Browsing by Author "BRANDAN, E"

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    A HIGH-MOLECULAR-WEIGHT PROTEOGLYCAN IS DIFFERENTIALLY EXPRESSED DURING DEVELOPMENT OF THE MOLLUSK CONCHOLEPAS-CONCHOLEPAS (MOLLUSCA, GASTROPODA, MURICIDAE)
    (1992) BRANDAN, E; GONZALEZ, M; INESTROSA, NC; TREMBLAY, C; URREA, R
    Incorporation of radioactive sulfate to hatched veliger larvae of the gastropod muricid Concholepas concholepas indicated that over 87% of the sulfated macromolecules were found in the detergent insoluble fraction, rich in extracellular matrix (ECM) components. The sulfated material was solubilized with guanidine salt followed by urea dialysis and fractionated by DEAE-Sephacel chromatography. Three sulfated compounds eluting at 0.7, 1.1, and 3.0 M NaCl, called peaks I, II, and III, respectively, were obtained. The sulfated compound present in peak I was degraded by pronase or sodium alkaline treatment to a small sulfated resistant material, suggesting the presence of a proteoglycan (PG). Filtration analysis on Sephacryl S-500 and SDS-PAGE of the intact PG indicates that it has a high molecular weight (360,000 to over 1 X 10(6)). Monoclonal antibodies (mAb) against this PG were produced. The specificity of one mAb, the 6H2, was demonstrated by size chromatography and ELISA analysis. The epitope recognized by this mAb seems to be present in the core protein of the PG. Both the extent of sulfation and the presence of different sulfated species of PGs were evaluated during the development of this mollusc. A twelvefold increase in the incorporation of sulfate to PGs per milligram of protein was found in veliger larvae compared to blastula-glastula stages. This change correlated well with the differential expression of the sulfated PG present in peak I. Biochemical and immunological analysis indicate that high levels of this PG are found in veliger and trocophore larvae in comparison with blastula-gastrula and early juveniles. These results indicate that a high molecular weight PG probably of the ECM is differentially expressed during the development of the gastropod Concholepas concholepas.
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    A LIPID-ANCHORED HEPARAN-SULFATE PROTEOGLYCAN IS PRESENT IN THE SURFACE OF DIFFERENTIATED SKELETAL-MUSCLE CELLS - ISOLATION AND BIOCHEMICAL-CHARACTERIZATION
    (1993) CAMPOS, A; NUNEZ, R; KOENIG, CS; CAREY, DJ; BRANDAN, E
    We have investigated the presence of hydrophobic membrane-associated heparan sulfate proteoglycans (HSPG) on the cell surface of differentiated skeletal muscle cells. A HSPG releasable by incubation with a phosphatidylinositol-specific phospholipase c (PtdIns-PLC) was obtained. HSPG were also isolated from Triton X-100 extracts of the cells. The hydrodynamic characteristics of the PtdIns-PLC-releasable and detergent-extracted HSPG were indistinguishable. SDS/PAGE analysis of the PtdIns-PLC-releasable HSPG indicated a molecular mass of 250 kDa. Analysis of proteins immunoprecipitated by specific antibodies against a HSPG isolated from Schwann cells demonstrated that the antisera precipitated an intact HSPG that was present in the pool of proteins released by PtdIns-PLC and by Triton X-100 from [S-35]sulfate labeled cells. Nitrous acid degradation of the immunoprecipitated proteins released by PtdIns-PLC from [S-35]methionine labeled cells produced a single 67-kDa core protein. Analysis of hydrophobicity of the purified HSPG revealed that only the HSPG obtained from the detergent extract were able to be incorporated into the liposomes whereas the PtdIns-PLC-released HSPG was not.
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    AXONAL SPROUTING INDUCED IN THE SCIATIC-NERVE BY THE AMYLOID PRECURSOR PROTEIN (APP) AND OTHER ANTIPROTEASES
    (1992) ALVAREZ, J; MORENO, RD; LLANOS, O; INESTROSA, NC; BRANDAN, E; COLBY, T; ESCH, FS
    Protease inhibition is the mechanism by which some trophic factors promote the extension of neurites. In the rat sciatic nerve, we assessed the ability to induce sprouts of the APP isoform that embodies the Kunitz antiprotease domain and other antiproteases. With the electron microscope, axonal sprouts were found when antiproteases were supplied but not after administration of inactive substances. We conclude that axons have a drive to sprout which can be released by the unbalance of an extracellular protease antiprotease system. We propose that this system is involved in the pathogenesis of Alzheimer's disease.
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    BINDING OF THE ASYMMETRIC FORMS OF ACETYLCHOLINESTERASE TO HEPARIN
    (1984) BRANDAN, E; INESTROSA, NC
    The interaction between acetylcholinesterase (EC 3.1.1.7) and heparin, a sulfated glycosaminoglycan, was studied by affinity chromatography. A specific binding of the asymmetric acetylcholinesterase to an agarose gel containing covalently bound heparin was demonstrated. This interaction required an intact collagenous tail, shown by the fact that the binding is abolished by pretreatment with collagenase. Globular forms did not bind to the column. Total and intracellular asymmetric acetylcholinesterase forms isolated from the endplate region of the rat diaphragm muscle showed higher affinity for the heparin than did the enzyme from the non-endplate region. Binding to the resin was destabilized with 0.55 M NaCl, and, among the various glycosaminoglycans tested, only heparin was able to displace the acetylcholinesterase bound to the column. Apparently, asymmetric acetylcholinesterase forms are immobilized on the synaptic basal lamina via interactions with heparin-like molecules, probably related to heparan sulfate proteoglycans.
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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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    DIFFERENT MEMBRANE-BOUND FORMS OF ACETYLCHOLINESTERASE ARE PRESENT AT THE CELL-SURFACE OF HEPATOCYTES
    (1989) PERELMAN, A; BRANDAN, E
    In the present study we have determinated the acetylcholinesterase molecular forms present in rat liver hepatocytes; we have also studied the association of acetylcholinesterase with the cell surface of the hepatocytes. Subcellular fractionation indicated that rough endoplasmic reticulum and plasma-membrane-enriched fractions contains G4 and G2 acetylcholinesterase forms bound to membranes. Hepatocytes incubated with phosphatidylinositol-specific phospholipase C released about 70% of the surface acetylcholinesterase. Sedimentation analysis showed that all the solubilized acetylcholinesterase activity comes exclusively from a G2 dimer. The G4 hydrophobic form of acetylcholinesterase accounts for the additional cell-surface activity. The existence of these two forms of acetylcholinesterase on the surface of hepatocytes was further established by analyzing the phosphatidylinositol-specific phospholipase C sensitivity of the acetylcholinesterase molecular forms present in isolated rat liver plasma membranes.
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    DIFFERENTIAL ASSOCIATION AND DISTRIBUTION OF ACETYLCHOLINESTERASE AND BUTYRYLCHOLINESTERASE WITHIN RAT-LIVER SUBCELLULAR ORGANELLES
    (1990) PERELMAN, A; ABEIJON, C; HIRSCHBERG, CB; INESTROSA, NC; BRANDAN, E
    Rat liver cholinesterases were found to share properties and characteristics with those expressed in cholinergic tissues. The distribution and presence of different molecular forms of cholinesterases in different subcellular organelles of rat liver were studied. The rough and smooth endoplasmic reticulum and Golgi apparatus were enriched in the G4 molecular form of acetylcholinesterase (AChE) (relative to the G2 molecular form), while the inverse was found in the plasma membrane. The interaction of these molecular forms of AChE with the Golgi membrane was studied in detail. Approximately one-half of the G4 form was free within the lumen while the remainder was an intrinsic membrane protein; all the G2 molecular form was anchored to the membrane via phosphatidylinositol. Only the G1 and G2 molecular forms of butyrylcholinesterase (BuChE) were found in the above subcellular organelles; both molecular forms were soluble within the lumen of Golgi vesicles. These results indicate that rat liver expresses several molecular forms of AChE which have multiple interactions with membranes and that liver is unlikely to be the source of the G4 form of BuChE present in high concentration in the plasma.
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    DIFFERENTIAL ASSOCIATION OF RAT-LIVER HEPARAN-SULFATE PROTEOGLYCANS IN MEMBRANES OF THE GOLGI-APPARATUS AND THE PLASMA-MEMBRANE
    (1989) BRANDAN, E; HIRSCHBERG, CB
    Heparan sulfate proteoglycans (HSPG) of rat liver are associated with the plasma membrane in a hydrophobic intrinsic and a hydrophilic extrinsic form. We were interested in determining whether or not these two forms could be detected in the Golgi apparatus, the subcellular site of addition of oligosaccharides and sulfate to HSPG. In vivo and in vitro radiolabeled HSPG from rat liver Golgi apparatus membranes could only be solubilized with detergents that disrupt the membrane lipid bilayer, suggesting that they are solely associated via hydrophobic interactions. Both forms of HSPG were detected in plasma membranes of rat liver and isolated rat hepatocytes. The detergent-solubilized HSPG bound to octyl-Sepharose columns, whereas the hydrophilic form did not; this latter form, however, was released from the membrane by heparin. The hydrophobic anchor of HSPG in the Golgi and plasma membranes was insensitive to treatment with phosphatidylinositol-specific phospholipase C under conditions in which alkaline phosphatase was sensitive; this suggests that the hydrophobic anchor of HSPG is the core protein itself. Preliminary experiments suggest that the subcellular site of processing of the hydrophobic to the hydrophilic form of HSPG is the plasma membrane. A specific processing activity, probably a protease of the plasma membrane not present in serum or the endoplasmic reticulum membrane, converted hydrophobic HSPG of the Golgi membrane to the hydrophilic form. In addition, pulse-chase experiments with [35S]Na2SO4 in rats demonstrated that at short times, the bulk of the radiolabeled cellular HSPG was in the Golgi apparatus; later on, the bulk of the radioactivity was found in the plasma membrane, the only subcellular site where the hydrophilic form of HSPG was detected.
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    EFFECT OF SALT CONCENTRATION ON THE SYNTHESIS OF SULFATED MACROMOLECULES IN THE BRINE SHRIMP (ARTEMIA-FRANCISCANA) - CHANGES OF SULFATION RATE DURING DEVELOPMENT
    (1993) BRANDAN, E; URREA, R; GAJARDO, G
    1. The effect of sodium chloride and sulphate concentrations on the rate of sulphation was evaluated in four developmental stages of the brine shrimp Artemia franciscana, a crustacean inhabiting extreme hypersaline environments.
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    HEPARIN-ACETYLCHOLINESTERASE INTERACTION - SPECIFIC DETACHMENT OF CLASS I-A FORMS AND BINDING OF CLASS I AND CLASS-II-A FORMS TO HEPARIN-AGAROSE
    (1986) BRANDAN, E; LLONA, I; INESTROSA, NC
    This study describes the specificity, time-course and characteristics of the solubilization of class I-A forms of AChE by heparin, from the endplate regions of rat diaphragm muscle. Heparin fractions which differed in charge size, anticoagulant activity and capacity to bind type I collagen, were probed in their ability to extract AChE. No differences were found among all the fractions tested. Affinity chromatography on heparin-agarose of class I- and class II-A forms of esterase showed that both classes were able to bind to the column with the same relative affinity. Our results establish the use of heparin, as a solubilizing agent for the class I-A. The existence of a heparin-binding domain in class I- and class II-A forms of AChE, opens the possibility, that heparan sulfate proteoglycans could be involved in the anchorage of both types of esterase to synaptic regions. Finally, our results suggest that class I and class II-A do not correspond to intrinsically distinct molecules, but rather to identical molecules engaged in different interactions in the tissue.
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    INCREASE OF MACROMOLECULE SYNTHESIS AFTER HATCHING OF CONCHOLEPAS-CONCHOLEPAS VELIGER LARVAE - EFFECT OF SULFATE IN THE SYNTHESIS OF PROTEOGLYCANS
    (1990) BRANDAN, E; GONZALEZ, M; GONZALEZPLAZA, R; INESTROSA, NC
    1. The synthesis of proteins, glycoproteins and sulfated macromolecules increase after hatching of the gastropod Concholepas concholepas larvae. 2. Sulfate present in the sea-water, stimulates over 30-fold the sulfation of sulfated macromolecules but not the proteins. 3. Analysis of the sulfated macromolecules indicates that almost 90% are localized in the larval insoluble material, corresponding to a high molecular weight macromolecule, probably a proteoglycan.
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    ISOLATION AND CHARACTERIZATION OF RAT SKELETAL-MUSCLE PROTEOGLYCAN DECORIN AND COMPARISON WITH THE HUMAN FIBROBLAST DECORIN
    (1991) ANDRADE, W; BRANDAN, E
    1. The extracellular matrix (ECM) of rat skeletal muscle contains several proteoglycans (PGs). The more abundant correspond to a chondroitin/dermatan sulfate PG or decorin.
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    ISOLATION AND PURIFICATION OF HUMAN BILIARY VESICLES WITH POTENT CHOLESTEROL-NUCLEATION-PROMOTING ACTIVITY
    (1992) MIQUEL, JF; RIGOTTI, A; ROJAS, E; BRANDAN, E; NERVI, F
    1. Cholesterol nucleation is a critical step in the formation of cholesterol gallstones. This nucleation takes place after aggregation and fusion of cholesterol-rich biliary vesicles, a process probably modulated by biliary proteins. The present study was conducted to identify specific proteins associated with native cholesterol-rich biliary vesicles and to explore their effect on the cholesterol-nucleation time of supersaturated artificial bile.
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    ISOLATION OF PROTEOGLYCANS SYNTHESIZED BY RAT-HEART - EVIDENCE FOR THE PRESENCE OF SEVERAL DISTINCT FORMS
    (1992) GONZALEZ, R; URREA, R; GONZALEZ, M; INESTROSA, NC; BRANDAN, E
    1. The proteoglycans (Ps) synthesized by auricle and ventricle from adult rat
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    ISOLATION OF THE HEPARAN-SULFATE PROTEOGLYCANS FROM THE EXTRACELLULAR-MATRIX OF RAT SKELETAL-MUSCLE
    (1987) BRANDAN, E; INESTROSA, NC
    We have previously shown that asymmetric collagen-tailed acetylcholinesterase (AChE) is anchored to the extracellular matrix (ECM) by heparan sulfate proteoglycans (HSPGs). Here we present our studies on the characterization of such PGs from the ECM of rat skeletal muscles. After radiolabeling with 35SO4 for 24 h, PGs were extracted from the muscle ECM with 4.0 M guanidine-HCl containing protease inhibitors. PGs were subsequently isolated using sequential DEAE-Sephacel chromatography, digestion with chondroitinase ABC, and Sepharose CL-4B. Two different hydrodynamic size species of HSPGs were found. One type had a Mr of 4-6 .times. 105 (Kav = 0.25) as estimated by gel chromatography in the presence of 1% SDS and accounted for 75% of the total HSPGs. The other HSPG had a Mr 1.5-2.5 .times. 105 (Kav = 0.41). The glycosaminoglycan (GAG) side chains (Mr 20,000 and 12,000) were found composed only of heparan sulfate as determined by nitrous acid oxidation and heparitinase treatment. The large-sized HSPG, which is concentrated in synaptic regions, contains only GAG chains of Mr 20,000, suggesting that each HSPG contains only one kind of heparan sulfate chain in its structure. Our results definitively establish by biochemical criteria that the basement membrane of mammalian skeletal muscle contains HSPGs, the likely matrix receptor for the immobilization of the asymmetric collagen-tailed AChE at the neuromuscular junction.
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    MOTOR-NERVE REGULATES MUSCLE EXTRACELLULAR-MATRIX PROTEOGLYCAN EXPRESSION
    (1990) FADIC, R; BRANDAN, E; INESTROSA, NC
    Denervation of rat leg muscles caused a 2-3 fold increase in 35S-sulfate and 3H-glucosamine incorporation into proteoglycans of the muscle extracellular matrix. The size of the proteoglycans and the glycosaminoglycan chain length and degree of sulfation were unchanged. Because the rate of degradation of proteolyglycans was also unchanged by denervation, we infer that denervation increases proteoglycan synthesis. Muscle reinnervation restored the original rate of synthesis of proteoglycans. Paralysis of innervated muscle caused increased incorporation of sulfate comparable to that seen in denervation. Thus motor nerve activity appears to regulate the level of proteoglycans in the muscle extracellular matrix.
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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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    SULFATION IS REQUIRED FOR MOBILITY OF VELIGER LARVAE OF CONCHOLEPAS-CONCHOLEPAS (MOLLUSCA, GASTROPODA, MURICIDAE)
    (1992) URREA, R; GONZALEZ, M; INESTROSA, NC; BRANDAN, E
    The sulfation reaction seems to be a critical biochemical process during early steps of development. We have evaluated the effect of sulfation on the mobility of veliger larvae of the gastropod Concholepas concholepas. It was found that incubation of larvae in low-sulfate artificial sea water had strong inhibitory effect on mobility. The use of sodium chlorate, a specific inhibitor of sulfation, also resulted in a strong inhibition of larval mobility. At the biochemical level, the synthesis of proteoglycans (PGs) and detergent-soluble sulfoproteins and sulfolipids was specifically inhibited by chlorate, without affecting either total protein synthesis or phosphorylation. Intracellular levels of the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) were decreased to 4% by chlorate treatment, indicating that this molecule is also involved in sulfation of marine invertebrates. Both effects of chlorate, the inhibition of sulfation and the larval mobility, were reversible. It is therefore concluded that sulfation is required for larval mobility in the mollusc C. concholepas.