Selective release of inner core proteins from intestinal microvillus membrane by lithium diiodosalicylate

Abstract

Lithium diiodosalicylate (LIS) was used to selectively solubilize proteins from purified intestinal brush border membrane vesicles. Incubation of the vesicles with increasing concentrations of LIS resulted in the progressive release of proteins with total disruption of the membranes being obtained at 200 mM. Maximum selectivity was observed at 20-30 mM LIS which preferentially released actin and other non-glycosylated proteins while all the glycoproteins remained associated with the membrane. Electron micrographs showed that, after LIS treatment, brush border vesicles are partially disrupted and have lost their inner core of microfilaments. Sucrase, trehalase, leucylnaphthylamide hydrolase, gamma-glutamyl transpeptidase and alkaline phosphatase all retained more than 70% of their activities and remained associated with the membrane fraction after LIS solubilization (30 mM). The results indicate that lithium diiodosalicylate treatment provides an efficient method for the separation of cytoskeletal proteins from intrinsic membrane glycoproteins and should be very useful for the purification of microvilli proteins and for the study of membrane-protein interactions.

Histones are exosome membrane proteins regulated by cell stress

“…NaCl 58 and Li-diiodosalicylate 59 were used for extraction of proteins from purified EVs (10 µg in 20 mM Tris-HCl, pH 7.5) using NaCl (250 mM-750 mM) or Li-diiodosalicylate (5-50 mM). The final volume was adjusted to 25 µl using Tris-HCl.…”

“…A high concentration of NaCl is commonly used to disrupt electrostatic interactions whilst still being a non-denaturing salt 58 . The chaotropic salt lithium 3,5-diiodosalicylate can solubilize membrane associated proteins at lower concentrations, while disrupting the membrane completely at higher concentrations 59 . In addition, we used the detergent TX-100 (0.1%) to lyse EVs 74 , or sonication to disrupt them.…”

Propriedades e secreção das enzimas digestivas e caracterização das membranas microvilares intestinais de insetos

“…Although this rnay result from arninopeptidase inactivation, other data suggest that behavior of insect microvilli in relation to those salts differs from mammalian microvilli. Thus, only 20 ± 2% (mean and SEM, n=3) of the M. domestica microvilli protein was solubilized in the presence of diiodosalicylate in contrast to 40% from mammalian microvilli (Riendeau et al, 1986), and only 35 ± 9% (mean and SEM, n=3, calculated from the total activity recovered) of the rnarker enzyrne rernains in the M. domestica microvillar membranes The enrichment of marker enzymes in microvillar membranes over the ini tial microvilli, as noted above, are similar among rnammals (Hopfer et al, 1983;Riendeau et al, 1986) and insects (Tables 2, 3 and 4). This suggests that our microvillar membranes are as pure as the best ones from mammals and that the ratio of 20 microvillar protein to core protein are similar among mammals and different insects.…”