Browsing by Author "FUENTES, ME"

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    A 13 KDA FRAGMENT IS RESPONSIBLE FOR THE HYDROPHOBIC AGGREGATION OF BRAIN G4 ACETYLCHOLINESTERASE
    (1988) FUENTES, ME; ROSENBERRY, TL; INESTROSA, NC
    Proteinase K treatment of the bovine brain acetylcholinesterase (AChE) releases a hydrophobic fragment of 13 kDa, which is entirely responsible entirely responsible for the aggregation of the G4 AChE in the absence of detergent. This observation provides evidence that the 13 kDa fragment, which comes from a previously identified 20 kDA subunit, is directly involved in the attachment of the G4 AChE to brain membranes. A model for the organization of the different sub-domains of the hydrophobic anchor of the G4 AChE is presented.
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    A COMPARISON OF THE XENOPUS-LAEVIS OOCYTE ACETYLCHOLINESTERASE WITH THE MUSCLE AND BRAIN ENZYME SUGGESTS VARIATIONS AT THE POSTTRANSLATIONAL LEVEL
    (1991) MOYA, MA; FUENTES, ME; INESTROSA, NC
    1. Xenopus laevis oocytes express endogenously two components of the cholinergic system: the muscarinic receptors and the acetylcholinesterase (AChE).
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    A MEMBRANE-ASSOCIATED DIMER OF ACETYLCHOLINESTERASE FROM XENOPUS SKELETAL-MUSCLE IS SOLUBILIZED BY PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHOLIPASE-C
    (1988) INESTROSA, NC; FUENTES, ME; ANGLISTER, L; FUTERMAN, AH; SILMAN, I
    The susceptibility to phosphatidylinositol-specific phospholipase C of the membrane associated acetylcholinesterase (AChE) forms of Xenopus laevis skeletal muscle was examined. This treatment released almost all the detergent-soluble AChE species from muscle homogenates. Sucrose gradient analysis showed that the released acetylcholinesterase form corresponds to a hydrophilic G2 dimer, indicating that this dimer has a glycolipid anchoring domain which contains phosphatidylinositol.
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    AMPHIPHILIC BEHAVIOR OF A BRAIN TETRAMERIC ACETYLCHOLINESTERASE FORM LACKING THE PLASMA-MEMBRANE ANCHORING DOMAIN
    (1992) FUENTES, ME; INESTROSA, NC
    We have studied the behavior of a mammalian brain tetrameric acetylcholinesterase (AChE) form released by proteinase K from a crude membrane fraction of bovine caudate nucleus. The solubilization of active AChE indicated the presence of a protease-sensitive site in the anchored protein. Unexpectedly, the solubilized AChE maintained its capacity to form aggregates in detergent-free gradients. We demonstrate here that this property was due neither to the presence of the hydrophobic membrane-anchoring domain still linked to the enzyme, nor to the presence of AChE activity trapped in small plasma membrane vesicles. Moreover, we found that the proteinase K-treated extract, devoid of AChE activity, induced the aggregation of purified hydrophilic AChE which usually does not form aggregates. Our results suggest the presence of an AChE aggregating factor in bovine brain extracts prepared in the presence of proteinase K. It is possible that this aggregation may reflect a process of AChE clustering on neurons.
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    BIOSYNTHESIS OF THE NEUROFILAMENT HEAVY SUBUNIT IN XENOPUS OOCYTES MICROINJECTED WITH RAT-BRAIN POLY(A)+ RNA
    (1987) CROSS, D; ALLENDE, ML; KRAUSS, RY; FUENTES, ME; KALTWASSER, G; ALVAREZ, J; INESTROSA, NC
    In order to study the expression of the major subunit of neurofilaments (NFs), rat brain poly(A)+ RNA was purified by three different procedures and was injected in Xenopus laevis oocytes. This system was able to translate efficiently the 200 kDa NF subunit as shown by a dot-blot immunoassay and by immunoprecipitation of labeled NF polypeptides.
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    CHARACTERIZATION OF A TETRAMERIC G4 FORM OF ACETYLCHOLINESTERASE FROM BOVINE BRAIN - A COMPARISON WITH THE DIMERIC G2 FORM OF THE ELECTRIC ORGAN
    (1988) FUENTES, ME; INESTROSA, NC
    Globular forms (G forms) of acetylcholinesterase (AChE) are formed by monomers, dimers and tetramers of the catalytic subunits (G1, G2 and G4). In this work the hydrophobic G2 and G4 AChE forms were purified to homogeneity from Discopyge electric organ and bovine caudate nucleus and studied from different points of view, including: velocity sedimentation, affinity to lectins and SDS-polyacrylamiide gel electrophoresis under reducing and non-reducing conditions. The polypeptide composition of Discopyge electric organ G2 is similar to Torpedo, however the pattern of the brain G4 AChE is much complex. Under non-reducing conditions the catalytic subunit possesses a molecular weight of 65 kDa, however this value increases to 68 kDa after reduction, suggesting that intrachain-disulfide bonds are important in the folding of the catalytic subunits of the AChE. Also it was found that after mild proteolysis; the (125I)-TID-20 kDa fragment decreased its molecular weight to approximately 10 kDa with little loss of AChE activity. Finally, we suggest a model for the organization of the different domains of the hydrophobic anchor fragment of the G4 form.
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    DECORIN, A CHONDROITIN DERMATAN SULFATE PROTEOGLYCAN IS UNDER NEURAL CONTROL IN RAT SKELETAL-MUSCLE
    (1992) BRANDAN, E; FUENTES, ME; ANDRADE, W
    Proteoglycans (PGs) are abundant components of the extracellular matrices (ECM) of skeletal muscle. We have previously found that the synthesis of skeletal muscle PGs present at the ECM increase after denervation. The experiments reported here were undertaken to identify which PG(s) increase after denervation of rat leg muscles. Incorporation of radioactive sulfate demonstrated the presence of a chondroitin/dermatan sulfate PG of 70-90 kDa in the skeletal muscle ECM, which increased after denervation. The PG has a core protein of 39-45 kDa after treatment with chondroitinase ABC. Antibodies against rat decorin, a chondroitin/dermatan sulfate PG synthesized by various cell types, specifically immunoprecipitated this PG from a mixture of PGs. Immunocytolocalization of this PG indicated that the chondroitin/dermatan sulfate PG accumulates at the perimysium of skeletal muscle after denervation. Finally, Northern blot analysis indicated an increase of muscle transcripts for decorin after denervation. The data reported here suggest that a chondroitin/dermatan sulfate PG present at the skeletal muscle ECM, very similar if not identical to decorin, increases after denervation.
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    NEUROTRANSMITTER-RELATED ENZYME ACETYLCHOLINESTERASE IN JUVENILES OF CONCHOLEPAS-CONCHOLEPAS (MOLLUSCA, GASTROPODA, MURICIDAE)
    (1990) GONZALEZ, M; PERELMAN, A; FUENTES, ME; CASTILLA, JC; LABARCA, R; BRANDAN, E; GONZALEZPLAZA, R; INESTROSA, NC
    With the aim of understanding the organization of the nervous systemn in the Prosobranchia gastropod Concholepas concholepas, we studied the properties, specificity, sedimentation coefficient, and solubility of the cholinergic enzyme, acetylcholinesterase (AChE). It was found that 95% of the esterase was inhibited by BW284c51 dibromide but not by iso-OMPA, which is consistent with the specificity of AChE. The calculated Km 0.22 mM is eight to ten times higher than are the Kms for AChE of other invertebrates and similar to the values reported for fish and vertebrates. The AChE shows a maximal activity around 22.degree. C, has a glycoprotein character and presents sedimentation coefficients of 6.5 S and 10.5 S. Most of this AChE activity is soluble under low ionic strength conditions; however, the enzyme aggregates in the absence of detergents. In conclusion, our evidence indicates the presence of a well-recognized molecular marker that could be useful for the study of the development of Concholepas concholepas.
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    PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHOLIPASE-C SOLUBILIZED G2 ACETYLCHOLINESTERASE FROM PLASMA-MEMBRANES OF CHROMAFFIN CELLS
    (1989) PRIETO, AL; FUENTES, ME; ARQUEROS, L; INESTROSA, NC
    Using whole homogenates and defined subcellular fractions of bovine adrenal medulla, we investigated the properties of the dimeric G2 molecular form of acetylcholinesterase (AChE), its distribution, and the mode of attachment to chromaffin cells. Our studies indicate that a substantial fraction of the G2 form is specifically susceptible to solubilization by phosphatidylinositol-specific phospholipase C (PIPLC) from subcellular fractions enriched with plasma membrane fragments. The results suggest that the G2 form of AChE is anchored in the plasma membrane to a glycolipid domain that contains phosphatidylinositol. Since a Ca+2-dependent PIPLC has been previously described in chromaffin granules, it is possible that the adrenal AChE could be released by a system reminiscent of that involved in the case of the surface glycoprotein of Trypanosoma brucei.
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    THE PROTEOGLYCAN DECORIN IS SYNTHESIZED AND SECRETED BY DIFFERENTIATED MYOTUBES
    (1991) BRANDAN, E; FUENTES, ME; ANDRADE, W
    Proteoglycans (PGs) are important components of the skeletal muscle extracellular matrix (ECM). Skeletal muscles are composed of muscle fibers and mononucleated cells. The latter are known to synthesize and secrete several PGs. Rat skeletal muscle ECM contains a chondrotin/dermatan sulfate PG which was immunoprecipitated by antibodies against rat decorin. The synthesis and secretion of PGs by a mouse cell line was analyzed during in vitro differentiation. PGs were characterized by biochemical and immunological techniques including immunocytolocalization experiments. At least three different PGs are synthesized and secreted by differentiated myotubes: a 220 to 460 kDa heparan sulfate, a 250 to 310 kDa chondroitin/dermatan sulfate, and a 75 to 130 kDa chondroitin/dermatan sulfate. This latter PG was specifically immunoprecipitated with antibodies against rat fibroblast decorin. Indirect immunocytolocalization analysis revealed that decorin was localized inside the cells, with a strong reaction around the nuclei. During differentiation the relative proportions of some PGs changed. Thus, a decrease in the relative proportion of the heparan sulfate PG was observed, whereas a significant increase in the relative proportion of decorin was detected. No change in the large chondroitin/dermatan PG was seen during the differentiation process. The possible cell sources of decorin found in rat skeletal muscle ECM are discussed.