to contain wild-type transthyretin as major amyloid fibril protein. Amino acid sequence analysis of tryptic peptides of this material revealed in addition to transthyretin sequences, also amino acid sequence corresponding to an N-terminal fragment of apolipoprotein A-IV. In immunohistochemistry, an antiserum to a synthetic apolipoprotein A-IV peptide labeled amyloid specifically. This peptide formed spontaneously amyloid-like fibrils in vitro and enhanced fibril formation from wild-type transthyretin. We conclude that several apolipoproteins, including apolipoprotein A-IV, may be important minor amyloid constituents, promoting fibril formation.


Assuntos
Amiloidose/patologia , Apolipoproteínas A/isolamento & purificação , Miocárdio/química , Pré-Albumina/isolamento & purificação , Fatores Etários , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Miocárdio/patologia
7.
Rev. cuba. endocrinol ; 8(3): 230-6, sept.-dic. 1997. graf
Artigo em Espanhol | LILACS | ID: lil-223041

RESUMO

El objetivo de nuestro trabajo fue desarrollar la metodología y la producción de los reactivos primarios para realizar un método inmunoenzimático (ELISA) tipo sandwich para determinar lipoproteina (a) en suero humano. La concentración final del antisuero policlonal antiapolipoproteína (a) (anti-apo [a]) obtenida fue de 1,3 mg/dL, purificado, por cromatografía de afinidad y de intercambio iónico. El antisuero antilipoproteína de baja densidad (anti LDL) de carnero purificado por cromatografía de afinidad fue utilizado en la obtención del conjugado policlonal perxodasa-IgG anti-LDL. La concentración óptima de recubrimiento con anti-apo (a) fue de 2 µg/mL y la dilución óptima del conjugado fue 1/7000, con lo cual obtuvimos una sensibilidad alta del ensayo. Poder producir en nuestro laboratorio reactivos primarios para el desarrollo de un sistema inmunoenzimático de determinación de Lp (a) hace posible la accesibilidad de la determinación con fines asistenciales e investigativos, así como un ahorro en moneda libremente convertible, pues estos reactivos tienen un elevado costo en el mercado


Assuntos
Coelhos , Apolipoproteínas A/isolamento & purificação , Apolipoproteínas A/imunologia , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Ensaio de Imunoadsorção Enzimática , Lipoproteína(a)/sangue , Lipoproteínas LDL/isolamento & purificação , Lipoproteínas LDL/imunologia , Kit de Reagentes para Diagnóstico
8.
Artif Organs ; 20(4): 311-7, 1996 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-8860712

RESUMO

Low-density lipoprotein (LDL) apheresis is applied in patients with coronary heart disease because of severe inherited forms of hypercholesterolemia, for which dietary and combined drug treatment cannot lower LDL cholesterol concentrations less than 130 mg/dl. The following article describes the changes in lipoprotein levels in a total of 19 patients undergoing weekly LDL apheresis. Immunoadsorption, operating with polyclonal antibodies against apolipoprotein B-100, was used in 6 patients. Five patients were put on heparin-induced extracorporeal LDL precipitation (HELP) therapy; 6 received dextran sulfate adsorption treatments. Under steady-state conditions a single treatment reduced LDL cholesterol by 149 + or - 3 mg/dl with immunoadsorption, 122 + or - 2 mg/dl with HELP, and 124 + or - 18 mg/dl with dextran sulfate adsorption. Lipoprotein (a) (Lp[a]) declined by 52 to 65%. Very low density lipoprotein (VLDL) cholesterol and VLDL triglycerides declined by 45 to 55% because of the activation of lipoprotein lipase and precipitation during the HELP procedure. In all procedures, there was a small reduction in the different high-density lipoprotein fractions, which had returned to normal after 24 h. The long-term HDL3 cholesterol levels increased significantly. During all procedures there was a decrease in the molar esterification rate of lecithin cholesterol acyltransferase activity. All changes in lipid fractions were paralleled by changes in the corresponding apolipoprotein levels. It is concluded that all three techniques described are powerful tools capable of lowering LDL cholesterol in severe hereditary forms of hypercholesterolemia. In HELP and dextran sulfate adsorption, the amount of plasma is limited by the elimination of other plasma constituents. Immunoadsorption may thus be preferred in very severe forms of hypercholesterolemia.


Assuntos
LDL-Colesterol/isolamento & purificação , VLDL-Colesterol/isolamento & purificação , Hiperlipoproteinemia Tipo II/terapia , Plasmaferese/métodos , Acil Coenzima A/antagonistas & inibidores , Adsorção , Apolipoproteínas A/sangue , Apolipoproteínas A/isolamento & purificação , LDL-Colesterol/sangue , VLDL-Colesterol/sangue , Angiografia Coronária , Sulfato de Dextrana/química , Sulfato de Dextrana/metabolismo , Humanos , Imunoadsorventes , Fosfatidilcolina-Esterol O-Aciltransferase/sangue , Plasmaferese/normas
9.
Acta bioquím. clín. latinoam ; 30(1): 59-65, mar. 1996. ilus, tab
Artigo em Espanhol | LILACS | ID: lil-177466

RESUMO

Las mujeres postmenospáusicas tienen un riesgo aumentado de padecer enfermedad coronaria en comparación con las premenospáusicas. Sin embargo, el riesgo disminuye cuando se realiza terapia hormonal sustitutiva. El objetivo de este estudio es investigar el posible efecto de la terapia con 17 ß Estradiol (E2) o de la combinación de 17 ß Estradiol y Acetato de Medroxiprogesterona (AMP), sobre las concentraciones plásmicas de colesterol total, colesterol HDL, colesterol LDL, colesterol VLDL y triglicéridos, fracciones de conocida participación en la aterogénesis. También se estudió la composición de ácidos grasos de fosfolípidos de la fracción no retenida obtenida por cromatografía de afinidad con Concanavalina A. Al cabo de 30 días de tratamiento con E2, el colesterol total disminuyó desde 226,0 ñ 54,4 mg/dl hasta 202,0 ñ 51,7 mg/dl; los niveles de triglicéridos descendieron desde 106,3 ñ 31,3 mg/dl hasta 80,6 ñ 13,9 mg/dl (p < 0,05), posiblemente a expensas de la fracción VLDL (21,3 ñ 6,2 mg/dl vs. 16,9 ñ 2,5 mg/dl); los fosfolípidos de la fracción no retenida de Concanavalina A mostraron una disminución de los ácidos grasos mirístico, palmítico y esteárico, y un aumento concomitante de los ácidos grasos oleico y linoleico. Los cambios observados con la administración de E2 tendieron a anularse cuando se agregó Acetato de Medroxiprogesterona


Assuntos
Humanos , Feminino , Pessoa de Meia-Idade , Colesterol , HDL-Colesterol , LDL-Colesterol/efeitos adversos , VLDL-Colesterol/efeitos adversos , Doença da Artéria Coronariana/etiologia , Doença das Coronárias/tratamento farmacológico , Estradiol/uso terapêutico , Acetato de Medroxiprogesterona , Menopausa/efeitos dos fármacos , Terapia de Reposição de Estrogênios/métodos , Triglicerídeos , Anticolesterolemiantes/uso terapêutico , Apolipoproteínas A/isolamento & purificação , Apolipoproteínas A , Apolipoproteínas A/sangue , Doença da Artéria Coronariana/fisiopatologia , Doença da Artéria Coronariana/prevenção & controle , Doença das Coronárias/etiologia , Doença das Coronárias/fisiopatologia , Ácidos Linoleicos , Ácidos Mirísticos , Resultado do Tratamento
10.
Acta bioquím. clín. latinoam ; 27(1): 75-85, mar. 1993. ilus, tab
Artigo em Espanhol | LILACS | ID: lil-124852

RESUMO

La utilización de la cromatografía de afinidad con concanavalina A o de cromatografía de inmunoafinidad con anticuerpos anti apoB permite obtener dos grupos de lipoproteínas: las que contienen apoA y las que contienen apoB. El subfraccionamiento por cromatografía de inmunoafinidad secuencial de las partículas lipoproteicas que contienen apoA permite obtener a su vez tres mayores partículas lipoproteicas: LP-A-I, LP-A-I:A-II y LP-A-II. El subfraccionamiento a través de inmunoprecipitación secuencial o cromatografía de inmunoafinidad secuencial de las partículas lipoproteicas que contienen apoB permite obtener cinco mayores grupos de partículas: LP-B, LP-B:C, LP-B:E, LP-B:C:E y LP-A-II:B. La diferencia entre normo e hiperlipoproteinémicos sería el resultado de cambios cuantitativos (y no cualitativos) de las partículas lipoproteicas. En hipercolesterolémicos se destaca un aumento de LP-B en tanto que en hipertrigliceridémicos aumentan la LP-B-C y LP-B:C:E. Las drogas hipolipemiantes, independientemente de su mecanismo de acción, afectan en diferentes sentidos las concentraciones de las partículas lipoproteicas que contienen apoA y apoB. Bajas concentraciones de LP-A-I y elevadas de LP-B:C y LP-B:C:E se asocian con riesgo aterogénico


Assuntos
Humanos , Masculino , Feminino , Apolipoproteínas A/isolamento & purificação , Apolipoproteínas B/isolamento & purificação , Apolipoproteínas/isolamento & purificação , Cromatografia de Afinidade/normas , Reação de Imunoaderência/normas , Apolipoproteínas A/classificação , Apolipoproteínas B/classificação , Apolipoproteínas B/metabolismo , Apolipoproteínas/classificação , Apolipoproteínas/sangue , Aterosclerose/fisiopatologia , Fracionamento Químico , Genfibrozila/uso terapêutico , Hiperlipoproteinemias/diagnóstico , Hiperlipoproteinemias/tratamento farmacológico
11.
Dev Biol ; 140(2): 430-6, 1990 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-1695585

RESUMO

Northern blot hybridization experiments showed that Apolipoprotein A-I (Apo A-I) mRNA is present at high concentration in chicken myotubes cultured in vitro, while it is virtually absent in fibroblasts and myoblasts. Myotubes are also capable of translating and secreting in the culture medium a protein which is specifically immunoprecipitated by anti-Apo A-I antibodies and has the same electrophoretic mobility as Apo A-I purified from circulating high-density lipoproteins. The appearance of Apo A-I mRNA in myotubes depends on the transcriptional activation of the corresponding gene, as it was shown by hybridizing 32P-labeled RNA synthesized in isolated nuclei to Apo A-I cDNA. The activation of the Apo A-I gene is regulated by the muscle cell coordinately with muscle-specific genes. In fact, treatment with TPA, a powerful inhibitor of differentiation, efficiently prevents myoblasts from producing Apo A-I mRNA, as well as muscle actin mRNA, and causes myotubes to quickly cease Apo A-I mRNA synthesis. The existence of a strict relationship between Apo A-I mRNA concentration and myogenic cell differentiation was also confirmed by experiments with quail myoblasts transformed with a temperature-sensitive mutant of the Rous Sarcoma Virus. Cells raised at the permissive temperature (undifferentiated phenotype) do not contain Apo A-I as well as alpha-actin mRNAs, while shifting to the nonpermissive temperature (differentiated phenotype) causes a rapid increase in Apo A-I and alpha-actin mRNA concentration.


Assuntos
Apolipoproteínas A/genética , Lipoproteínas HDL/genética , Músculos/metabolismo , Actinas/genética , Actinas/isolamento & purificação , Animais , Apolipoproteína A-I , Apolipoproteínas A/biossíntese , Apolipoproteínas A/isolamento & purificação , Northern Blotting , Diferenciação Celular , Células Cultivadas , Embrião de Galinha , Peso Molecular , Músculos/citologia , Hibridização de Ácido Nucleico , Plasmídeos , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro/genética , Transcrição Gênica
12.
Arteriosclerosis ; 10(4): 625-32, 1990.
Artigo em Inglês | MEDLINE | ID: mdl-2114867

RESUMO

To explore the potential of the common marmoset monkey (Callithrix jacchus) as a model for human plasma lipoprotein metabolism, several marmoset apolipoproteins were isolated and characterized in this study. Based on several properties, including molecular weight, amino acid composition, and sequence, the marmoset apolipoproteins are strikingly similar to human apolipoprotein (apo) A-I, A-II, C-III, and A-IV. The first 54 residues of marmoset apo A-I showed 87% sequence identity with the corresponding region of human apo A-I. Amino-terminal sequence analysis of a minor basic apo A-I isoform revealed that it contained an amino-terminal hexapeptide extension (Arg-His-Phe-Gln-Gln-) identical to that found in human proapo A-I. Like apo A-II in most nonhuman primates, marmoset apo-A-II differed from human apo A-II in that it did not contain cysteine and therefore existed as a monomer. The complete amino acid sequence of marmoset apo A-II was deduced. The protein contains 77 amino acids, as does human apo A-II, and showed an 82% identity with its human equivalent. In both species, apo C-III and E had similar amino-terminal sequences and amino acid compositions. Like human apo E, marmoset apo E contained minor sialylated isoforms. However, unlike human apo C-III, no sialyated isoforms of marmoset apo C-III were observed. In addition, the marmoset possessed an apolipoprotein whose molecular weight and amino acid composition were similar to those of human apo A-IV. The close structural similarities between corresponding marmoset and human apolipoproteins indicate that the marmoset monkey will be useful as a model for human lipoprotein metabolism.


Assuntos
Apolipoproteínas/isolamento & purificação , Callithrix/sangue , Callitrichinae/sangue , Sequência de Aminoácidos , Animais , Apolipoproteínas/sangue , Apolipoproteínas/metabolismo , Apolipoproteínas A/sangue , Apolipoproteínas A/isolamento & purificação , Apolipoproteínas E/sangue , Apolipoproteínas E/isolamento & purificação , Callithrix/metabolismo , Modelos Biológicos , Dados de Sequência Molecular
13.
Biotecnol. apl ; 7(1): 58-65, 1990. ilus
Artigo em Espanhol | LILACS | ID: lil-96015

RESUMO

Se describe la producción de anticuerpos monoclonales (AcMs) de ratón contra la apolipoproteína A1 (APO A1), a partir de la inmunización de ratones BALB/c con APO A1 purificada de plasma humano. Los anticuerpos obtenidos son de la clase IgG1 y fueron purificados a partir de líquido ascítico mediante cromatografía de afinidad con Proteína A Sepharosa. Dos de ellos, que reconocen sitios diferentes de la molécula de APO A1 en muestras de suero. Los coeficientes de variación intra e interensayo fueron de 3,4 y 10 % respectivamente. Con este sistema se detectaron niveles disminuídos de APO A1 en pacientes con infarto agudo del miocardio entre 45 y 80 años, y en grupo con aterosclerosis periférica entre 40 y 49 años, con relación a sujetos controles de igual rango de edad. En los pacientes con aterosclerosis periférica de mayor edad (entre 60 y 80 años), aunque se encontraron valores de APO A1 más bajos que el grupo control respectivo, esta diferencia no fue estadísticamente significativa


Assuntos
Camundongos , Anticorpos Monoclonais , Apolipoproteínas A/isolamento & purificação , Cromatografia de Afinidade
14.
DNA ; 8(6): 429-36, 1989.
Artigo em Inglês | MEDLINE | ID: mdl-2673706

RESUMO

A human liver cDNA library was used to isolate a clone coding for apolipoprotein A-I (Apo A-I). The clone carries the sequence for the prepeptide (18 amino acids), the propeptide (6 amino acids), and the mature protein (243 amino acids). A coding cassette for the proapo A-I molecule was reconstructed by fusing synthetic sequences, chosen to optimize expression and specifying the amino-terminal methionine and amino acids -6 to +14, to a large fragment of the cDNA coding for amino acids 15-243. The module was expressed in pOTS-Nco, an Escherichia coli expression vector carrying the regulatable lambda PL promoter, leading to the production of proapolipoprotein A-I at up to 10% of total soluble proteins. The recombinant polypeptide was purified and characterized in terms of apparent molecular mass, isoelectric point, and by both chemical and enzymatic peptide mapping. In addition, it was assayed in vitro for the stimulation of the enzyme lecithin: cholesterol acyltransferase. The data show for the first time that proapo A-I can be produced efficiently in E. coli as a stable and undegraded protein having physical and functional properties indistinguishable from those of the natural product.


Assuntos
Apolipoproteínas A/genética , DNA/genética , Escherichia coli/genética , Precursores de Proteínas/genética , Sequência de Aminoácidos , Apolipoproteína A-I , Apolipoproteínas A/isolamento & purificação , Apolipoproteínas A/farmacologia , Sequência de Bases , Clonagem Molecular , Humanos , Fígado/metabolismo , Dados de Sequência Molecular , Mapeamento de Peptídeos , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Plasmídeos , Precursores de Proteínas/isolamento & purificação , Precursores de Proteínas/farmacologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Mapeamento por Restrição
15.
J Cell Physiol ; 128(3): 413-20, 1986 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-3018002

RESUMO

The lipid-free apolipoproteins of human high density lipoprotein (HDL) have been assayed for their ability to substitute for native HDL in promoting the growth of a SV40-transformed REF52 cell line in serum-free medium. Total HDL-apolipoproteins (apoHDL) were found to mimic almost exactly the growth promoting effects of whole HDL. The apoHDL-associated growth promoting activity eluted from a Sephacryl S-200 column in two separate fractions coinciding with the protein peaks of apolipoprotein A-I and the C group of apolipoproteins. These two fractions, designated S-II and S-IV, respectively, acted additively in promoting WT1A cell growth when tested at saturating concentrations. The active component in the S-II fraction maximally stimulated WT1A cell growth at 40-60 micrograms/ml and was identified as apolipoprotein A-1 by NaDodSO4 polyacrylamide gel electrophoresis and affinity chromatography on anti-(apoA-I). The active component in the S-IV fraction was maximally active at 1-2 micrograms/ml and was identified as apolipoprotein C-III by DEAE ion exchange high pressure liquid chromatography and polyacrylamide gel electrophoresis (at pH 8.3) in 6 M urea. These results indicate that the growth promoting effect of HDL on WT1A cells is mediated via the HDL-apolipoproteins, A-I and C-III, and that the mechanism responsible does not necessarily involve their participation in the uptake (or utilization) of HDL-associated lipids.


Assuntos
Apolipoproteínas A/farmacologia , Apolipoproteínas C/farmacologia , Divisão Celular/efeitos dos fármacos , Lipoproteínas HDL/farmacologia , Animais , Apolipoproteína A-I , Apolipoproteína C-III , Apolipoproteínas A/isolamento & purificação , Apolipoproteínas C/isolamento & purificação , Linhagem Celular , Transformação Celular Viral , Embrião de Mamíferos , Lipídeos/farmacologia , Ratos , Vírus 40 dos Símios
16.
J Biol Chem ; 260(18): 10256-62, 1985 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-4019511

RESUMO

The four peptide analogs of the amphipathic helix whose interactions with dimyristoyl phosphatidylcholine were described in the preceding paper were compared with apolipoproteins (apo) A-I and A-II in ability to displace native apolipoprotein from high density lipoprotein (HDL) and in ability to activate lecithin:cholesterol acyltransferase. The rank order of the ability of the four peptide analogs to displace apo-A-I from intact HDL was 18A-Pro-18A greater than 18A greater than des-Val10-18A greater than reverse-18A, the same order suggested in the preceding paper for relative lipid affinities. Modified HDL from which 40% of the apo-A-I had been displaced by 18A was indistinguishable from unmodified HDL in its ability to act as a lecithin:cholesterol acyltransferase substrate. This suggests that the easily displaced apo-A-I molecules in polydisperse HDL are relatively ineffectual as lecithin:cholesterol acyltransferase activators and/or 18A replaces the lecithin:cholesterol acyltransferase activity lost. The peptide analog 18A-Pro-18A was found to be a powerful activator of lecithin:cholesterol acyltransferase when incubated with unilamellar egg phosphatidylcholine (PC) vesicles, reaching 140% of the activity of apo-A-I at a 1:1.75 peptide-to-egg PC ratio. In another experiment, it was found that discoidal egg PC complexes of 18A-Pro-18A, 18A, and des-Val10-18A, formed by cholate dialysis, had 30-45% of the activity of apo-A-I/egg PC discoidal complexes, also formed by cholate dialysis, at the same peptide/lipid weight ratio. Examination of the structures formed when the 18A-Pro-18A peptide was incubated with unilamellar egg PC vesicles indicated that the ability of 18A-Pro-18A to exceed apo-A-I in lecithin:cholesterol acyltransferase activating ability is due to the spontaneous conversion by 18A-Pro-18A of egg PC vesicles to small protein annulus-bilayer disc structures. Apo-A-I, apo-A-II, nor any of the other three peptide analogs of the amphipathic helix studied were able to convert a significant fraction of egg PC unilamellar vesicles to discoidal structures.


Assuntos
Apolipoproteínas A , Dimiristoilfosfatidilcolina , Peptídeos , Sequência de Aminoácidos , Apolipoproteínas A/sangue , Apolipoproteínas A/isolamento & purificação , Humanos , Cinética , Lipoproteínas HDL/sangue , Lipoproteínas HDL/isolamento & purificação , Fosfatidilcolina-Esterol O-Aciltransferase/sangue , Relação Estrutura-Atividade
17.
Biol Chem Hoppe Seyler ; 366(2): 181-7, 1985 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-3921042

RESUMO

Apolipoprotein AI of human high-density lipoproteins is secreted by hepatocytes as a proapolipoprotein with a N-terminal hexapeptide sequence (Arg-His-Phe-Trp-Gln-Gln-) which differs from the prosequence of rat apolipoprotein AI (Trp-Asp-Phe-Trp-Gln-Gln). The two proteins have in common the unusual cleavage site -Gln-Gln-Asp-Glu-. It is hydrolysed by a specific serum proteinase with the release of mature apo AI. We synthesized a model substrate for the study of the final processing of pro-apo AI by the serum proteinase. It is an undecapeptide embracing the human pro-hexapeptide sequence and the first five N-terminal residues of apo AI, covalently linked to a hydrophilic resin. The N-terminal arginine residue was 3H-labelled. [formula; see text] This sequence was not cleaved by human serum under the conditions under which rat serum processes the pro-form of apo AI secreted by rat hepatocytes. Pepsin and chymotrypsin fragmented the undecapeptide at sites characteristic for these proteinases. We conclude that the proteolytic cleavage at the specific site (-Gln-Gln-Asp-Glu-) requires the correct conformation in addition to the specific amino-acid sequence.


Assuntos
Apolipoproteínas A/isolamento & purificação , Precursores de Proteínas/isolamento & purificação , Sequência de Aminoácidos , Apolipoproteína A-I , Apolipoproteína A-II , Apolipoproteínas A/análise , Fenômenos Químicos , Química , Quimotripsina , Humanos , Hidrólise , Hipolipoproteinemias/sangue , Pepsina A , Peptídeos/análise , Conformação Proteica , Precursores de Proteínas/análise , Resinas Vegetais
18.
J Biol Chem ; 259(24): 15556-63, 1984 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-6096378

RESUMO

The two principal high-density lipoprotein apolipoproteins A-I and A-II are both initially synthesized as preproproteins. The prosegment of apo-A-I is unusual: it ends with paired glutamine residues and is removed extracellularly. The apo-A-II prosegment resembles the propeptides of prohormones and proalbumin: it ends with paired basic amino acids. We have studied the processing of proapo-A-II in a human hepatoma cell line (Hep G2) which is known to accurately and efficiently remove the prosegment from proalbumin prior to secretion. Pulse-chase experiments were performed in order to determine if the apo-A-II prosegment is removed prior to or after secretion. Apo-A-II was purified from cell lysates and media at various times during the chase and subjected to automated sequential Edman degradation. The results indicate that proteolytic processing of proapo-A-II is largely an extracellular event. These cells secrete the protease responsible for prosegment removal. The converting activity present in media is not blocked by serine protease inhibitors (phenylmethanesulfonyl fluoride, aprotinin, and furoyl saccharin) or by a metalloprotease inhibitor (o-phenanthroline). It is inhibited by the thiol protease reagents p-chloromercuribenezene-sulfonic acid and leupeptin. Prosegment removal changes the pI of the dominant apo-A-II isoform from 6.61 to 4.95. The presence of the propeptide does not prevent specific in vitro recombination of apo-A-II with high-density lipoprotein3 particles present in normolipemic serum. Extracellular processing after a single basic amino acid has been described for a variety of precursor proteins. Extracellular cleavage of the apo-A-II propeptide after paired COOH-terminal basic residues represents a novel processing pathway.


Assuntos
Apolipoproteínas A/metabolismo , Carcinoma Hepatocelular/enzimologia , Endopeptidases/metabolismo , Neoplasias Hepáticas/enzimologia , Precursores de Proteínas/metabolismo , Sequência de Aminoácidos , Apolipoproteínas A/isolamento & purificação , Linhagem Celular , Meios de Cultura , Cisteína Endopeptidases , Humanos , Focalização Isoelétrica , Cinética , Inibidores de Proteases/farmacologia , Precursores de Proteínas/isolamento & purificação
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