RESUMEN
Piglets born with spread-leg syndrome, a congenital weakness of the hindlimb adductors, were investigated to determine the site of lesion leading to limb impairment. Histological and immunohistochemical studies of the motor neuron unit showed no alterations but quantitative analysis revealed a reduction of axonal diameter and myelin sheath-thickness of the fibres innervating the adductors of the affected limbs. In the lumbar spinal cord a lack of myelination was observed in the tracts descending to the lower motor neurons. Recovery from the syndrome was accompanied by a catching-up of myelination with that of the controls. The spread-leg syndrome is due to a nutritional deficiency in the sow; thus it is assumed that the deficient maternal substances, mainly choline and methionine, are essential for the normal myelin production by spinal white matter oligodendrocytes of the fetus.
Asunto(s)
Deformidades Congénitas de las Extremidades/etiología , Músculo Esquelético/anomalías , Músculo Esquelético/inervación , Fibras Nerviosas Mielínicas/patología , Fibras Nerviosas Mielínicas/ultraestructura , Médula Espinal/anomalías , Porcinos/anomalías , Factores de Edad , Animales , Animales Recién Nacidos , Enfermedades Carenciales/complicaciones , Enfermedades Carenciales/patología , Enfermedades Carenciales/fisiopatología , Vías Eferentes/anomalías , Vías Eferentes/patología , Vías Eferentes/ultraestructura , Deformidades Congénitas de las Extremidades/patología , Deformidades Congénitas de las Extremidades/fisiopatología , Vértebras Lumbares , Placa Motora/citología , Placa Motora/embriología , Placa Motora/metabolismo , Enfermedad de la Neurona Motora/embriología , Enfermedad de la Neurona Motora/patología , Enfermedad de la Neurona Motora/fisiopatología , Neuronas Motoras/patología , Neuronas Motoras/ultraestructura , Músculo Esquelético/patología , Nervios Periféricos/anomalías , Nervios Periféricos/patología , Nervios Periféricos/ultraestructura , Médula Espinal/patología , Médula Espinal/ultraestructura , Porcinos/metabolismoRESUMEN
Immunoreactivity of dystrophin family proteins was observed in the astrocytes of the adult and immature rat hippocampus and cerebral cortex by using Dys2, a monoclonal antibody recognizing both 427 kDa and short dystrophin isoforms. As revealed by light and electron microscopy, immunostaining of the ribosomal apparatus and of pericapillary endfeet was particularly pronounced in the adult. In the pericapillary astrocyte processes immunostaining appeared between postnatal days 10 and 20, and reached the intensity seen in the adult by postnatal day 30. In the pericapillary astrocyte process, the membrane facing the endothelial basal lamina was the earliest structure to show the immunoreaction. At later stages, the pericapillary astrocyte process was gradually filled up with immunoprecipitate. Findings suggest that dystrophins are expressed coinciding with the development of the blood-brain barrier, and it is assumed that they contribute to the formation of this system.
Asunto(s)
Astrocitos/química , Corteza Cerebral/química , Distrofina/análisis , Hipocampo/química , Animales , Barrera Hematoencefálica/fisiología , Capilares/química , Capilares/crecimiento & desarrollo , Corteza Cerebral/anatomía & histología , Corteza Cerebral/crecimiento & desarrollo , Femenino , Hipocampo/anatomía & histología , Hipocampo/crecimiento & desarrollo , Inmunohistoquímica , Masculino , RatasRESUMEN
Dystrophin, utrophin and dystroglycan are present not only in muscle but also in brain. In muscle, they link the extracellular matrix to the cytoskeleton. Their function in brain is not understood. Here we show their presence in the hypothalamo-neurohypophysial system which secretes the neurohormones oxytocin and vasopressin. Using immunocytochemistry, we showed that dystrophins are present in the neurohypophysis of control rats. After water deprivation, immunoreactivity dramatically decreased and appeared in axonal swellings in the hypothalamic tract. Dystrophin immunostaining can be ascribed to dystrophin and/or utrophin as well as the DMD (Duchenne Muscular Dystrophy) gene short products Dp140 and Dp71 as revealed by Western immunoblots of synaptosomes isolated from neurohypophyses of control rats. In synaptosomes isolated from rats under water deprivation, the immunoreactivity entirely disappeared. Further biochemichal characterization of isolated neurosecretory granules (NSG) showed that Dp140 and Dp71 are enriched in the NSG stored in the swellings of the neurohypophysis whereas the NSG of the nerve endings are devoid of these proteins. In addition we observed that the presence of beta-dystroglycan and actin correlates with the presence of dystrophins. Our data favor a direct implication of the dystrophins and/or utrophin, dystroglycan and actin in the neurosecretory processes of the hypothalamo-neurohypophysial system.
Asunto(s)
Deshidratación/metabolismo , Distrofina/análogos & derivados , Neurohipófisis/metabolismo , Animales , Anticuerpos Monoclonales , Western Blotting , Gránulos Citoplasmáticos/química , Gránulos Citoplasmáticos/metabolismo , Proteínas del Citoesqueleto/análisis , Proteínas del Citoesqueleto/inmunología , Distrofina/análisis , Distrofina/inmunología , Electroforesis , Hipotálamo/química , Hipotálamo/metabolismo , Immunoblotting , Masculino , Proteínas de la Membrana/análisis , Proteínas de la Membrana/inmunología , Sistemas Neurosecretores/química , Sistemas Neurosecretores/metabolismo , Neurohipófisis/química , Ratas , Ratas Wistar , Sinaptosomas/química , Utrofina , Privación de Agua/fisiologíaRESUMEN
Vimentin contributes to the cytoskeleton of different cell-types, among them glial cells. We report here that different forms of this protein, distinguishable by the monoclonal antibodies Vim3B4 and V9, are species-specifically expressed in cultures of glial fibrillary acidic protein (GFAP) positive, primary astrocytes of the chicken and rat. Most cells in the cultures co-expressed GFAP and one of the two vimentin epitopes. The Vim3B4 positive epitope was present in chicken astrocytes, while the V9 positive was not. Inverse situation was found in the astrocytes of rat. In vitro age of the cells did not influence the hierarchy of vimentin epitope expression with respect to species-specificity. Our result shows that the different vimentin expression program of cultured astrocytes of the chicken and rat is preserved under in vitro conditions. The presented data support the concept of the species-specific regulation of vimentin forms in glial cells of the central nervous system.
Asunto(s)
Astrocitos/inmunología , Epítopos/análisis , Vimentina/inmunología , Animales , Western Blotting , Células Cultivadas , Embrión de Pollo , Técnica del Anticuerpo Fluorescente , Ratas , Ratas Sprague-Dawley , Especificidad de la EspecieRESUMEN
Dystrophin immunocytochemistry was carried out using monoclonal antibody against the C-terminal part of dystrophin (Dys2) in rat cerebral cortex, cerebellum and hippocampus containing a high number of spine synapses of similar morphological character. Dys2 immunoreactivity was found in the spine component of spine synapses. Particularly heavy label was observed on the postsynaptic densities. In the cerebral and cerebellar cortices all spines were immunopositive. In the hippocampus some postsynaptic densities were intensely immunostained, whereas those of adjacent synapses remained unstained. The findings suggest that dystrophin is an integral protein of the postsynaptic component of spine synapses but is lacking in a subpopulation of hippocampal spine synapses. The heterogeneity of input to the hippocampal spiny sites by contrast to the homogeneous population of fibres synapsing with cerebral and cerebellar cortical spines is suggested to account for differences.
Asunto(s)
Química Encefálica/fisiología , Encéfalo/ultraestructura , Distrofina/metabolismo , Fibras Nerviosas/metabolismo , Sinapsis/metabolismo , Sinapsis/ultraestructura , Animales , Encéfalo/citología , Femenino , Hipocampo/citología , Hipocampo/metabolismo , Hipocampo/ultraestructura , Técnicas para Inmunoenzimas , Masculino , Fibras Nerviosas/ultraestructura , Pruebas de Precipitina , RatasRESUMEN
In order to study structural alterations which occur after a defined unilateral cortical infarct, the hindlimb region of the rat cortex was photochemically lesioned. The infarcts caused edema restricted to the perilesional cortex which affected allocortical and isocortical areas differently. Postlesional changes in cytoskeletal marker proteins such as microtubule-associated protein 2, non-phosphorylated (SMI32) and phosphorylated (SMI35, SMI31 and 200,000 mol. wt) neurofilaments and 146,000 mol. wt glycoprotein Py as well as changes in proteoglycans visualized with Wisteria floribunda lectin binding (WFA) were studied at various time points and related to glial scar formation. The results obtained by the combination of these markers revealed six distinct regions in which transient, epitope-specific changes occurred: the core, demarcation zone, rim, perilesional cortex, ipsilateral thalamus and contralateral homotopic cortical area. Within the core immunoreactivity for microtubule-associated protein 2 and SMI32 decreased and the cellular components showed structural disintegration 4 h post lesion, but partial recovery of somatodendritic staining was seen after 24 h. Microtubule-associated protein 2 and SMI32 persisted up to days 7 and 5 respectively in the core, whereas the number of glial fibrillary acidic protein- and WFA-positive cells decreased between days 7 and 14. The demarcation zone showed a dramatic loss of immunoreactivity for all epitopes 4 h post lesion which was not followed by a phase of recovery. In the inner region of the demarcation zone there was an invasion and accumulation of non-neuronal WFA-positive cells which formed a tight capsule around the core. Neuronal immunoreactivities for microtubule-associated protein 2, SMI31 and Py as well as astrocytic glial fibrillary acidic protein increased strongly within an approximately 0.4-1.0 mm-wide rim region directly bordering the demarcation zone. Py immunoreactivity increased significantly in the perilesional cortex, whereas glial fibrillary acidic protein-positive astrocytes became transiently more numerous in the entire lesioned hemisphere including strongly enhanced immunoreactivity in the thalamus by days 5-7 post lesion. Glial fibrillary acidic protein immunoreactivity increased in the corpus callosum and the homotopic cortical area of the unlesioned hemisphere by days 5-7. In this homotopic area additional changes in SMI31 immunoreactivity occurred. Our results showed that a cortical infarct is not only a locally restricted lesion, but leads to a variety of cytoskeletal and other structural changes in widely-distributed functionally-related areas of the brain.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Ataque Isquémico Transitorio/metabolismo , Proteoglicanos/metabolismo , Animales , Biomarcadores , Proteínas del Citoesqueleto/química , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Proteína Ácida Fibrilar de la Glía/química , Proteína Ácida Fibrilar de la Glía/metabolismo , Masculino , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Peso Molecular , Proteínas de Neurofilamentos/química , Proteínas de Neurofilamentos/metabolismo , Neuroglía/metabolismo , Ratas , Ratas Wistar , Trombosis/metabolismo , Trombosis/patologíaRESUMEN
Microtubule-associated tau proteins are likely candidates to interfere with axonal transport of membranous organelles. We studied that tau proteins influenced the enzyme activity of kinesin, known to drive anterograd transport along microtubules. An in vitro reconstituted system was applied; microtubules were assembled from purified tubulin with or without tau proteins. Both types of reconstituted microtubules stimulated MgATPase activity of purified kinesin in a concentration dependent, saturable manner. The extent of maximal stimulation by tau-coated microtubules was lower than that of microtubules without tau proteins. Analysis of kinetic data, on the other hand, suggests that tau-coated microtubules apparently bind kinesin with higher affinity then microtubules not associated with tau proteins. Tau proteins, similarly to tubulin dimers, seem to bind to the heavy chain of kinesin. These data support the notion that tau proteins could act as regulators of kinesin-driven processes.
Asunto(s)
Cinesinas/metabolismo , Microtúbulos/metabolismo , Proteínas tau/metabolismo , Animales , Química Encefálica , ATPasa de Ca(2+) y Mg(2+)/aislamiento & purificación , ATPasa de Ca(2+) y Mg(2+)/metabolismo , Bovinos , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Immunoblotting , Cinesinas/aislamiento & purificación , Cinesinas/farmacología , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/enzimología , Unión Proteica , Proteínas tau/aislamiento & purificaciónAsunto(s)
Anticuerpos , Encéfalo/citología , Proteínas Asociadas a Microtúbulos/análisis , Proteínas Asociadas a Microtúbulos/inmunología , Células de Purkinje/citología , Médula Espinal/citología , Animales , Cerebelo/citología , Dendritas/ultraestructura , Técnicas para Inmunoenzimas , Inmunohistoquímica , Bulbo Raquídeo/citología , RatasRESUMEN
Remote astroglial response was evoked in the adult rat visual cortex by unilateral destruction of the corpus geniculatum laterale. Homogenates of ipsi- and contralateral (operated and control) visual cortices were analysed by immunoblotting. A peak increase in glial fibrillary acidic protein (GFAP) was observed 2 weeks after lesion which then declined but did not return to normal showing a time course similar to that of previously observed immunohistochemical changes. Findings prove that the mechanism underlying the enhanced immunoreactivity during remote astroglial response is an up-regulation of GFAP synthesis in the astroglial processes surrounding degenerating synapses.
Asunto(s)
Astrocitos/fisiología , Proteína Ácida Fibrilar de la Glía/biosíntesis , Animales , Astrocitos/metabolismo , Desnervación , Potenciales Evocados Visuales/fisiología , Femenino , Inmunohistoquímica , Masculino , Degeneración Nerviosa/fisiología , Ratas , Sinapsis/fisiología , Regulación hacia Arriba , Corteza Visual/anatomía & histología , Corteza Visual/metabolismo , Corteza Visual/fisiologíaRESUMEN
Two monoclonal antibodies directed against vimentin, Vim 3B4 and V9 could distinguish between vimentins originating from certain species, when tested on cell lines (Bohn et al, 1992). Our comparative immunohistochemical studies in the rat and chicken brain with the same antibodies suggest the coexistence of two vimentin forms in the glial cells of both species. One of these forms bearing the epitope present in the respective non-glial cell lines is present in astrocytes and Bergmann glia independently of the ontogenic state of the animal. The other epitope appeared also mutually in both species, albeit its expression was more restricted. These patterns suggest that in these two species, the expression of the different vimentin forms might be differently regulated.
Asunto(s)
Anticuerpos Monoclonales , Encéfalo/citología , Neuroglía/citología , Vimentina/análisis , Vimentina/inmunología , Envejecimiento/metabolismo , Animales , Especificidad de Anticuerpos , Astrocitos/citología , Vasos Sanguíneos/citología , Encéfalo/crecimiento & desarrollo , Línea Celular , Cerebelo/irrigación sanguínea , Cerebelo/citología , Pollos , Técnica del Anticuerpo Fluorescente Indirecta , Inmunohistoquímica/métodos , Fibras Nerviosas/ultraestructura , Ratas , Especificidad de la EspecieRESUMEN
Kinesin, a mechanochemical enzyme that translocates membranous organelles, was initially identified and purified from soluble extracts from vertebrate brains. However, immunocytochemical and morphological approaches have demonstrated that kinesin could be associated to intracellular membranous organelles. We used an antibody raised against the head portion of the Drosophila kinesin heavy chain to reveal the presence of this protein in membranous organelles from rat brain. By using differential centrifugation and immunoblotting we observed a 116 kDa protein that crossreacts with this antibody in microsomes, synaptic vesicles, and mitochondria. This protein could be extracted from mitochondria with low salt concentrations or ATP. The 116 kDa solubilized protein has been identified as conventional kinesin based on limited sequence analysis. We also show that a polyclonal antibody raised against mitochondria-associated kinesin recognizes soluble bovine brain kinesin. The soluble and mitochondrial membrane-associated kinesins show a different isoform pattern. These results are consistent with the idea that kinesin exists as multiple isoforms that might be differentially distributed within the cell. In addition digitonin fractionation of mitochondria combined with KI extraction revealed that kinesin is a peripheral protein, preferentially located in a cholesterol-free outer membrane domain; this domain has the features of contact points between the mitochondrial outer and inner membranes. The significance of these observations on the functional regulation of the mitochondria-associated kinesin is discussed.
Asunto(s)
Química Encefálica , Cinesinas/química , Proteínas de la Membrana/química , Mitocondrias/química , Adenosina Trifosfatasas/análisis , Adenosina Trifosfato , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Fraccionamiento Celular , Digitonina , Cinesinas/genética , Cinesinas/aislamiento & purificación , Cinesinas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Microsomas/química , Microtúbulos/metabolismo , Mitocondrias/enzimología , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos , Ratas , Alineación de Secuencia , Análisis de Secuencia , Vesículas Sinápticas/químicaRESUMEN
In isolated, intact liver mitochondria from hyperthyroid rats, the L-glycerol-3-phosphate binding site(s) of the L-glycerol-3-phosphate dehydrogenase was (were) found to be influenced by the nature of the electron acceptor, as well as by the pH and the presence of calcium ions. With the hydrophobic electron acceptor menadione a single L-glycerol-3-phosphate binding site was detected kinetically at bulk pH values between 6.5 and 9.0. With the hydrophilic phenazine methosulfate, on the other hand, two L-glycerol-3-phosphate binding sites were distinguishable at pH > or = 7.5 and pH > or = 7.0, in the presence and absence of Ca2+, respectively. The kinetic mechanism of the reaction catalyzed by L-glycerol-3-phosphate dehydrogenase is ping pong Bi Bi with a hydrophilic electron acceptor, where as with the hydrophobic substance, a sequential Bi Bi mechanism was observed. We suggest that the latter mechanism has physiological relevance.
Asunto(s)
Deshidrogenasas de Carbohidratos/metabolismo , Hipertiroidismo/enzimología , Mitocondrias Hepáticas/enzimología , 2,6-Dicloroindofenol/metabolismo , Animales , Sitios de Unión , Calcio/metabolismo , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Concentración de Iones de Hidrógeno , Cinética , Masculino , Ratas , Vitamina K/metabolismoRESUMEN
Clinical data of 70 patients, treated and observed with myelodysplastic syndrome between 1977 and 1989 were analysed. Two-thirds of the patients belonged to the elder age-group and a mild female predominance was registered. With the application of complex cytochemical-histological and cytogenetical methods, correct diagnosis could be established. The clinical material included patients from different morphologic subtypes: 19 with refractory anaemia (with a longer course of the illness). 20 with sideroblastic anaemia, 26 with chronic myelomonocytic leukaemia and the remaining 5 with refractory anaemia with excess of blasts (a more progressive type of the myelodysplastic syndrome, with a short duration). The mean survival of all patients were 42 months. 45 (69%) died during this period and 12 (18.5%) among them in acute myelogenous leukaemia (mean survival: 16 month). Megakaryoblastic leukaemic transformation was observed in three patients with sideroblastic refractory anaemia. Haemorrhage and infection-sepsis, due to thrombocytopenia and/or granulocytopenia, was fatal in 30 cases. In the treatment of the myelodysplastic syndrome an appropriate supportive therapy (blood transfusion, antibiotics) has a decisive importance. A more aggressive treatment with cytostatic drugs is suggested in the progressive form of the disease of younger patients and in patients with overt acute leukaemia.
Asunto(s)
Síndromes Mielodisplásicos/mortalidad , Adulto , Anciano , Femenino , Humanos , Hungría , Masculino , Persona de Mediana EdadRESUMEN
FAD-linked L-glycerol-3-phosphate dehydrogenase purified from liver mitochondria of hyperthyroid rats was incorporated into unilamellar phospholipid liposomes. The incorporation influenced both Vmax,app and Km,app values of the enzyme for its substrate, L-glycerol 3-phosphate. The Km,app for the electron acceptor remained unchanged with a simultaneous slight enhancement of the corresponding Vmax,app value. The steady-state fluorescence anisotropies of the fluorescein isothiocyanate and trimethylammoniumdiphenylhexatriene labels were affected by sodium oleate and calcium ions in the case of both solubilized and liposome-incorporated L-glycerol-3-phosphate dehydrogenase. These results indicate that calcium ions cause a significant alteration of the enzyme conformation. Sodium oleate (used as a model of free fatty acids), besides its direct action on the enzyme itself, affects the enzyme indirectly as well, via alteration of the physical properties of the membrane.
Asunto(s)
Calcio/farmacología , Glicerolfosfato Deshidrogenasa/metabolismo , Liposomas/metabolismo , Mitocondrias Hepáticas/enzimología , Ácido Oléico , Ácidos Oléicos/farmacología , Animales , Cardiolipinas/análisis , Cardiolipinas/farmacología , Difenilhexatrieno/análogos & derivados , Fluoresceína-5-Isotiocianato , Fluoresceínas , Colorantes Fluorescentes , Glicerofosfatos/metabolismo , Hipertiroidismo/enzimología , Cinética , Liposomas/análisis , Masculino , Fosfolípidos/análisis , Conformación Proteica/efectos de los fármacos , Ratas , Espectrometría de Fluorescencia , TiocianatosRESUMEN
Recently, analysis of protein distribution in rat brain mitochondria suggested the existence of distinct cholesterol domains in the outer membrane (Dorbani et al., 1987, Arch. Biochem. Biophys. 252, 188-196) while such domains were not detected in rat liver mitochondria (Jancsik et al., 1988, Arch. Biochem. Biophys. 264, 295-301). We studied cholesterol distribution in both types of mitochondria by analyzing the kinetics of filipin-cholesterol complex formation, using the stopped-flow technique. In liver mitochondria, the kinetics are characterized by a biphasic curve which presumably corresponds to the two membranes. This was confirmed by the finding that pretreatment with digitonin abolished one of the kinetic components. Sonication of the mitochondria increased the rate of the filipin-cholesterol complex formation and also abolished one of the two components. In the case of brain mitochondria, several distinct cholesterol domains could be revealed: one of them was cholesterol-free and it was directly accessible to filipin. Two other domains were revealed by differences found in the rate of the cholesterol-filipin complex formation. It is noteworthy that only a part of the cholesterol is accessible to filipin. Sonication of mitochondria decreased the proportion of cholesterol molecules accessible to filipin. This suggests specific interactions of cholesterol with other mitochondrial components, which occur only in brain mitochondria.
Asunto(s)
Antifúngicos , Química Encefálica , Colesterol/análisis , Filipina , Membranas Intracelulares/análisis , Lípidos de la Membrana/análisis , Mitocondrias Hepáticas/análisis , Mitocondrias/análisis , Partículas Submitocóndricas/análisis , Animales , Cinética , Liposomas , RatasRESUMEN
Adriamycin was used in situ, in isolated liver mitochondria of hyperthyroid rats to study the role of cardiolipin in the functioning of FAD-linked L-glycerol-3-phosphate dehydrogenase. The apparent kinetic parameters of the reaction catalyzed by the enzyme were affected by adriamycin. The effect of adriamycin was dependent on the electron acceptor, suggesting the existence of distinct binding sites for hydrophobic and hydrophilic acceptors. Assuming a correlation between the two plateaus observed upon binding of adriamycin to the mitochondria and the penetration of the drug into the two leaflets of the inner membrane [Cheneval et al. (1985) J. Biol. Chem. 260, 13003-13007], we can deduce that cardiolipin in both leaflets influences predominantly the electron acceptor binding site(s).
Asunto(s)
Cardiolipinas/metabolismo , Glicerolfosfato Deshidrogenasa/metabolismo , Mitocondrias Hepáticas/enzimología , Animales , Sitios de Unión , Doxorrubicina/metabolismo , Doxorrubicina/farmacología , Glicerofosfatos/metabolismo , Hipertiroidismo/enzimología , Cinética , Masculino , Metosulfato de Metilfenazonio/metabolismo , Mitocondrias Hepáticas/efectos de los fármacos , Oxidación-Reducción , Ratas , Ubiquinona/metabolismo , Vitamina K/metabolismoRESUMEN
Two major brain microtubule-associated proteins (MAPs), MAP2 and tau, were found to be able to bind to purified rat brain mitochondria. The apparent dissociation constants of the binding of thermostable 32P-labeled MAP2 and tau are 0.9 +/- 0.04 x 10(-7) and 3.8 +/- 0.7 x 10(-7) M, respectively. 32P-labeled MAP2 and tau bound to the mitochondria can be displaced by phosphorylated, nonradioactive MAP2. The binding parameters of MAP2 prepared without heat treatment and those of the thermostable MAP2 were of the same order of magnitude. Microtubule-binding and projection domains of MAP2 were obtained by chymotryptic digestion of rat brain microtubules (Vallee, Proc. Natl. Acad. Sci. USA, 77:3206-3210, 1980). Displacement studies with these two domains show that MAP2 bound to mitochondria can be displaced by the microtubule-binding domain, whereas the projection domain does not displace MAP2. The two domains of MAP2 bind to the mitochondria with similar affinity constants; however, the Bmax for the projection domain was 10 times and 35 times lower than the Bmax of the binding of the intact MAP2 and the microtubule-binding domain, respectively. Chymotryptic digestion of MAP2 bound to the mitochondria yielded peptide fragments with molecular masses similar to those obtained by the digestion of MAP2 bound to the microtubules. The fragments corresponding to the projection domain were released into the extramitochondrial supernatant, whereas the fragments originating from the microtubule-binding domain remained bound to the mitochondria. These results suggest that MAP2 binds to mitochondria preferentially via its microtubule-binding domain.
Asunto(s)
Encéfalo/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitocondrias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Sitios de Unión , Quimotripsina , Fosforilación , Unión Proteica , Ratas , Ratas Endogámicas , Proteínas tauRESUMEN
The effect of thyroid status on the physical properties of rat liver mitochondrial membranes and on the lipid microenvironment of proteins was investigated. The steady-state fluorescence anisotropy of diphenyl-1,3,5-triene and 1-[4-(trimethylaminophenyl)phenyl]-6-phenylhexa-1,3,5-triene revealed an increase of the order of the membranes with the increase of hormone level. Protein arrangement in the inner mitochondrial membrane altered with the thyroid status, which was reflected by digitonin subfractionation of mitochondria. The microenvironment of FAD-linked L-glycerol-3-phosphate dehydrogenase was dramatically influenced by thyroxine.
Asunto(s)
Glicerolfosfato Deshidrogenasa/metabolismo , Membranas Intracelulares/metabolismo , Mitocondrias Hepáticas/metabolismo , Enfermedades de la Tiroides/metabolismo , Hormonas Tiroideas/fisiología , Animales , Digitonina , Difenilhexatrieno/análogos & derivados , Polarización de Fluorescencia , Colorantes Fluorescentes , Masculino , Ratas , SolubilidadRESUMEN
The present investigation has attempted to define in rat liver mitochondria the distribution of outer membrane proteins in relation to the inner membrane by fractionation with digitonin and phospholipase A2. Porin, the channel-forming protein in the outer membrane, was measured quantitatively by immunological methods. Neither monoamine oxidase nor porin could be released by phospholipase A2 treatment, but both were released by digitonin, at the same detergent concentration. Thus, the release of monoamine oxidase and porin requires the disruption of the cholesterol but not the phospholipid domains of the membrane and the two polypeptides exist in the same, or similar, membrane environment with regard to cholesterol. Changes in the energy state, or binding of brain hexokinase to rat liver mitochondria prior to fractionation with digitonin, did not alter the release patterns of porin and monoamine oxidase. The uptake of Ca2+, however, resulted in the concomitant release of the outer membrane markers together with the matrix marker, malate dehydrogenase. The present findings with liver differ from those obtained recently with brain mitochondria (L. Dorbani et al. (1987) Arch. Biochem. Biophys. 252, 188-196) in which two populations of porin were located in two different cholesterol domains. The significance of these differences in the location of porin in liver and brain mitochondria is discussed.