J.P. Zanetta

Occurrence of ceramides and neutral glycolipids with unusual long-chain base composition in purified rat liver mitochondria.

The free ceramide content of rat liver mitochondria was found to be 1.7 nmol/mg protein and outer membranes contained a three‐fold higher concentration than inner membranes. The mitochondrial content in neutral glycolipids was 0.6 nmol/mg protein. The long‐chain bases found in free ceramides were d18:1 sphingosine, d18:0 3‐ketosphinganine and t21:1 phytosphingosine in increasing order. In contrast, 3‐ketosphinganine was the only base of glucosylceramide and lactosylceramide of inner membranes, whereas d18:1 sphingosine was the major long‐chain base of glucosylceramide of outer membranes.

Purification and properties of the membrane-bound acetylcholinesterase from adult rat brain

The membrane-bound acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) from adult rat brain has been purified to homogeneity using sequential affinity chromatography on Con A-Sepharose and on dimethyl-aminoethylbenzoic acid-Sepharose 4B followed by DEAE-cellulose chromatography. The yield of the purified enzyme (specific activity: 3068 U/mg protein) is higher than 50%. Polyacrylamide gel electrophoresis in the presence of Triton X-100 gives only one band with acetylcholinesterase activity. With the exception of electrofocusing and pore gradient electrophoresis, where a multiple band pattern was detected (which seems to be artefactual), the enzyme appears to be homogeneous. Gel filtration and sucrose density gradient centrifugation in the presence of Triton X-100 give only one symmetrical peak, with a calculated molecular weight of 328 000. Since polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) and mercaptoethanol gives only one band with a molecular weight of 74 500, a tetrameric structure can be postulated for the membrane-bound acetylcholinesterase from rat brain.

Isolation and Immunochemical Study of a Soluble Cerebellar Lectin Delineating Its Structure and Function

Abstract: A procedure of sequential extractions of cerebellar tissue was set up, which allowed specific solubilization of endogenous lectins by mannose. Two cerebellar soluble lectins, CSL1 (Mr= 33,000) and CSL2 (Mr= 31,500), were isolated. They appeared to consist of structurally and immunologically related polypeptides chains. By immunoaffinity, another minor component (Mr= 45,000) was isolated. Immunological studies suggested that the minor component is the precursor of the two other, i.e., CSL1 and CSL2, subunits. CSL1 (mainly lysosomal) possesses an additional peptide compared with CSL2 (mainly cytoplasmic and extracellular), which seems to be implicated in the signal for secretion and release.

Isolation and immunohistochemical localization of a lectin-like molecule from the rat cerebellum

A lectin with a mannose specificity was isolated from the cerebellum of young rats. The method of purification was based on the observation that during homogenization of the tissue, the lectin binds to a class of mannose-rich glycoproteins highly insoluble in Triton X-100. Sequential extractions in saline buffer devoid of, then containing, 0.5% Triton X-100 allowed the elimination of a great part of other proteins. Using the same buffer containing 0.5 M mannose, a specific class of protein can be solubilized. This fraction was enriched by affinity adsorption on insolubilized mannose-rich glycoproteins followed by specific detachment with mannose. One of the protein subunits, of molecular weight (MW) 130,000, was isolated by preparative gel electrophoresis. Upon re-electrophoresis, this compound gives two bands of MW 65,000 and 130,000, which appear to be a monomer and a dimer of a molecule called R1. Antibodies were raised against R1 which react with the monomer and the dimer and not against other proteins of the rat cerebellum. The immunohistochemical localization of this lectin was performed in cerebella of 20-day-old rats. The antigen is concentrated in endothelial cells and in large and intermediate size neurons (Purkinje, Golgi, basket and deep nuclei neurons). Granule cell bodies are lightly stained and no label at all was found in glial cells. At the level of electron microscopy, the antigen was found to be very concentrated in multivesicular bodies and lysosomes of large neurons, on parts of the endoplasmic reticulum, on some mitochondrial outer membranes and on the plasma membrane of the dendrites. The possible role of this lectin in cerebella of young rats is discussed in relation to its interaction with a specific class of mannose-rich glycoproteins.

Immunohistochemical localization of a lectin-like molecule, R1, during the postnatal development of the rat cerebellum

The immunohistochemical localization of an endogenous lectin R1 isolated from the rat cerebellum was studied during its postnatal development. The lectin is present in the cerebellum from birth to adulthood, essentially in lysosomes, multivesicular bodies, and parts of the endoplasmic reticulum, principally of large and intermediate size neurons. During the period of massive synaptogenesis in the molecular layer, there is a sprouting of R1 in some distal dendrites of Purkinje cells. The lectin appears to be particularly concentrated on their plasma membranes, in coated pits, in coated vesicles, multivesicular bodies and lysosomes. At the same period, in cerebella of rats treated with chloroquine (an inhibitor of lysosomal function), both the lectin and mannose-rich glycoproteins of newly formed parallel fibres (able to bind specifically this lectin) are found in the same non-functional lysosomes of Purkinje cells. It is thus suggested that both this lectin (with a high-affinity for the glycans of the mannose-rich glycoproteins of the membrane of the newly formed parallel fibres) and these glycoproteins could be the recognition molecules allowing a specific contact between parallel fibres and Purkinje cells at the period of synaptogenesis.

Gangliosides from rat cerebellum: Demonstration of considerable heterogeneity using a new solvent for thin layer chromatography

Using a new solvent (methyl acetate/n-propanol/chloroform/methanol/0.25% aqueous KCl, 25∶20∶20∶20∶17, v/v) and high performance silica gel thin layer chromatographic plates, all common gangliosides found in brain can be easily separated with 1 hr. This system is reproducible and “tailing” is negligible compared with previous solvents. When such a system is applied to separate the gangliosides found during the development of the rat cerebellum, a considerable heterogeneity is observed. Data are presented (using rechromatography experiments, fractionation on DEAE-Sephadex, treatment with neuraminidase or alkaline medium and carbohydrate analysis) suggesting that the complex profiles obtained with this chromatographic system are not due to chromatographic artifacts but result from the high resolving power of the system. After separation by ion-exchange chromatography, 28 gangliosides can be detected.

Con A-binding glycoproteins in the developing cerebellum of control and hypothyroid rats

Concanavalin A (Con A)-binding glycoproteins were studied during the postnatal development of the cerebellum of control and hypothyroid rats. Only 4 glycoprotein bands have a transient behavior in control animals. They progressively increase until the 13th day and markedly decline between the 15th and the 18th postnatal day. In the cerebellum of hypothyroid rats, the level of these compounds is greatly reduced and the previous decrease observed in controls is not found again. This defect of Con A-binding glycoproteins mainly localized on the plasma membrane of parallel fibers might be related to the reduced synaptogenesis observed in the molecular layer of hypothyroid rats between parallel fibers and Purkinje cell dendritic spines.

Immunohistochemical Localization of α-Mannosidase During Postnatal Development of the Rat Cerebellum

Abstract: The localization of α‐d‐mannosidase in the rat cerebellum was studied by using indirect immunohistochemistry at both optical and electron microscopic levels. In the adult the enzyme is particularly concentrated in the dendrites and cell bodies of Purkinje cells, basket cells, and Golgi neurons in the cerebellar cortex and in the cytoplasm and dendrites of deep nuclei neurons. The cytoplasm of granule cells is poorly stained, whereas parallel fibers, white matter, Bergman fibers, and Golgi epitheloid cell perikarya show virtually no staining. Electron microscopy suggests that most of the staining is found in the cytosol, although some staining is found in the postsynaptic densities of the synapses between parallel fibers and Purkinje dendrites. The pattern of staining was followed throughout the postnatal development of the rat cerebellum. At birth an intense and diffuse staining is found in all cells except those of the external germinative layer. At the 6th postnatal day, Purkinje cell bodies and apical cones are strongly labeled. From the 13th day on, the pattern is very similar to that found in the adult. However, at the 18th postnatal day (when compared with the other structures), the staining of Purkinje cell dendrites seems to be higher than at all other ages. These data are correlated with biochemical studies and discussed in relation to the possible role of this enzyme during the postnatal development of the rat cerebellum.

Evidence for the Presence of Lectins with Mannose Specificity in the Rat Cerebellum

Abstract: Two different methods were set up to detect the possible presence of lectin‐like molecules with a specificity for mannose‐rich glycans in the rat cerebellum. The first, affinity histochemistry, involved the isolation of a particular class of glycoproteins from the cerebella of 11‐day‐old rats followed by the formation of covalent complexes with horseradish peroxidase and then incubation with cerebellar slices. The second used in vitro interactions between [3H]leucine‐labeled proteins, kept in solution, with insolubilized [14C]glucosamine‐labeled glyco proteins. The results of both methods are compatible with the presence of lectin‐like activities inhibited by high mannose concentrations, but not other sugars. However, the binding sites preferred by these molecules seem to be more than a single mannose residue.

Antibodies against the membrane-bound acetylcholinesterase from adult rat brain

As in most other tissues [2-6,10,12,14,17,18, 21-23,2&27,30,32,36,37];review [35],mammalian brain acetylcholinesterase exists in several molecular forms [1,7,15,16,19,20,24,29,31,33,34,38-40]. Depending on the method of extraction of the enzyme between 2 and 4 forms have been detected in the rat brain [ 1 ,15,24,29,3 1,34,38,40] either by gel filtration or sucrose gradient centrifugation or polyacrylamide gel electrophoresis. It is generally assumed that the various forms are different states of aggregation of a single monomer (review [39]) but no clear proof has been reported of this hypothesis for the rat brain enzyme. In [29], we compared by gel filtration and sucrose gradient centrifugation the membrane-bound molecular species of the rat brain AChE with the various ‘soluble’ species and reported that the structural relationships as well between the membrane-bound enzyme and the 3 soluble species as between the 3 soluble species are not evident. This paper describes the preparation of a monospecific antibody against the purified membrane-bound enzyme [28] which allows further progress in this research and permits the study of possible cross immunoreactivity between the soluble and the membrane-bound species of rat brain AChE.