Calmodulin Localization and its Effects on Endocytic and Phagocytic Membrane Trafficking in Paramecium multimicronucleatum

ABSTRACT. In ciliates, calmodulin (CaM), as in other cells, has multiple functions, such as activation of regulatory enzymes and modulating calcium‐dependent cellular processes. By immunogold localization, CaM is concentrated at multiple sites in Paramecium. It is seen scattered over the cytosol, but bound to its matrix, and is concentrated at the pores of the contractile vacuole complexes and with at least three microtubular arrays. It was localized peripheral to the nine‐doublet microtubules of the ciliary axonemes. The most striking localization was on the akinetic side only of the cytopharyngeal microtubular ribbons opposite the side where the discoidal vesicles, acidosomes and the 100‐nm carrier vesicles bind and move. CaM was also present at the periphery of the postoral microtubular bundles along which the early vacuole moves and was associated with the cytoproct microtubules that guide the spent digestive vacuoles to the cytoproct. It was not found on the membranes of, or in the interior of nuclei, mitochondria, phagosomes, and trichocysts, and was only sparsely scattered over the cytosolic sides of discoidal vesicles, acidosomes, lysosomes, and digestive vacuoles. Together the associations with specific microtubular arrays and the effects of trifluoperazine and calmidazolium indicate that CaM is involved (i) in vesicle transport to the cytopharynx area for vacuole formation and subsequent vacuole acidification, (ii) in early vacuole transport along the postoral fiber, and (iii) in transporting the spent vacuole to the cytoproct. Higher CaM concentrations subjacent to the cell's pellicle and close to the decorated tubules of the contractile vacuole complex may support a role for CaM in ion traffic.     

Properties of Brush Border Vesicles Isolated from Rat Kidney Cortex by Calcium Precipitation

Brush border membrane vesicles were isolated from rat kidney cortex by differential centrifugation in the presence of 10 mM calcium. Their properties were compared to brush border vesicles isolated by free-flow electrophoresis. By the calcium precipitation method membrane vesicles were obtained in a shorter time with a similar enrichment of brush border marker enzymes (11- to 12-fold for alkaline phosphatase and maltase), with a similarly reduced activity of the marker enzyme for basal-lateral plasma membranes and an almost identical protein composition as revealed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The transport properties of the two membrane preparations for D-glucose, L-phenylalanine, and phosphate are essentially the same; there is some indication for a lower sodium permeability of the vesicles prepared by the calcium precipitation method. The latter vesicles were also shown to exhibit sodium gradient stimulated uptake of L-glutamate.     

Membrane vesicles from brown adipose tissue: A tool for the study of amino acid transport. The case of L-alanine

A density gradient method is used to isolate membrane vesicles from brown adipose tissue. These respond to changes in osmolarity and show the classical overshoot pattern when L-alanine uptake is assayed. Transport is shown to be effected by two components: a linear (K_d=0.498 min⁻¹) and Na⁺-dependent saturable component (K_m=2.3 mM) and a V_max=19.9 pmol/μg protein·min). This pattern is similar to that shown by cells isolated from brown adipose tissue.     

Lactate transport by skeletal muscle sarcolemmal vesicles

Recent studies have indicated that lactate traversal of the sarcolemmal membrane of skeletal muscle could be a carrier mediated process. In the present study, the initial rates of L(+)-lactate flux (J_lact) were measured in highly purified rat hindlimb skeletal muscle sarcolemmal vesicles. Fluxes were determined by the vesicle uptake of L(+)-[U-¹⁴C] lactate from the extra-vesicular medium. J_lact was saturable with respect to increasing concentrations of L(+)-lactate. Regression of these data to the Michaelis-Menten equation yielded a Km of 12.5 mM. J_lact was inhibited 81% by 10 mM pyruvate and 83% by 5mM alpha-cyano 4 hydroxycinnamate (p<0.05), but not by D-lactate indicating the presence of a stereoselective monocarboxylate transporter in the sarcolemmal membrane. Preincubation of the vesicles with the protein modifier, N-ethylmaleimide (20mM), inhibited J_lact by 86% (p<0.05). An inhibitor of the inorganic anion exchanger, SITS (1mM), had no effect on J_lact. However, J_lact was markedly sensitive to an inwardly directed proton gradient (p<0.05), and the flux was more closely related to the concentration of external ionic L(+)-lactate than to the protonated (HLa) form. These studies suggest that skeletal muscle sarcolemmal membranes possess a specific transport system for L-lactate and other monocarboxylates, which has similar properties to the lactate carrier described for several other tissues.     

Two-compartment behavior during transport of folate compounds in L1210 cell plasma membrane vesicles

Summary The transport of [³H] 1,l 5-formyltetrahydrofolate, [³H] folic acid, and [³H]methotrexate by L1210 cell plasma membrane vesicles exhibited multicompartmental behavior. Two separate vesicular compartments (parallel relationship) of approximately equal volume were revealed during measurements of influx and efflux. Flux in one compartment was rapid, saturable, highly temperature-sensitive, and inhibited by pCMBS. Flux in the other compartment exhibited all of the characteristics of passive diffusion. These results imply that our plasma membrane vesicle preparations consist of a mixture of two functional species. Transport of folate into one of these species occurs by passive diffusion alone, whereas transport into the other kind of vesicle occurs by both passive diffusion and carrier-facilitated transport.     

Mechanisms of transmembrane cation transport studied by nuclear magnetic resonance spectroscopy

Several molecules like ionophores, vitamins, ion-binding cyclic peptides, acidic phospholipids, surfactants are known to expose the inner side of vesicles, to the externally added cations. Whereas ionophores and certain other systems bring about these changes by a selective transport (influx) of the cation by specialized mechanisms known as the carrier and channel mechanism, other systems cause lysis and vesicle fusion. These systems have been successfully studied using¹H,³¹ P and¹³C nuclear magnetic resonance spectroscopy after the demonstration, fifteen years ago, of the ability of paramagnetic lanthanide ions to distinguish the inside of the vesicle from the outside. The results of these ’nuclear magnetic resonance kinetics’ experiments are reviewed.     

Differences in L-alanine uptake by livers of Wistar and lean Zucker rats

The L-alanine uptake by livers of Wistar and lean Zucker rats has been studied. The hepatic uptake and fractional extraction rates of alanine were estimated in 50–55 day old rats. No significant differences in amino acid concentrations and blood flows in afferent and efferent liver vessels were seen in lean Zucker rats when compared with Wistar rats. However, the hepatic uptake (1.6±0.1 and 0.7±0.1 mol/min/100 g bw, p<0.01) and the fractional extraction (26.8±2.1 and 15.2±3.1%, p<0.05) were much lower in Zucker than in Wistar rats. The hepatic active transport of L-alanine was determinedin vitro using isolated plasma membrane vesicles. Vesicles isolated from livers of lean Zucker rats showed similar values of Km (2.5±0.7 vs 2.0±0.5 mM for Wistar and Zucker respectively, N.S.), but lower values of Vmax when compared with Wistar rats (1.1±0.1 vs 0.6±0.005 nmol/mg prot 5 s, p<0.01, for Wistar and lean Zucker rats respectively). These results indicate that, the liver of lean Zucker rats concentrates alanine less efficiently than the liver of Wistar rats. This fact correlates well with a lower capacity of the Na⁺-dependent L-alanine trasport in liver plasma membrane vesicles from lean Zucker rats.     

Stoichiometric studies of the renal outer cortical brush border membraned-glucose transporter

Summary The stoichiometric properties of the renal outer cortical brush-border membraned-glucose transporter are studied. Experiments which establish the glucose/sodium, glucose/phlorizin and phlorizin/sodium stoichiometries are reported. Three independent methods of determining the substrate/activator (glucose/sodium) stoichiometry for coupled transport systems are presented and discussed. One of these, the Static Head Method, is introduced here for the first time. This type of experiment appears to be more generally applicable than the usual procedure of directly measuring the coupled fluxes of substrate and activator to determine stoichiometric coupling ratios. The results presented in this paper demonstrate that the glucose/sodium/phlorizin stoichiometry of the renal outer cortical brush-border membraned-glucose transport system is 111.     

Folate Transport by Prawn Hepatopancreas Brush-Border Membrane Vesicles

The transport system of folic acid (Pte-Glu) by brush-border membrane vesicles (BBMV) isolated from prawn (Penaeus japonicm) hepatopancreas, was studied by measuring the uptake of Pte-Glu. This uptake was found to have two components, intravesicular transport and membrane binding. Membrane binding was not affected by the presence of a transmembrane pH-gradient at a short incubation period. However, a transmembrane pH-gradient increased membrane binding at 60 min. The transport of Pte-Glu appeared to be carrier-mediated, was stimulated by an inwardly proton gradient (pH 5.5 outside, 7.4 inside) and was unaffected by a sodium-gradient. The relationship between pH gradient-driven Pte-Glu uptake and medium Pte-Glu concentration followed saturating Michaelis–Menten kinetics. Eadie–Hofstee representation of the pH gradient-driven Pte-Glu uptake indicated a single transport system with a Km of 0.37 M and Vmax of 1.06 pmol/mg protein/15 s. These findings indicate that BBMV isolated from prawn hepatopancreas possesses a Pte-Glu transport system similar to that described in mammalian intestine.     

The Na+-independentd-glucose transporter in the enterocyte basolateral membrane: Orientation and cytochalasin B binding characteristics

Summary Phloridzin-insensitive, Na⁺-independentd-glucose uptake into isolated small intestinal epithelial cells was shown to be only partially inhibited by trypsin treatment (maximum 20%). In contrast, chymotrypsin almost completely abolished hexose transport. Basolateral membrane vesicles prepared from rat small intestine by a Percoll^® gradient procedure showed almost identical susceptibility to treatment by these proteolytic enzymes, indicating that the vesicles are predominantly oriented outside-out. These vesicles with a known orientation were employed to investigate the kinetics of transport in both directions across the membrane. Uptake data (i.e. movement into the cell) showed aK _t of 48mm and aV _max of 1.14 nmol glucose/mg membrane protein/sec. Efflux data (exit from the cell) showed a lowerK _t of 23mm and aV _max of 0.20 nmol glucose/mg protein/sec.d-glucose uptake into these vesicles was found to be sodium independent and could be inhibited by cytochalasin B. TheK _t for cytochalasin B as an inhibitor of glucose transport was 0.11 m and theK _D for binding to the carrier was 0.08 m.d-glucose-sensitive binding of cytochalasin B to the membrane preparation was maximized withl- andd-glucose concentrations of 1.25m. Scatchard plots of the binding data indicated that these membranes have a binding site density of 8.3 pmol/mg membrane protein. These results indicate that the Na⁺-independent glucose transporter in the intestinal basolateral membrane is functionally and chemically asymmetric. There is an outward-facing chymotrypsin-sensitive site, and theK _t for efflux from the cell is smaller than that for entry. These characteristics would tend to favor movement of glucose from the cell towards the bloodstream.     

Leucine transport in membrane vesicles from Chironomus riparius larvae displays a mélange of crown-group features

Leucine uptake into membrane vesicles from larvae of the midge Chironomus riparius was studied. The membrane preparation was highly enriched in typical brush border membrane enzymes and depleted of other membrane contaminants. In the absence of cations, there was a stereospecific uptake of l-leucine, which exhibited saturation kinetics. Parameters were determined both at neutral (Km 33 +/- 5 microM and Vmax 22.6 +/- 6.8 pmol/7s/mg protein) and alkaline (Km 46 +/- 5 microM and Vmax 15.5 +/- 2.5 pmol/7s/mg protein) pH values. At alkaline pH, external sodium increased the affinity for leucine (Km 17 +/- 1 microM) and the maximal uptake rate (Vmax 74.0 +/- 12.5 pmol/7s/mg protein). Stimulation of leucine uptake by external alkaline pH agreed with lumen pH measurements in vivo. Competition experiments indicated that at alkaline pH, the transport system readily accepts most L-amino acids, including branched, unbranched, and alpha-methylated amino acids, histidine and lysine, but has a low affinity for phenylalanine, beta-amino acids, and N-methylated amino acids. At neutral pH, the transport has a decreased affinity for lysine, glycine, and alpha-methylleucine. Taken together, these data are consistent with the presence in midges of two distinct leucine transport systems, which combine characters of the lepidopteran amino acid transport system and of the sodium-dependent system from lower neopterans.     

l- andd-alanine transport in brush border membrane vesicles from lepidopteran midgut: Evidence for two transport systems

Summary In brush border membrane vesicles from the midgut ofPhilosamia cynthia larvae (Lepidoptera) thel- andd-alanine uptake is dependent on a potassium gradient and on transmembrane electrical potential difference. Each isomer inhibits the uptake of the other form: inhibition ofl-alanine uptake byd-alanine is competitive, whereas inhibition ofd-alanine uptake byl-alanine is noncompetitive. Transstimulation experiments as well as the different pattern of specificity to cations suggest the existence of two transport systems. Kinetic parameters for the two transporters have been calculated both when K_out>K_in and K_out=K_in.d-alanine is actively transported also by the whole midgut, but it is not metabolized by the intestinal tissue.     

The Relation between Membrane Potential and the Transport Activity of Systems a and L in Plasma Membrane Vesicles of the Ehrlich Cell

Plasma membrane vesicles isolated from Ehrlich ascites tumor cells have been used to investigate the role of the transmembrane potential in the energetics of Systems A and L. As expected, Na⁺-dependent System A was responsive to changes in membrane potential. System L activity, as measured by transport of 2-aminonorbornane-2-carboxylic acid (BCH), was shown to be Na⁺-independent and was not altered by changes in the membrane potential. The combination of valinomycin and nigericin decreased accumulation of MeAIB but not that of BCH. The presence of nigericin alone caused a significant decrease in uptake by System A and a decrease in uptake by System L to a lesser degree. The inhibitory action of nigericin might reflect its ability to dissipate the Na⁺ gradient rather than an effect on K⁺ or H⁺ flows. The results indicate that modes of energization not produced through the transmembrane potential must account for any uphill operation of System L.     

The orientation of insulin receptors in the red cell membrane

High-affinity binding of insulin to receptors in human erythrocyte membranes occurred at the external surface, but not at the cytoplasmic surface of the plasma membrane, as assessed by insulin binding to right-side-out and inside-out membrane vesicles. Even after prolonged (3 h) incubation at 22°C, binding at the cytoplasmic membrane aspect remained negligible. The data indicate that the insulin receptor displays its hormone-binding site exclusively toward the extracellular space and that transmembrane mobility (“flip-flop”) of the receptor from one to the other membrane leaflet is severely restricted.     

Nitrogen transfer in the interface between the symbionts in pea root nodules

Transport mechanisms for transfer of nitrogen from the bacteroid side across the symbiosome membrane of pea (Pisum sativum L.) root nodules were identified by the use of energised bacteroid side-out symbiosome membrane vesicles. Such membrane vesicles were used to study a mechanism with high capacity for transport of ammonium and another mechanism capable of transporting aspartate. Both transport mechanisms are voltage driven and the rate of transport relates positively to the magnitude of the imposed membrane potentials. Competition for transport between ammonium and aspartate was not observed. The ammonium transporter has been identified as a voltage-driven channel whereas the symbiosome membrane aspartate transporter appears to be a H⁺/aspartate symport. The results suggest that nitrogen transfer between the symbionts in pea root nodules involves transfer of amino acids as well as ammonium. In the symbiosome subfraction, which represents the interface between the symbionts, specific aspartate aminotransferase activity was more than four times as high as in the bacteroid cytosol. This finding supports a hypothesis that transamination cycles operating between the symbionts may constitute a component of the transfer of nitrogen between the symbionts.