Plasma membrane (PM) sphingolipid pattern and content are the result of a complex network of metabolic pathways, including those potentially involving the activity of PM sphingolipid hydrolases and sphingolipid synthases (1, 2). In human fibroblasts it has been observed a crosstalk between the expression and the activity of membrane associated glycohydrolases (3), furthermore, qualitative and quantitative changes in glycosphingolipids, together with changes in the expression of the corresponding glycosyltransferases, have been reported along neuronal differentiation and aging (2, 4-8). Using human fibroblasts we set up an HTA (high throughput assay) method able to measure the activity of beta-galactosidase, CBE sensitive beta-glucosidase, GBA2 beta-glucosidase, SMase and beta-hexosaminidases, enzymes working directly at the plasma membrane level using MUB derivates as substrate. Afterwards we applied this assays in order to study these PM associated activities in rat cerebellar granule cells along differentiation and aging in culture. We started to characterize the kinetic parameters of these enzymes working directly on the external leaflet of the PM in living cells: first of all, we performed an assay using cell cultured medium at different pH. Our data showed higher activity of CBE sensitive -glucosidase at pH 6.2, GBA2 beta-glucosidase at pH 5.7, beta-hexosaminidase at pH 5.10 while beta-galactosidase, SMase and beta-hexosaminidase A at pH 5.45. We could distinguish between CBE sensitive beta-glucosidase and GBA2 beta-glucosidase using specific inhibitors that are CBE and AMP-DNM respectively, therefore we determined Km and Vmax of CBE sensitive beta-glucosidase and GBA2. Thought the pH conditions are not those typically associated with the PM, at least 7 different families of transporters/exchangers are present at this level and are capable of varying the pH locally. In order to verify whether the varying of the membrane potential acting on these pumps can influence also the activity of these enzymes, we carried out preliminary experiments using EIPA, an inhibitor of NHEs (Na+/H+) exchangers. What we achieve is that exists a correlation between the action of the pump and the enzymes. So, all our data confirm the presence of this PM associated activities. This could play an important role in the cell social life and in particular our data show an involvement of sphingolipid hydrolases during the neuronal differentiation.
|Titolo:||PLASMA MEMBRANE ASSOCIATED GLYCOHYDROLASES: KINETICS PARAMETER CHARACTERIZATION AND BIOLOGICAL FUNCTION|
|Supervisori e coordinatori interni:||BONOMI, FRANCESCO|
|Data di pubblicazione:||9-dic-2010|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
|Citazione:||PLASMA MEMBRANE ASSOCIATED GLYCOHYDROLASES: KINETICS PARAMETER CHARACTERIZATION AND BIOLOGICAL FUNCTION / P. Lanteri ; docente guida: Vanna Luigia Chigorno ; coordinatore: Franco Bonomi. - Milano : Università degli studi di Milano. Universita' degli Studi di Milano, 2010 Dec 09. ((23. ciclo, Anno Accademico 2010.|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.13130/lanteri-patrizia_phd2010-12-09|
|Appare nelle tipologie:||Tesi di dottorato|