RESUMEN
Extracellular matrices (ECMs) in the intervertebral disc (IVD), lung and artery are thought to undergo age-dependant accumulation of damage by chronic exposure to mechanisms such as reactive oxygen species, proteases and glycation. It is unknown whether this damage accumulation is species-dependant (via differing lifespans and hence cumulative exposures) or whether it can influence the progression of age-related diseases such as atherosclerosis. Peptide location fingerprinting (PLF) is a new proteomic analysis method, capable of the non-targeted identification of structure-associated changes within proteins. Here we applied PLF to publicly available ageing human IVD (outer annulus fibrosus), ageing mouse lung and human arterial atherosclerosis datasets and bioinformatically identified novel target proteins alongside common age-associated differences within protein structures which were conserved between three ECM-rich organs, two species, three IVD tissue regions, sexes and in an age-related disease. We identify peptide yield differences across protein structures which coincide with biological regions, potentially reflecting the functional consequences of ageing or atherosclerosis for macromolecular assemblies (collagen VI), enzyme/inhibitor activity (alpha-2 macroglobulin), activation states (complement C3) and interaction states (laminins, perlecan, fibronectin, filamin-A, collagen XIV and apolipoprotein-B). Furthermore, we show that alpha-2 macroglobulin and collagen XIV exhibit possible shared structural consequences in IVD ageing and arterial atherosclerosis, providing novel links between an age-related disease and intrinsic ageing. Crucially, we also demonstrate that fibronectin, laminin beta chains and filamin-A all exhibit conserved age-associated structural differences between mouse lung and human IVD, providing evidence that ECM, and their associating proteins, may be subjected to potentially similar mechanisms or consequences of ageing across both species, irrespective of differences in lifespan and tissue function.
Asunto(s)
Aterosclerosis , Degeneración del Disco Intervertebral , Disco Intervertebral , Ratones , Animales , Humanos , Fibronectinas/metabolismo , Filaminas/análisis , Filaminas/metabolismo , Proteómica/métodos , Disco Intervertebral/metabolismo , Degeneración del Disco Intervertebral/metabolismo , Colágeno/metabolismo , Envejecimiento/metabolismo , Laminina/metabolismo , Péptidos/metabolismo , Aterosclerosis/genética , Aterosclerosis/metabolismo , Macroglobulinas/análisis , Macroglobulinas/metabolismoAsunto(s)
Proteína de Bence Jones/análisis , Macroglobulinas/análisis , Mieloma Múltiple/diagnóstico , Proteínas de Mieloma/análisis , Macroglobulinemia de Waldenström/diagnóstico , gammaglobulinas/análisis , Técnica del Anticuerpo Fluorescente/métodos , Humanos , Mieloma Múltiple/patología , Células Plasmáticas/patología , Macroglobulinemia de Waldenström/patologíaRESUMEN
To evaluate the effectiveness and safety of autologous cytokine-induced killer (CIK) cells in elderly patients with diffuse large B-cell lymphoma. Peripheral blood mononuclear cells (PBMC) were isolated from nine elderly patients with diffuse large B-cell lymphoma. PBMCs were augmented by priming with interferon gamma (IFN-γ) followed by IL-2 and monoclonal antibody (mAb) against CD3. Autologous CIK cells (range 5 × 10(9)-1 × 10(10)) were then infused back to individual patients; infusion was repeated every 4 weeks for 32 weeks (eight cycles). Patients were assessed for changes in lymphocyte subgroup, tumor-related biological parameters, imaging characteristics, the condition of remission, quality of life (QOL), and survival. Prior to CIK infusion, two patients were in complete remission and seven patients were in partial remission. After autologous CIK cell transfusions, the proportion of CD3+, CD3+CD8+, and CD3+CD56+ cells were significantly increased compared with baseline (P < 0.05); whereas serum levels of ß2-microglobulin and LDH were significantly decreased (P < 0.05). The lymphoma symptoms were reduced and QOL was improved (P < 0.05) in all patients. All patients achieved complete remission at study endpoint. No adverse reactions were reported. Autologous CIK cell immunotherapy is safe and efficacious for the treatment of elderly patients with diffuse large B-cell lymphoma.
Asunto(s)
Células Asesinas Inducidas por Citocinas/trasplante , Inmunoterapia , Linfoma de Células B Grandes Difuso/terapia , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Complejo CD3/inmunología , Complejo CD3/metabolismo , Antígeno CD56/metabolismo , Antígenos CD8/metabolismo , Células Cultivadas , Células Asesinas Inducidas por Citocinas/inmunología , Femenino , Humanos , Interferón gamma/farmacología , Interleucina-2/farmacología , L-Lactato Deshidrogenasa/sangre , Linfoma de Células B Grandes Difuso/diagnóstico por imagen , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/mortalidad , Macroglobulinas/análisis , Masculino , Persona de Mediana Edad , Imagen Multimodal , Tomografía de Emisión de Positrones , Análisis de Supervivencia , Tomografía Computarizada por Rayos X , Trasplante Autólogo , Resultado del TratamientoRESUMEN
PURPOSE: There has been significant criticism of how technologies such as SELDI have been used in biomarker discovery and how the data have been analysed. We initiated a proof-of-principle pilot study using SELDI with stringent pre-analytic and analytical procedures with robust statistical analysis, to determine whether, under such conditions, using different degrees of renal dysfunction as a model, useful data could be obtained. EXPERIMENTAL DESIGN: SELDI-TOF-MS profiling with stringent quality control measures was used to examine the proteomic profile of serum from healthy controls (n=30), patients with end-stage renal failure being treated by dialysis (n=30) and renal transplant patients (n=50) with varying degrees of graft stability. RESULTS: Principal component analysis of the data suggests that the continuum from normality to end-stage renal failure through 'stable' and 'unstable' transplant may be detected by SELDI profiling. Serum ß2 microglobulin was identified as a major component and this was validated using immunonephelometry. CONCLUSIONS AND CLINICAL RELEVANCE: This pilot study suggests that stringently controlled SELDI analysis is able to detect proteins which may be useful in the stratification of patients post-renal transplant. Further studies using a larger cohort of patients with chronic allograft dysfunction, defined by protocol biopsies, are indicated.
Asunto(s)
Biomarcadores/sangre , Insuficiencia Renal Crónica/sangre , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Trasplante de Riñón , Macroglobulinas/análisis , Macroglobulinas/inmunología , Masculino , Persona de Mediana Edad , Nefelometría y Turbidimetría , Proyectos Piloto , Análisis de Componente Principal , Diálisis Renal , Insuficiencia Renal Crónica/patologíaRESUMEN
The well-characterized small heat-shock protein, alphaB-crystallin, acts as a molecular chaperone by interacting with unfolding proteins to prevent their aggregation and precipitation. Structural perturbation (e.g., partial unfolding) enhances the in vitro chaperone activity of alphaB-crystallin. Proteins often undergo structural perturbations at the surface of a synthetic material, which may alter their biological activity. This study investigated the activity of alphaB-crystallin when covalently bound to a support surface; alphaB-crystallin was immobilized onto a range of solid material surfaces, and its characteristics and chaperone activity were assessed. Immobilization was achieved via a plasma-deposited thin polymeric interlayer containing aldehyde surface groups and reductive amination, leading to the covalent binding of alphaB-crystallin lysine residues to the surface aldehyde groups via Schiff-base linkages. Immobilized alphaB-crystallin was characterized by X-ray photoelectron spectroscopy, atomic force microscopy, and quartz crystal microgravimetry, which showed that 300 ng cm(-2) (dry mass) of oligomeric alphaB-crystallin was bound to the surface. Immobilized alphaB-crystallin exhibited a significant enhancement (up to 5000-fold, when compared with the equivalent activity of alphaB-crystallin in solution) of its chaperone activity against various proteins undergoing both amorphous and amyloid fibril forms of aggregation. The enhanced molecular chaperone activity of immobilized alphaB-crystallin has potential applications in preventing protein misfolding, including against amyloid disease processes, such as dialysis-related amyloidosis, and for biodiagnostic detection of misfolded proteins.
Asunto(s)
Proteínas de Choque Térmico Pequeñas/química , Proteínas Inmovilizadas/química , Chaperonas Moleculares/química , Cadena B de alfa-Cristalina/química , Amiloide/antagonistas & inhibidores , Amiloide/química , Caseínas/análisis , Macroglobulinas/análisis , Microscopía de Fuerza Atómica , Espectroscopía de Fotoelectrones , Unión Proteica , Pliegue de Proteína , Tecnicas de Microbalanza del Cristal de Cuarzo , Soluciones , Propiedades de SuperficieRESUMEN
BACKGROUND: The Clara cell 10 (CC10) protein is produced by the airway epithelium. Reduced levels of CC10 are associated with allergic rhinitis and asthma. In experimental models, treatment with the CC10 protein may reduce features of airway inflammation. OBJECTIVES: To examine whether or not topical treatment with recombinant human CC10 (rhCC10) affects symptoms and signs of allergic rhinitis in a pollen season model. METHODS: Out of the pollen season, patients with allergic rhinitis received treatment with rhCC10, 0.56 mg per nasal cavity, once daily for 7 days in a double-blinded, placebo-controlled, crossover design. During this period, individualized allergen challenges were given once daily. Symptoms and peak nasal inspiratory flow (PNIF) were recorded daily in the morning, 10 minutes after challenge, and in the evening. Mean recordings of the last 3 days of the challenge series were used in the analysis. Nasal lavages were performed at the end of each challenge period, and eosinophil cationic protein, myeloperoxidase, and alpha2-macroglobulin levels were measured as indices of eosinophil and neutrophil activity and plasma exudation, respectively. RESULTS: Recombinant human CC10 did not affect allergen-induced morning, postchallenge, or evening symptoms compared with placebo. Morning, postchallenge, and evening PNIF were not improved by rhCC10. No statistically significant differences were observed between rhCC10 and placebo for any of the lavage fluid indices. CONCLUSIONS: Repeated nasal administrations of rhCC10 protein, in the present dose, do not exert antiallergic effects in seasonal allergic rhinitis.
Asunto(s)
Polen/inmunología , Proteínas Recombinantes/uso terapéutico , Rinitis Alérgica Estacional/tratamiento farmacológico , Uteroglobina/uso terapéutico , Administración Intranasal , Adulto , Alérgenos/inmunología , Alérgenos/farmacología , Estudios de Casos y Controles , Estudios Cruzados , Método Doble Ciego , Proteína Catiónica del Eosinófilo/análisis , Proteína Catiónica del Eosinófilo/inmunología , Humanos , Macroglobulinas/análisis , Macroglobulinas/inmunología , Masculino , Persona de Mediana Edad , Lavado Nasal (Proceso) , Nariz/inmunología , Peroxidasa/análisis , Peroxidasa/inmunología , Proteínas Recombinantes/administración & dosificación , Rinitis Alérgica Estacional/inmunología , Uteroglobina/administración & dosificaciónRESUMEN
BACKGROUND: Adenomas have the highest potential or clinical value from among colonic polyps of developing into adenocarcinoma. The aims of this paper are: to establish criteria to identify the high risk group of patients in a group of patients with colonic polyps, to work out a simple scheme for follow-up care after endoscopic polypectomy, and to establish indications for surgery. The usefulness of determination of electrophoresis of serum proteins has been specially analysed to detect early development of malignant growths in patients with colonic polyps regarding alfa-1/alfa-2 and alfa/beta. 67 cases - 21 women, 46 men were tested. Follow-up endoscopy with the electrophoresis was performed after 6 weeks, 6 and 12 months after polypectomy. 97 polyps were resected with endoscopy in 67 patients. 38 patients (39.17%), those constituting the high risk group, were selected. Included were all polyps with grade II and III of cellular differentiation. CONCLUSIONS: 1) alfa-1/alfa-2 and alfa/beta is a helpful test in identifying the high risk group among patients with colonic polyps and it can be used as a screening test, 2) the determination of beta-2-macroglobuline is not useful in the diagnosis of this group of patients, 3) the electrophoresis of proteins should be the first test to perform on patients with colonic polyps. The relation of electrophoresis to endoscopic polypectomy aids evaluations of patients specially predisposed to malignant.
Asunto(s)
Pólipos Adenomatosos/metabolismo , Biomarcadores de Tumor/análisis , Electroforesis de las Proteínas Sanguíneas/métodos , Transformación Celular Neoplásica/metabolismo , Pólipos del Colon/metabolismo , Macroglobulinas/análisis , Adenocarcinoma/metabolismo , Adenocarcinoma/prevención & control , Adenocarcinoma/cirugía , Pólipos Adenomatosos/clasificación , Pólipos Adenomatosos/patología , Pólipos Adenomatosos/cirugía , Adulto , Anciano , Neoplasias del Colon/metabolismo , Neoplasias del Colon/prevención & control , Neoplasias del Colon/cirugía , Pólipos del Colon/clasificación , Pólipos del Colon/patología , Pólipos del Colon/cirugía , Colonoscopía , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Medición de RiesgoRESUMEN
BACKGROUND: The aim of the study was to examine the role of endothelin in radiocontrast-induced nephropathy (RCN) in patients with chronic renal failure. METHODS: We measured plasma endothelin-1 (ET) and the urinary excretion of endothelin-like immunoreactivity before and after infusion of radio contrast medium (CM) in patients with normal renal function (group N; n = 6; mean serum creatinine concentration, 0.8 +/- 0.1 (SEM) mg/dl), and in another group, with renal dysfunction (group R; n = 6; 2.7 +/- 0.5 mg/dl). Half-normal saline (0.45% NaCl solution) was continuously infused in all patients for 25 h, at a rate of 100 ml/h; starting from 5 h before the infusion of CM. RESULTS: Plasma ET in group R (5.2 +/- 1.4 pg/ml) was significantly higher than in group N (0.9 +/- 0.3; P << 0.01). Urinary endothelin excretion corrected by creatinine concentration (uET/Cr) in group R (7.9 +/- 2.4 mg/g Cr) was significantly higher than in group N (1.5 +/- 0.4 mg/g Cr; P << 0.05). Urinary excretion levels of N-acetyl-Beta- d-glucosaminidase (NAG) and Beta2-microglobulin (Beta2M) were also significantly higher in group R (0.8 +/- 0.2 mU/g Cr and 670 +/- 400 mg/g Cr, respectively) than in group N (0.3 +/- 0.1 and 7.5 +/- 2.2, respectively). After CM infusion, uET/Cr in group R significantly increased, to 10.7 +/- 2.6 mg/g Cr on the next day and returned to baseline level on the third day. NAG and Beta2M showed a similar pattern, but a significant change in NAG was observed on the second day in group R. In group N, uET/Cr, NAG, and Beta2M did not change after CM infusion. Plasma ET remained unchanged throughout the observation period of 4 days in both groups. No patient developed pulmonary edema or a significant rise in serum creatinine (more than 0.5 mg/dl), caused by infusion of the amount of half-normal saline used. CONCLUSIONS: In the present study, uET/Cr increased after the administration of CM only in the patients with renal impairment. This difference in endothelin reaction may be a causal one, in that patients with renal insufficiency readily develop RCN. The infusion of half-normal saline starting before CM infusion causes no side effects and is safe for the prevention of CM-induced acute renal failure. The aim of the study was to examine the role of endothelin in radiocontrast-induced nephropathy (RCN) in patients with chronic renal failure.
Asunto(s)
Medios de Contraste/efectos adversos , Endotelinas/orina , Fallo Renal Crónico/orina , Acetilglucosaminidasa/sangre , Adulto , Anciano , Creatinina/sangre , Endotelinas/sangre , Femenino , Humanos , Pruebas de Función Renal , Macroglobulinas/análisis , Macroglobulinas/metabolismo , Masculino , Persona de Mediana EdadAsunto(s)
Antineoplásicos/efectos adversos , Enfermedad de Hodgkin/complicaciones , Sarcoidosis/inducido químicamente , Antineoplásicos/uso terapéutico , Sedimentación Sanguínea , Diagnóstico Diferencial , Enfermedad de Hodgkin/diagnóstico , Enfermedad de Hodgkin/tratamiento farmacológico , Humanos , Macroglobulinas/análisis , Masculino , Persona de Mediana Edad , Radiografía , Sarcoidosis/diagnóstico , Sarcoidosis/etiología , Sarcoidosis Pulmonar/diagnóstico por imagen , Sarcoidosis Pulmonar/patologíaRESUMEN
A protein with the apparent molecular mass of 720 kDa which hydrolyzes anilide substrates of p-guanidino-L-phenylalanine was purified from ascites and pleural effusion of patients with pulmonary, breast, gastric, and ovarian cancers by chromatographic techniques. When this protein was separated on SDS-PAGE on nonreducing conditions, two bands corresponding to 720 and 360 kDa were seen to have gelatin-digestive activity in zymography assay. Moreover, when it separated by SDS-PAGE on reducing conditions, it migrated as several bands up to 180 kDa. The N-terminal amino acid sequence and immunoreactivity of anti-alpha2-macroglobulin polyclonal antibody revealed that the 180-kDa band was intact alpha2-macroglobulin. The hydrolytic activity of this complex was completely inhibited by diisopropyl fluorophosphate (DFP) and p-amidinophenylmethanesulfonyl fluoride. In addition, the 65-kDa protein observed under reducing conditions bound 3H-labeled DFP. These results suggest that the purified protein is a complex of the plasma proteinase inhibitor alpha2-macroglobulin and a serine proteinase. Several monoclonal antibodies were obtained when the purified complex was used as an antigen. One of these antibodies, which was immunoreactive to this complex but not to alpha2-macroglobulin, gave a positive band corresponding to 65 kDa on SDS-PAGE under reducing conditions. Use of this antibody in immunohistochemical studies revealed immunoreactivities in numerous neoplastic tissues with strong activity in advanced gastric cancers (e.g., poorly differentiated adenocarcinoma). In addition, strong cross-reactivity was detected in glandular cells of the fetus intestine.
Asunto(s)
Anticuerpos Monoclonales , Ascitis , Inmunohistoquímica , Macroglobulinas/química , Derrame Pleural/química , Serina Endopeptidasas/química , Adenocarcinoma/patología , Western Blotting , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Humanos , Intestinos/anatomía & histología , Macroglobulinas/análisis , Macroglobulinas/aislamiento & purificación , Serina Endopeptidasas/análisis , Serina Endopeptidasas/aislamiento & purificación , Distribución Tisular , Células Tumorales CultivadasRESUMEN
A study was made of the influence of plasmin (PL) complexes with alpha 2-macroglobulin (MG) and alpha 2-antiplasmin on secretion of MG, pregnancy-associated alpha 2-glycoprotein (PAG), and pregnancy-associated plasma protein A (PAPP-A) by mononuclear cells (MC), obtained from human peripheral blood. It has been shown that incubation of the MG-PL complexes with MC resulted in the increase in concentrations of all these macroglobulins (MG, PAG, and PAPP-A) in supernatants of cultured MC. We revealed the same effects of AP-PL with regard to the secretion of PZP and PAPP-A. However, this complex was found to suppress MG production by cultured MC. The effects found were dose dependent and some of them differed greatly in men and women, resp.
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Fibrinolisina/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Macroglobulinas/efectos de los fármacos , Inhibidores de Proteasas/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Técnicas para Inmunoenzimas , Leucocitos Mononucleares/metabolismo , Macroglobulinas/análisis , Macroglobulinas/metabolismo , Masculino , Caracteres Sexuales , Estadísticas no ParamétricasAsunto(s)
Biomarcadores de Tumor , Macroglobulinas/análisis , Neoplasias/sangre , Animales , Femenino , Humanos , Macroglobulinas/biosíntesis , Macroglobulinas/fisiología , Masculino , Neoplasias/inmunología , Neoplasias/patología , Neoplasias Experimentales/sangre , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Embarazo , Proteínas Gestacionales/análisis , Proteínas Gestacionales/fisiología , Ratas , alfa-Macroglobulinas/análisis , alfa-Macroglobulinas/biosíntesis , alfa-Macroglobulinas/fisiologíaRESUMEN
Studies have been made on physicochemical and immunochemical properties of macroglobulins, as well as of associated with pregnancy glycoproteins, from human subjects, mammals, birds, fishes and invertebrates. It was shown that these proteins exhibit similar composition, structure and capacity to bind proteinases inhibiting the latter. Using immunochemical methods, reactions of antigenic identity of these proteins were investigated. A hypothesis of evolutionary formation of macroglobulin family is discussed.
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Evolución Biológica , Macroglobulinas/análisis , Animales , Western Blotting , Cromatografía en Gel , Femenino , Humanos , Inmunodifusión , Inmunoelectroforesis Bidimensional , Focalización Isoeléctrica , Macroglobulinas/aislamiento & purificación , Peso Molecular , Embarazo , Especificidad de la EspecieRESUMEN
The presence of melanin macroglobules, and sometimes that of melanosome complexes also, in epidermal melanocytes has been considered a feature of various skin diseases. Opinions differ as to whether these structures can occur in normal skin. We have studied these melanin inclusions in normal Caucasian skin in the entire soma of 116 melanocytes and the occurrence of melanosomes in phagosomes of 77 Langerhans' cells obtained in different seasons. During winter the melanocytes contained few melanosomes but many melanosome complexes and melanin macroglobules. These melanosome inclusions were in 86%, localized in the most basal part of the melanocytes, particularly in the dermal protrusions. It is suggested that these structures can be transferred from epidermal melanocytes to dermal cells and that melanin macroglobules derive from melanosome complexes. Irrespective of the season, most of the Langerhans' cells contained melanosomes in their phagosomes, which suggests a phagocytic capacity of these cells and a role in the elimination of the melanin.
Asunto(s)
Células de Langerhans/química , Macroglobulinas/análisis , Melaninas/análisis , Melanocitos/química , Biopsia , Femenino , Humanos , Células de Langerhans/ultraestructura , Masculino , Melanocitos/ultraestructura , Fagosomas/química , Fagosomas/ultraestructura , Estaciones del Año , Método Simple CiegoAsunto(s)
Donantes de Sangre , Macroglobulinas/aislamiento & purificación , Proteínas Gestacionales/sangre , Adulto , Electroforesis de las Proteínas Sanguíneas/métodos , Contrainmunoelectroforesis/métodos , Femenino , Humanos , Macroglobulinas/análisis , Masculino , Persona de Mediana Edad , Embarazo , Proteínas Gestacionales/análisisRESUMEN
Gel filtration chromatography of maternal plasma from late pregnant hamsters demonstrated that approximately 90% of hamster placental lactogen-II (PL-II) is present as high mol wt (Mr) forms. A major peak of immunoactive hamster PL-II with a Mr of 600,000 and a smaller peak (Mr, 210,000) were observed. The major high Mr form of hamster PL-II in plasma had a Mr of 360,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). High Mr forms also predominate in placental extracts, but differ from the plasma forms. 125I-Labeled monomeric hamster PL-II (Mr, 22,000) formed a disulfide-bonded complex (Mr, 720,000 by gel filtration and 360,000 by SDS-PAGE) when incubated in vitro with serum of male, nonpregnant female, or pregnant hamsters. The in vitro complex was also formed with unlabeled hamster PL-II. Similar high Mr complexes were formed, but to a lesser extent, when 125I-labeled mouse PL-II and human PL were incubated with the homologous late pregnant serum. High Mr complexes (Mr, 360,000 by SDS-PAGE) were also formed when the three 125I-labeled PLs were incubated with purified human alpha 2-macroglobulin. The most prominent circulating high Mr form of hamster PL-II had a Mr similar to that of alpha 2-macroglobulin by both gel filtration and SDS-PAGE. In addition, it had a mobility similar to that of alpha 2-macroglobulin on nondenaturing polyacrylamide gels, and it was about 10% asparagine-linked carbohydrate by weight, as is alpha 2-macroglobulin. These similarities and the ability of hamster PL-II to form a disulfide-bonded complex with alpha 2-macroglobulin in vitro suggest that the major circulating form of PL-II in the hamster may be a disulfide-bonded complex of one or more PL monomers with alpha 2-macroglobulin or a related plasma protein. Similar complexed forms of PL may be present in mice and humans.
Asunto(s)
Macroglobulinas/metabolismo , Lactógeno Placentario/análisis , Animales , Cromatografía en Gel , Cricetinae , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Macroglobulinas/análisis , Mesocricetus , Ratones , Ratones Endogámicos , Peso Molecular , Placenta/metabolismo , Lactógeno Placentario/metabolismo , EmbarazoRESUMEN
The plasma alpha-macroglobulin and egg white ovomacroglobulin were purified from the sea turtle, Chelonia mydas japonica, and their structural and functional properties were studied with the aim of clarifying the degree of evolutional divergence of two homologous proteins specific to different tissues of the same animal. The concentration of alpha-macroglobulin in green turtle plasma was about 4 mg/ml. The protein was purified from the plasma by precipitation with polyethylene glycol 6000, followed by zinc chelate chromatography and gel chromatography on Sepharose CL-6B. The concentration of ovomacroglobulin in green turtle egg white was about 0.4 mg/ml. Ovomacroglobulin was purified by gel chromatography on Sepharose CL-6B. The two proteins had similar molecular weights and amino acid compositions, and both inhibited proteinases such as trypsin, chymotrypsin, papain, and thermolysin. The amino terminal sequences of the two proteins were homologous to each other but higher homologies were found between the ovomacroglobulin of turtle and chicken, and between the serum macroglobulins of the same animals. The functional difference between turtle alpha-macroglobulin and ovomacroglobulin became clear when they were treated with methylamine, which is known to destroy the inhibitory activity of human alpha 2-macroglobulin by splitting internal thiolester bonds. The inhibitory activity of the turtle plasma protein was completely destroyed by methylamine but that of ovomacroglobulin was only partially affected. The number of sulfhydryl groups as titrated with 5,5'-dithiobis(2-nitrobenzoate) before and after treatment with proteinases or methylamine was different for the two proteins. The amount of radioactive methylamine that was incorporated was also different between the two proteins. The two proteins purified in this study had no immunological cross-reactivity.
Asunto(s)
Proteínas del Huevo/aislamiento & purificación , Macroglobulinas/aislamiento & purificación , Tortugas/metabolismo , alfa-Macroglobulinas/aislamiento & purificación , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Proteínas del Huevo/análisis , Electroforesis en Gel de Poliacrilamida , Inmunodifusión , Macroglobulinas/análisis , Metilaminas/metabolismo , Péptido Hidrolasas/metabolismo , Compuestos de Sulfhidrilo/análisis , Inhibidores de Tripsina , Tortugas/sangre , Ultracentrifugación , alfa-Macroglobulinas/análisisRESUMEN
Green turtle plasma alpha-macroglobulin and ovomacroglobulin underwent conformational changes when they were treated with proteinases or methylamine. Their conformational changes were studied by HPLC gel chromatography, circular dichroism, and electron microscopy. The Stokes radii of native green turtle alpha-macroglobulin and ovomacroglobulin were estimated to be 84.3 +/- 0.5 A, and 93.0 +/- 0.5 A, respectively, by means of an HPLC experiment. After reaction with methylamine or proteinases, the Stokes radius of alpha-macroglobulin changed to 83.0 +/- 0.5 A or 85.4 +/- 0.5 A, respectively, and that of ovomacroglobulin to 93.0 +/- 0.5 A or 87.1 +/- 0.5 A. The circular dichroic spectra of native alpha-macroglobulin and ovomacroglobulin exhibited a negative band at around 215 nm, indicating the presence of beta-structure. Reaction of the two macroglobulins with methylamine resulted in a slight decrease in the ellipticity and reaction with proteinases led to a slight increase. The electron micrographic images of native alpha-macroglobulin and ovomacroglobulin can be described as deformed rings for the former and rugby balls for the latter. A common characteristic feature of the two molecules was that the central parts of the molecules were only thinly occupied by subunit. After reaction of macroglobulins with proteinases, the void spaces became partially filled and their overall shape more rectangular. Methylamine treatment caused a structural change only in alpha-macroglobulin but not in ovomacroglobulin. The difference in the susceptibility of the macroglobulins to methylamine was taken as an indication of evolutional divergence of the two homologous proteins within the last 300 million years.