Effect of factors of favism on the protein and lipid components of rat erythrocyte membrane

Erythrocytes prepared from riboflavin- and tocopherol-deficient (RT^?) and from control rats were used to investigate the mechanism of oxidative hemolysis by the factors of favism. RT^? erythrocytes have a defense system against the oxidative stress which is blocked either where regeneration of GSH occurs or the scavenging of the radicals from the membrane is prevented. The oxidative factors used were isouramil, divicine and diamide. When RT^? erythrocytes were treated with isouramil, GSH decreased to undetectable levels and was not regenerated. Complete hemolysis occurred, but no oxidation of SH groups of membrane proteins or formation of spectrin polymers was detected. A similar effect was observed with diamide. However, SH groups of membrane proteins were completely oxidized and spectrin polymers were formed. Extensive lipid peroxidation was also detected together with a 30% fall in the arachidonic acid level. Control erythrocytes treated with either isouramil or diamide were not hemolyzed. When treated with isouramil, after a fall in the first few minutes, the GSH level was completely regenerated after 20 min. Incubation with diamide caused extensive oxidation of SH groups of membrane proteins and formation of spectrin polymers. No lipid peroxidation was detected after treatment with isouramil, but the same decrease of arachidonic acid occurred as in RT^? erythrocytes. These results support the hypothesis that oxidative hemolysis by the factors of favism is caused by uncontrolled peroxidation of membrane lipids.     

Comparison of protein components of the plasma membrane fraction from rat liver and Morris hepatomas 5123D and 7777.

Isolated plasma membrane fractions from rat liver and Morris hepatoma 5123D and 7777 were labelled with radioiodine 125I by a chemical or enzymatic procedure and then were solubilized in 2 per cent solution of sodium dodecyl sulphate containing 1 per cent 2-mercaptoethanol. Solubilized proteins were separated into 20--22 zones staining with Coomassie Brilliant Blue R-250 after disc gel electrophoresis (7.5 per cent polyacrylamide gel). The high similarity of electrophoretic patterns of polypeptide components of all three preparations of cellular membranes was found in distinction to apparent differnces in the amount and disposition of substances stained with Schiff's reagent. Some tentative conclusions were drawn on the disposition of proteins within membrane structure studied by the method of labelling by chemical and enzymatic procedures (distinguishing between extrinsic and integral proteins).     

The presence of 17K Mr protein,a major specific substrate for kinase C,found in the Triton-insoluble fraction of synaptosome prepared from rat brain

Cytoskeletal preparation obtained from synaptosome fractions of rat cerebrum contained the activity of kinase C, which phosphorylated 17K Mr protein endogenous to the preparation. The kinase C activity associated with the synaptosome cytoskeletons is greater in the cerebellum and hippocampus than in the cerebrum. The enhancement rates of phosphorylation of the 17K Mr protein were 293%, 544%, and 526% in the Triton X-100-insoluble fractions of synaptosomes prepared from cerebral cortex, hippocampus, and cerebellum, respectively. The 17K Mr protein was distinct from myelin basic protein (MBP) for the following reasons: 1) The electrophoretic mobility of the protein was slightly smaller than that of major MBP of rat in the polyacrylamide gel of 10–20% linear gradient, and the protein was not contained in the purified rat myelin. 2) The isoelectric point of the protein was in neutral range, whereas that of MBP was in alkaline one. 3) The 17K Mr protein did not cross-react with anti-MBP antibody. The protein was shown to be a major substrate contained in the cytoskeletal preparation of synaptosome obtained from cerebrum except for contaminating MBP. Only serine residue of the 17K Mr protein was phosphorylated by the kinase C endogenous to the preparation. The results suggest strongly that the synaptic role of protein kinase C through phosphorylation of the 17K Mr protein.Abbreviations used EGTA ethyleneglycol-bis(-aminoethyl ether) - HEPES N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid - MBP myelin basic protein - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - SPM synaptic plasma membrane     

The spatial and temporal expression of delta-like protein 1 in the rat pituitary gland during development

An analysis of secreted proteins by the signal sequence trap method using a cDNA library of the rat pituitary anlage at embryonic days (E) 13.5 revealed the abundant expression of delta-like protein 1 (Dlk1) in the pituitary gland. Dlk1, an epidermal growth factor-like repeat protein in preadipocytes, functions in maintaining the preadipose state. Expression of Dlk1 mRNA in the pituitary at E13.5 and in the adult pituitary was confirmed by in situ hybridization. The expression pattern of Dlk1 during pituitary development was also studied by immunohistochemistry. Dlk1 protein first appeared in Rathke’s pouch and the infundibulum at E11.5; as development proceeded, expression became restricted to the pars distalis and pars tuberalis (PT). Dlk1 was expressed in most ACTH cells during the embryonic stages, but its expression was limited to only a few ACTH cells in the adult pituitary. It was also expressed in a small population of TSH, GTH, and PRL cells throughout development, whereas it was present in the cytoplasm of most GH cells at all developmental stages. Similarly, Dlk1 was localized in the cytoplasm of PT cells during development. These findings provide new insights into the mechanism of Dlk1 regarding its regulation of pituitary hormone-secreting cells during development.     

Identification and characterization of the pore-forming protein in the outer membrane of rat liver mitochondria

The proteins of the outer membrane from rat liver mitochondria have been subfractionated by means of density gradient centrifugation. The different polypeptides of the membrane were incorporated into asolectin vesicles and black lipid membranes. It was observed that a polypeptide of M_r 32 000 renders asolectin vesicles permeable to ADP and forms pores in bilayer membrane. These pores showed the same properties as the channels which are formed in the lipid membrane after addition of Triton X-100 solubilized complete outer membrane. The properties of the pore are as follows: (1) The formation of pores depends on the type of phospholipid used for the preparation of the black membranes. (2) The pore is inserted asymmetrically into the membrane. (3) The pore is voltage gated but does not switch off completely at higher voltages. The pore seems to show different conductance states decreasing conductance being observed at increasing voltage. The implications of these findings for the regulation of transport processes across the outer membrane are discussed.     

The activation of protein kinase A by cyclic AMP is influenced by oestradiol and progesterone in supernatants from the anterior pituitary but not from hypothalamus of the female rat

Summary— The activation of protein kinase A (PKA) by cAMP was estimated in supernatant fractions from the hypothalamus (Hyp) and anterior pituitary (AP) of the female rat during the oestrous cycle and in ovariectomized and ovariectomized, ovarian steroid hormone treated animals. In both structures, the largest activation of PKA was found in dioestrus-2, while the lowest one was in Hyp in dioestrus-1 and in AP in oestrus. Ovariectomy had no influence on cAMP-dependent activation of PKA from Hyp and AP. Treatment of ovariectomized rats with 17-β-oestradiol (E₂), progesterone (P) or both abolished the activation of PKA by cAMP from AP and had no effect on hypothalamic PKA. These results indicate that ovarian steroids act specifically on AP processes via cAMP dependent pathway and regulation of PKA activity.     

P400 protein is one of the major substrates for Ca2+/calmodulin-dependent protein kinase II in the postsynaptic density-enriched fraction isolated from rat cerebral cortex, hippocampus and cerebellum.

Concanavalin A-binding glycoprotein with 250 K M(r) found in the postsynaptic density (PSD)-enriched preparation (or synaptic cytoskeleton) from rat cerebellum was identified with P400 protein from the physicochemical properties and enrichment in the cerebellum. Proteins homologous to the cerebellar 250 K M(r) protein occurred in the PSD-enriched preparations from rat cerebral cortex and from hippocampus, although the contents in the preparations were very low. The 250 K M(r) proteins in the PSD-enriched preparations from cerebellum and from cerebrum were highly phosphorylated by Ca2+/calmodulin (CaM)-dependent protein kinase II. The protein of synaptic plasma membrane (SPM) and PSD-enriched fractions prepared from cerebral cortex were not phosphorylated by the cAMP-dependent protein kinase endogenous to the fractions, whereas the protein from cerebellum was done in SPM and PSD-enriched fractions. The facts suggest that P400 or P400-like protein is closely associated with Ca2+/CaM-dependent protein kinase II in the PSD-enriched preparations, especially in the preparation from cerebral cortex. Phosphorylation of the protein by Ca2+/CaM-dependent protein kinase II may play an important role in the postsynaptic function in both cerebellum and at least in some areas of cerebrum.     

The localization change of Ybr078w/Ecm33, a yeast GPI-associated protein,from the plasma membrane to the cell wall,affecting the cellular function

The YBR078W/ECM33 gene of Saccharomyces cerevisiae encodes a glycosylphosphatidylinositol (GPI)-attached protein and its disruptant strain exhibited a temperature-sensitive (ts) growth defect. A HA-tagged Ybr078w protein, which complemented the ts growth phenotype of the ybr078wdelta strain, was predominantly located on the plasma membrane by GPI anchoring. To examine the requirement of the GPI anchoring on the plasma membrane for the function, the omega-minus region of Ybr078w was replaced with those of Ydr534c/Fit1 and Ynl327w/Egt2, which are known as GPI-dependent cell wall proteins. The replacement induced the change in localization of the mutant proteins from the plasma membrane to the cell wall and the mutant proteins lost the function to complement the ts cell growth defect of the ybr078wdelta strain. In addition, a similar result was obtained in a mutant protein, where the authentic SKKSK sequence at the omega-5 to omega-1 site of Ybr078w was replaced with a synthetic ISSYS sequence. It is concluded that the GPI anchoring on the plasma membrane is required for the Ybr078w function.     

Particle density and protein composition of the peribacteroid membrane from soybean root nodules is affected by mutation in the microsymbiont Bradyrhizobium japonicum

Particle frequency of the peribacteroid membrane (PBM) from nodules of Glycine max (L.) Merr. cv. Maple Arrow infected with Bradyrhizobium japonicum 61-A-101 (wild-type strain) was determined by freeze-fracturing to be about 2200·m^-2 in the protoplasmic fracture face and 700·m^-2 in the exoplasmic fracture face. In membranes isolated from nodules infected with the mutant RH 31-Marburg of B. japonicum, the particle frequency was similar in both fracture faces with 1200–1300 particles·m^-2. Analysis of particlesize distribution on peribacteroid membranes showed a loss, especially of particle sizes larger than 11 nm, in the mutant-infected nodules. Two-dimensional gel electrophoresis (isoelectric focussing and sodium dodecyl sulfate-polyacrylamide) showed 27 different polypeptides in the PBM from nodules infected with the wild-type strain, four of which were absent from the PBM of nodules infected with the mutant RH 31-Marburg, which also exhibited one extra small-molecular-weight polypeptide. At least 14 of the 27 polypeptides in the PBM from the wild-type-infected nodule were glycoproteins. In three of these glycoproteins, post-translational modifications were either lacking or different when the membrane was derived from mutant-infected nodules.Abbreviations EF exoplasmatic fracture face - HRPO horse radish peroxidase - IEF Isoelectric focussing - PBM peribacteroid membrane - PF protoplasmatic fracture face - PNA peanut agglutinin - PSA Pisum sativum agglutinin - SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

The ESCRT‐III protein CeVPS‐32 is enriched in domains distinct from CeVPS‐27 and CeVPS‐23 at the endosomal membrane of epithelial cells

Background information. Within the endocytic pathway, the ESCRT (endosomal sorting complex required for transport) machinery is essential for the biogenesis of MVBs (multivesicular bodies). In yeast, ESCRTs are recruited at the endosomal membrane and are involved in cargo sorting into intralumenal vesicles of the MVBs. Results. In the present study, we characterize the ESCRT‐III protein CeVPS‐32 (Caenorhabditis elegans vacuolar protein sorting 32) and its interactions with CeVPS‐27, CeVPS‐23 and CeVPS‐4. In contrast with other CevpsE (class E vps) genes, depletion of Cevps‐32 is embryonic lethal with severe defects in the remodelling of epithelial cell shape during organogenesis. Furthermore, Cevps‐32 animals display an accumulation of enlarged early endosomes in epithelial cells and an accumulation of autophagosomes. The CeVPS‐32 protein is enriched in epithelial tissues and in residual bodies during spermatid maturation. We show that CeVPS‐32 and CeVPS‐27/Hrs (hepatocyte‐growth‐factor‐regulated tyrosine kinase substrate) are enriched in distinct subdomains at the endosomal membrane. CeVPS‐27‐positive subdomains are also enriched for the ESCRT‐I protein CeVPS‐23/TSG101 (tumour susceptibility gene 101). The formation of CeVPS‐27 subdomains is not affected by the depletion of CeVPS‐23, CeVPS‐32 or the ATPase CeVPS‐4. Conclusion. Our results suggest that the formation of membrane subdomains is essential for the maturation of endosomes.     

Interaction of membrane/lipid rafts with the cytoskeleton: Impact on signaling and function : Membrane/lipid rafts,mediators of cytoskeletal arrangement and cell signaling

The plasma membrane in eukaryotic cells contains microdomains that are enriched in certain glycosphingolipids, gangliosides, and sterols (such as cholesterol) to form membrane/lipid rafts (MLR). These regions exist as caveolae, morphologically observable flask-like invaginations, or as a less easily detectable planar form. MLR are scaffolds for many molecular entities, including signaling receptors and ion channels that communicate extracellular stimuli to the intracellular milieu. Much evidence indicates that this organization and/or the clustering of MLR into more active signaling platforms depends upon interactions with and dynamic rearrangement of the cytoskeleton. Several cytoskeletal components and binding partners, as well as enzymes that regulate the cytoskeleton, localize to MLR and help regulate lateral diffusion of membrane proteins and lipids in response to extracellular events (e.g., receptor activation, shear stress, electrical conductance, and nutrient demand). MLR regulate cellular polarity, adherence to the extracellular matrix, signaling events (including ones that affect growth and migration), and are sites of cellular entry of certain pathogens, toxins and nanoparticles. The dynamic interaction between MLR and the underlying cytoskeleton thus regulates many facets of the function of eukaryotic cells and their adaptation to changing environments. Here, we review general features of MLR and caveolae and their role in several aspects of cellular function, including polarity of endothelial and epithelial cells, cell migration, mechanotransduction, lymphocyte activation, neuronal growth and signaling, and a variety of disease settings. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.