Studies on the submicrosomal fractions of bovine oligodendroglia: Lipid composition and glycolipid biosynthesis

Oligodendroglia were isolated from bovine brain, and a crude, microsomal fraction obtained from cell homogenates was subfractionated into myelin (MP), plasma membranes (PM), Golgi (GF), smooth (SER) and rough (RER) endoplasmic membranes using discontinuous-sucrose gradient centrifugation. The submicrosomal fractions were characterized by ultrastructural examination and analysis of the specific organelle markers. The myelin and plasma membrane rich fractions contained characteristically the highest amounts of the lipid with lower mole percentages of total phospholipids and phosphatidylcholine, and higher concentrations of phosphatidylethanolamine (+plasmalogens), cholesterol and galactolipids. Considerable amounts of the typical myelin galactolipids (galacto-cerebrosides, sulfatides and monogalactosyl diglycerides) were also found in the Golgi fraction (GF). The GF fraction had the greatest enrichment of glycolipid-forming galactosyltransferases, and the distribution of these enzymes correlated well with that of the Golgi marker enzymes. The results give evidence that intracellular Golgi apparatus of oligodendroglia is rich in the myelin-specific lipids, and suggest its involvement in the synthesis and processing of myelin lipids.     

From cilia and flagella to intracellular motility and back again: a review of a few aspects of microtubule-based motility

Ciliary or flagellar movement is the model of microtubule-dependent motility, the best studied at the molecular level. It is based on the relative sliding of outer doublets of microtubules that are linked at their proximal end to the basal structure and interconnected by associated proteins, among which dynein ATPase is at the origin of the movement. It is regulated from inside and outside media by various diffusible factors such as Ca2+, cyclic adenosine monophosphate (cAMP), polypeptides and so on (see other conferences presented during this meeting). Other motility processes are based on microtubules: vesicle and organelle transport through the cytoplasm (axonal flow in neurons, pigment granule movements in fish chromatophores, movements of particles along heliozoan axopods, etc.) could be mediated by microtubule motors such as kinesin or MAP 1C. Kinesin and MAP 1C, like dynein, are proteins that bind to microtubules and show an ATPase activity associated with force production. They differ from each other by their structure, and biochemical and pharmacological properties. The movements of chromosomes during mitosis and meiosis have long been studied, but are still poorly understood at the molecular level; this topic will be discussed in the light of recent data. Other constituents of the cytoskeleton are certainly involved in cellular motility: actin microfilaments and their motor myosin, intermediate filaments, non-actin filaments, all organized around the Microtubule Organizing Center (MTOC). As more information becomes available, it seems increasingly obvious that these various networks are closely interconnected and that each component probably modulates, resists, or favors properties of its partners, contributing to cellular and intracellular motility.     

pH Dependence of Histidine Affinity for Blood-Brain Barrier Carrier Transport Systems for Neutral and Cationic Amino Acids

The effects of pH (3.5-7.5) on the brain uptake of histidine by the blood-brain barrier (BBB) carriers for neutral and cationic amino acids were tested, in competition with unlabeled histidine, arginine, or phenylalanine, with the single-pass carotid injection technique. Cationic amino acid ( [14C]arginine) uptake was increasingly inhibited by unlabeled histidine as the pH of the injection solution decreased. In contrast, the inhibitory effect of unlabeled histidine on neutral amino acid ( [14C]phenylalanine) uptake decreased with decreasing pH. Brain uptake indices with varying histidine concentrations indicated that the neutral form of histidine inhibited phenylalanine uptake whereas the cationic form competed with arginine uptake. Since phenylalanine decreased [14C]histidine uptake at all pH values whereas arginine did not, it was concluded that the cationic form of histidine had an affinity for the cationic carrier, but was not transported by it. We propose that the saturable entry of histidine into brain is, under normal physiological circumstances, mediated solely by the carrier for neutral amino acids.     

Separation of insect hemolymph proteins by cascade-mode multi-affinity chromatography.

The hemolymph of the adult female Manduca sexta was fractionated by cascade-mode multi-affinity chromatography (CASMAC) on a main-line tandem column chain containing Zn(2+)-TED, T-gel, Ni(2+)-DPA, and phenylsepharose and a side-line column containing Zn(2+)-DPA. The technique separated some of the previously described major hemolymph proteins, and yielded a number of fractions with simple composition. Some of these fractions contained only less abundant proteins of Manduca hemolymph. Thus, it appears that CASMAC would be a very useful fractionation technique for purification and characterization of the minor proteins of insect hemolymph.     

Reconstitution of the tonoplast amino-acid carrier into liposomes

The tonoplast amino-acid transporter of barley (Hordeum vulgare L.) mesophyll cells was functionally reconstituted by incorporating solubilized tonoplast membranes, vacuoplast membranes or tonoplast-enriched microsomal vesicles into phosphatidylcholine liposomes. (i) Time-, concentration- and ATP-dependence of amino-acid uptake were similar to results with isolated vacuoles. Although the orientation of incorporation could not be controlled, the results indicate that the transporter functions as a uniport system which allows regulated equilibration by diffusion between the cytosolic and vacuolar amino-acid pools. (ii) The ATP-modulated amino-acid carrier was also successfully reconstituted from barley epidermal protoplasts and Valerianella or Tulipa vacuoplasts, indicating its general occurrence. (iii) Fractionation of solubilized tonoplasts by size-exclusion chromatography followed by reconstitution of the fractions for glutamine transport gave two activity peaks: the first eluted in the region of high-molecular-mass vesicles and the second at a size of 300 kDa for the Triton-protein micelle.Abbreviation SDS-PAGE sodium dodecyl sulfate-polyacryl-amide gel electrophoresis This work was part of our research efforts within the Sonderforschungsbereich 176 of the University. We gratefully acknowledge experimental support by Marion Betz and valuable discussions with Professors U. Heber and U.-I. Flügge and Dr. Armin Gross (University of Würzburg) and Dr. E. Martinoia (ETH, Zürich, Switzerland).     

Changes in haemolymph ion concentrations of Astacus astacus L. and Pacifastacus leniusculus (Dana) after exposure to low pH and aluminium

During exposure to soft water, acidified to pH 4.0, the haemolymph concentrations of Na⁺, K⁺, and Cl^– decreased whereas the Ca²⁺ concentration fluctuated in Astacus astacus. The haemocyte content of K⁺ decreased from 9% to 2% of the total haemolymph K⁺ content after exposure to pH 3.7 for 3 days. Within 14 days, 250 µg Al³⁺ l^–1, as Al₂(SO₄)₃ at pH 5.0, reduced the haemolymph Na⁺ content in Astacus astacus and Pacifastacus leniusculus, however, the effects were less pronounced than earlier reported for fish. Disturbed ion regulation, mainly depending on low pH, is thought to contribute to the absence of these species in acid waters.     

Reduced pyridine nucleotides in Pseudomonas carboxydovorans are formed by reverse electron transfer linked to proton motive force

In cell suspensions of Pseudomonas carboxydovorans pulsed with lithotrophic substrates (CO or H₂) in the presence of oxygen, formation of reduced pyridine nucleotides and of ATP could be demonstrated using the bioluminescent assay. Experiments employing base-acid transition, an uncoupler and inhibitors of ATPase or electron transport enabled us to propose a model for the formation of NAD(P)H in chemolithotrophically growing P. carboxydovorans.The protonophor FCCP (carbonly-p-trifluormethoxyphenylhydrazon) inhibited both, formation of NAD(P)H and of ATP. In the absence of oxygen, a chemical potential imposed by base-acid transition resulted in the formation of NAD(P)H and ATP when electrogenic substrates (CO or H₂) were present. This suggests proton motive force-driven NAD(P)H formation. The proton motive force was generated by oxidation of substrate, and not by ATP hydrolysis, as obvious from NAD(P)H formation during inhibition of ATP synthesis by oligomycin and N,N-dicyclohexylcarbodiimide.That the CO-born electrons are transferred via the ubiquinone 10-cytochrome b region to NADH dehydrogenase functioning in the reverse direction, was indicated by inhibition of NAD(P)H formation by HQNO (2-n-heptyl-4-hydroxyquinoline-N-oxide) and rotenone, and by resistance to antimycin A.We conclude that in P. carboxydovorans, growing with CO or H₂, electrons and a proton motive force, generated by respiration, are required to drive an reverse electron transfer for the formation of reduced pyridine nucleotides.Abbreviations CODH carbon monoxide dehydrogenase - DCCD N,N-dicyclohexylcarbodiimide - FCCP carbonyl-p-trifluormethoxyphenylhydrazon - HQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - pmf proton motive force     

Modulation of glucose transport in human red blood cells by ATP

Depletion of energy stores of human red cells decreases the maximum transport capacity, $\text{J}{}_{\mathrm{<mtext>m</mtext>}}$, for glucose transport to a value one-third or less of that found in red cells from freshly drawn blood. There is no change in $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. Hemolysis and resealing of red cells with ATP or ADP reverses the decrease in $\text{J}{}_{\mathrm{<mtext>m</mtext>}}$. The maximum effect occurs at concentrations of ATP in the normal range for red cells, however, there is little effect from ADP concentrations in its normal range in freshly drawn red cells. Hemolysis and resealing with ATP gives an increase in $\text{J}{}_{\mathrm{<mtext>m</mtext>}}$ and an increase in differential labeling by photolytic labeling with tritiated cytochalasin B. Most of the activation is lost after a second hemolysis-reseal without ATP but about 25% of the activation remains.     

Junctions in the central nervous system of the cat

Summary Fixed and unfixed astrocytic membranes from the CNS of the cat were studied by means of the freeze-etching technique.A variable number of gap junctions was detected in astrocytic membranes. They are characterized by the well known hexagonal composition of their subunits. Besides this type of highly ordered membrane-bound particles, a second one was found. It is composed of four single particles (diameter 5 nm) which form an orthogonal subunit with a side length of about 10 nm. These membrane-associated orthogonal particle complexes (MOPC) could be observed in different stages of aggregation and expansion. They reveal an accumulation in membranes of the marginal glia layers and in the perivascular astrocytic end-feet. Unfixed, glycerol treated membranes, however, do not show these structures. After glycerol treatment of the unfixed membranes by immersion, the MOPC disintegrate to single particles which form clusters of various extension. The clustering phenomenon is dependent on the length of the time of exposure to glycerol. Shortening of the glycerol treatment by intravasal perfusion of the cryoprotectant agent causes an decrease of the clusters. Fragments and transient forms of the MOPC become visible. By variation of different physico-chemical parameters of the washing solution a similar effect on the MOPC was not achieved. The discussion deals with probable functional aspects of the MOPC. They are considered to act as membrane-bound functional multienzyme complexes which a) might play a role in mediating transmembrane passage of metabolites, or b) are essential for CSF control mechanisms, or c) have a functional relation to the nexus.Presented in part at the IIIth European Congress of Anatomists, Manchester, September 1973.Supported by Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 114(Bionach).     

Two substrate sites in the renal Na+-d-glucose cotransporter studied by model analysis of phlorizin binding and-d-glucose transport measurements

Summary Time courses of phlorizin binding to the outside of membrane vesicles from porcine renal outer cortex and outer medulla were measured and the obtained families of binding curves were fitted to different binding models. To fit the experimental data a model with two binding sites was required. Optimal fits were obtained if a ratio of low and high affinity phlorizin binding sites of 1:1 was assumed. Na⁺ increased the affinity of both binding sites. By an inside-negative membrane potential the affinity of the high affinity binding site (measured in the presence of 3 mM Na⁺) and of the low affinity binding site (measured in the presence of 3 or 90 mM Na⁺) was increased. Optimal fits were obtained when the rate constants of dissociation were not changed by the membrane potential. In the presence of 90 mM Na⁺ on both membrane sides and with a clamped membrane potential,K _D values of 0.4 and 7.9 M were calculated for the low and high affinity phlorizin binding sites which were observed in outer cortex and in outer medulla. Apparent low and high affinity transport sites were detected by measuring the substrate dependence ofd-glucose uptake in membrane vesicles from outer cortex and outer medulla which is stimulated by an initial gradient of 90 mM Na⁺(out>in). Low and high affinity transport could be fitted with identicalK _m values in outer cortex and outer medulla. An inside-negative membrane potential decreased the apparentK _m ofhigh affinity transport whereas the apparentK _m of low affinity transport was not changed. The data show that in outer cortex and outer medulla of pighigh and low affinity Na⁺-d-glucose cotransporters are present which containlow and high affinity phlorizin binding sites, respectively. It has to be elucidated from future experiments whether equal amounts of low and high affinity transporters are expressed in both kidney regions or whether the low and high affinity transporter are parts of the same glucose transport moleculc.