Interaction of rat liver lysosomal membranes with actin

Membranes were prepared from lysosomes purified 80-fold by centrifugation in a discontinuous metrizamide gradient. When salt- washed membranes were combined with rabbit muscle actin, an increase in viscosity could be measured using a falling ball viscometer. The lysosomal membrane-actin interaction was actin- and membrane- concentration dependent and appeared to be optimal under presumed physiological conditions (2 mM MgCl2, 1 mM MgATP, neutral pH, and free calcium concentration less than 10(-8) M). The actin cross-linking activity of the membrane was optimal at pH 6.4. The interaction was maximal between 10(-7) and 10(-9) M free calcium ions and inhibited by approximately 50% at concentrations of calcium greater than 0.5 x 10(- 7) M. The actin-lysosomal membrane interaction was destroyed if the membranes were pretreated with Pronase, or if the membranes were purified in the absence of protease inhibitors. The interaction was not destroyed if membranes were washed with high salt or extracted with KCl and urea. In addition, a sedimentation assay for the actin-lysosomal membrane interaction was also performed to corroborate the viscometry data. The results suggest the existence of an integral lysosomal membrane actin-binding protein.     

Model of epithelial morphogenesis based on elastic forces and cell contact polarization

It is well known that in embryonic tissues at the key stages of morphogenesis there arise stable, stage--specific tension fields. These fields occur due to particular pattern of morphologically polarized cells. Some basic properties have been understood previously. 1. Morphologically polarized and isotropic shapes correspond to the alternative stable states of embryonic cells. 2. Polarization can be transmitted between the adjacent cells via intercellular contacts. 3. The tension fields at particular stage of development determine the pattern of morphogenetic movements. In this paper the physical model is suggested which interprets the selforganization of tension fields in embryonic tissues. The polarization in some region of tissue is assumed to generate the elastic tension in the surrounding cells thus restricting the propagation of cell polarization. It is shown that the properties underlined are sufficient to provide spontaneous subdivision of the cellular layer into the domains of polarized and unpolarized stretched cells. The proportion of polarized and unpolarized areas is determined and size--invariant.     

Kinetics of the Reconstituted Tricarboxylate Carrier from Eel Liver Mitochondria

The tricarboxylate carrier from eel liver mitochondria was purified by chromatography on hydroxyapatite and Matrix Gel Blue B and reconstituted into liposomes by removal of the detergent with Amberlite. Optimal transport activity was obtained by using a phospholipid concentration of 11.5 mg/ml, a Triton X-114/phospholipid ratio of 0.9, and ten passages through the same Amberlite column. The activity of the carrier was influenced by the phospholipid composition of the liposomes, being increased by cardiolipin and phosphatidylethanolamine and decreased by phosphatidylinositol. The reconstituted tricarboxylate carrier catalyzed a first-order reaction of citrate/citrate or citrate/malate exchange. The maximum transport rate of external [¹⁴C]citrate was 9.0 mmol/min per g of tricarboxylate carrier protein at 25°C and this value was virtually independent of the type of substrate present in the external or internal space of the liposomes. The half-saturation constant (K _m) was 62 M for citrate and 541 M for malate. The activation energy of the citrate/citrate exchange reaction was 74 kJ/mol from 5 to 19°C and 31 kJ/mol from 19 to 35°C. The rate of the exchange had an external pH optimum of 8.     

Covariance of tricarboxylate carrier activity and lipogenesis in liver of polyunsaturated fatty acid (n-6) fed rats.

The mitochondrial tricarboxylate (citrate) carrier plays an important role in hepatic intermediary metabolism because, among other functions, it supplies the cytosol with acetyl units for fatty-acid synthesis. In this study, the effect of polyunsaturated fatty acids (PUFA, n-6) on the function of this mitochondrial transporter and on lipogenic enzyme activities was investigated by feeding rats for 4 weeks with a 15%-fat diet composed of high linoleic safflower oil. Citrate transport was strongly reduced in liver mitochondria isolated from PUFA-treated rats. A reduced transport activity was also observed when solubilized mitochondrial citrate carrier from PUFA-treated rats was reconstituted into liposomes. In the same animals, a decrease of cytosolic lipogenic enzyme activities was observed. These results indicate a coordinated modulation of citrate carrier and of lipogenic enzyme activities by PUFA feeding. Kinetic analysis of the carrier activity showed that only V(max) decreased, whereas K(m) was almost virtually unaffected. The PUFA-mediated effect is most likely due to the reduced mRNA level and lower content of the citrate carrier protein observed in the safflower oil-fed rats.     

Transmembrane topology,genes, and biogenesis of the mitochondrial phosphate and oxoglutarate carriers

Phosphate and oxoglutarate carriers transport phosphate and oxoglutarate across the inner membranes of mitochondria in exchange for OH^– and malate, respectively. Both carriers belong to the mitochondrial carrier protein family, characterized by a tripartite structure made up of related sequences about 100 amino acids in length. The results obtained on the topology of the phosphate and oxoglutarate carriers are consistent with the six -helix model proposed by Saraste and Walker. In both carriers the N- and C-terminal regions are exposed toward the cytosol. In addition, the oxoglutarate carrier has been shown to be a dimer by means of cross-linking studies. The bovine and human genes coding for the oxoglutarate carrier are split into eight and six exons, respectively, and five introns are found in the same position in both genes. The bovine and human phosphate carrier genes have the same organization with nine exons separated by eight introns at exactly the same positions. The phosphate carrier of mammalian mitochondria is synthesized with a cleavable presequence, in contrast to the oxoglutarate carrier and the other members of the mitochondrial carrier family. The precursor of the phosphate carrier is efficiently imported, proteolytically processed, and correctly assembled in isolated mitochondria. The presequence-deficient phosphate carrier is imported with an efficiency of about 50% as compared with the precursor of the phosphate carrier and is correctly assembled, demonstrating that the mature portion of the phosphate carrier contains sufficient information for import and assembly into mitochondria.     

The aminoglycoside G418 hinders de novo prion infection in cultured cells

The study of prions and the discovery of candidate therapeutics for prion disease have been facilitated by the ability of prions to replicate in cultured cells. Paradigms in which prion proteins from different species are expressed in cells with low or no expression of endogenous prion protein (PrP) have expanded the range of prion strains that can be propagated. In these systems, cells stably expressing a PrP of interest are typically generated via coexpression of a selectable marker and treatment with an antibiotic. Here, we report the unexpected discovery that the aminoglycoside G418 (Geneticin) interferes with the ability of stably transfected cultured cells to become infected with prions. In G418-resistant lines of N2a or CAD5 cells, the presence of G418 reduced levels of protease-resistant PrP following challenge with the RML or 22L strains of mouse prions. G418 also interfered with the infection of cells expressing hamster PrP with the 263K strain of hamster prions. Interestingly, G418 had minimal to no effect on protease-resistant PrP levels in cells with established prion infection, arguing that G418 selectively interferes with de novo prion infection. As G418 treatment had no discernible effect on cellular PrP levels or its localization, this suggests that G418 may specifically target prion assemblies or processes involved in the earliest stages of prion infection.