Propidium uptake and ATP release in A549 cells share similar transport mechanisms

The lipid bilayer of eukaryotic cells’ plasma membrane is almost impermeable to small ions and large polar molecules, but its miniscule basal permeability in intact cells is poorly characterized. This report describes the intrinsic membrane permeability of A549 cells toward the charged molecules propidium (Pr²⁺) and ATP^4?. Under isotonic conditions, we detected with quantitative fluorescence microscopy, a continuous low-rate uptake of Pr (～150 × 10^?21 moles (zmol)/h/cell, [Pr]_o = 150 μM, 32°C). It was stimulated transiently but strongly by 66% hypotonic cell swelling reaching an influx amplitude of ～1500 (zmol/h)/cell. The progressive Pr uptake with increasing [Pr]_o (30, 150, and 750 μM) suggested a permeation mechanism by simple diffusion. We quantified separately ATP release with custom wide-field-of-view chemiluminescence imaging. The strong proportionality between ATP efflux and Pr²⁺ influx during hypotonic challenge, and the absence of stimulation of transmembrane transport following 300% hypertonic shock, indicated that ATP and Pr travel the same conductive pathway. The fluorescence images revealed a homogeneously distributed intracellular uptake of Pr not consistent with high-conductance channels expressed at low density on the plasma membrane. We hypothesized that the pathway consists of transiently formed water pores evenly spread across the plasma membrane. The abolition of cell swelling-induced Pr uptake with 500 μM gadolinium, a known modulator of membrane fluidity, supported the involvement of water pores whose formation depends on the membrane fluidity. Our study suggests an alternative model of a direct permeation of ATP (and other molecules) through the phospholipid bilayer, which may have important physiological implications.     

Dialectics in carrier research: The ADP/ATP carrier and the uncoupling protein

A concise review is given of the research in our laboratory on the ADP/ATP carrier (AAC) and the uncoupling protein (UCP). Although homologous proteins, their widely different functions and contrasts are stressed. The pioneer role of research on the AAC, not only for the mitochondrial but also for other carriers, and the present state of their structure-function relationship is reviewed. The function of UCP as a highly regulated H⁺ carrier is described in contrast to the largely unregulated ADP/ATP exchange in AAC. General principles of carrier catalysis as derived from studies on the AAC and UCP are elucidated.     

Selective protein transport: Identity of the solubilized phosvitin receptor from chicken oocytes

By two independent methods, the solubilized receptor for phosvitin (PV) has a subunit MW of 116K. Affinity chromatography, showed that only 2 of the more than 25 proteins present in the total detergent solubilized oocyte membrane extract were retained on a PV–agarose column. These proteins of MW of 116K and 100K could be eluted from PV–agarose with free PV. By gel exclusion chromatography, the receptor-¹²⁵I-PV complexes elute in the void volume of a Biogel A-1.5 column. When these void fractions were assayed by SDS-PAGE only a single protein of MW of 116K was observed in addition to ¹²⁵I-PV.     

Effect of membrane potential and internal pH on active sodium-potassium transport and on ATP content in high-potassium sheep erythrocytes

Ouabain-sensitive Na⁺ and K⁺ fluxes and ATP content were determined in high potassium sheep erythrocytes at different values of membrane potential and internal pH. Membrane potential was adjusted by suspending erythrocytes in media containing different concentrations of MgCl₂ and sucrose. Concomitantly either the external pH was changed sufficiently to maintain a constant internal pH or the external pH was kept constant with a resultant change of internal pH. The erythrocytes were preincubated before the flux experiment started in a medium which produced increased ATP content in order to avoid substrate limitation of the pump. p] It was found that an increased cellular pH reduced the rates of active transport of Na⁺ and K⁺ without significantly altering the ratio of pumped $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$. This reduction was not due to limitation in the supply of ATP although ATP content decreased when internal pH increased. Changes of membrane potential in the range between ?10 and +60 mV at constant internal pH did not affect the rates of active transport of Na⁺ or K⁺.     

Energy coupling of membrane transport and efficiency of sucrose dissimilation in yeast

Proton coupled transport of α-glucosides via Mal11 into Saccharomyces cerevisiae costs one ATP per imported molecule. Targeted mutation of all three acidic residues in the active site resulted in sugar uniport, but expression of these mutant transporters in yeast did not enable growth on sucrose. We then isolated six unique transporter variants of these mutants by directed evolution of yeast for growth on sucrose. In three variants, new acidic residues emerged near the active site that restored proton-coupled sucrose transport, whereas the other evolved transporters still catalysed sucrose uniport. The localization of mutations and transport properties of the mutants enabled us to propose a mechanistic model of proton-coupled sugar transport by Mal11. Cultivation of yeast strains expressing one of the sucrose uniporters in anaerobic, sucrose-limited chemostat cultures indicated an increase in the efficiency of sucrose dissimilation by 21% when additional changes in strain physiology were taken into account. We thus show that a combination of directed and evolutionary engineering results in more energy efficient sucrose transport, as a starting point to engineer yeast strains with increased yields for industrially relevant products.     

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.     

A simple method for the isolation of basolateral plasma membrane vesicles from rat kidney cortex Enzyme activities and some properties of glucose transport

A procedure for preparing basolateral membrane vesicles from rat renal cortex was developed by differential centrifugation and Percoll density gradient centrifugation, and the uptake of d-[³H]glucose into these vesicles was studied by a rapid filtration technique. (Na⁺ + K⁺)-ATPase, the marker enzyme for basolateral membranes, was enriched 22-fold compared with that found in the homogenate. The rate of d-glucose uptake was almost unaffected by Na⁺ gradient (no overshoot).     

Conserved structural domains among species and tissues-specific differences in the mitochondrial phosphate-transport protein and the ADP/ATP carrier

Peptide maps were generated of the CNBr-digested mitochondrial phosphate-transport protein and ADP/ATP carrier from bovine and rat heart, rat liver and blowfly flight muscle. Total mitochondrial proteins from the same sources plus pig heart were separated by SDS-polyacrylamide gel electrophoresis. The peptide maps and the total mitochondrial proteins were electroblotted onto nitrocellulose membranes and reacted with rabbit antisera raised against the purified bovine heart phosphate-transport protein and the ADP/ATP carrier. On the basis of antibody specificity, mobility in SDS-polyacrylamide gel electrophoresis, and peptide maps the following was concluded. (1) Phosphate-transport protein and phosphate-transport protein β (pig and bovine heart) react equally with the first and also with the second of two independent phosphate-transport protein-antisera. (2) Tissue-specific structural domains exist for both the phosphate-transport protein and the ADP/ATP carrier, i.e., one phosphate-transport protein-antiserum reacts with the phosphate-transport protein from all assayed sources, the other only with the cardiac phosphate-transport protein. These differences may reflect tissue-specific regulation of phosphate and adenine nucleotide transport. (3) Homologies among the different species are found for the phosphate transport protein and the ADP/ATP carrier, except for the flight muscle ADP/ATP carrier. These conserved structural domains of the phosphate-transport protein may relate directly to catalytic activity. (4) Alkylation of the purified phosphate-transport proteins and the ADP/ATP carriers by the transport inhibitor N-ethylmaleimide affects electrophoretic mobilities but not the antibody binding. (5) Neither of the two phosphate-transport protein-antisera nor the ADP/ATP-carrier antiserum react with both phosphate transport protein and ADP/ATP carrier, even though these two proteins possess similarities in primary structure and function. Possible mechanisms for generating tissue-specific structural differences in the proteins are discussed.     

ADP and ATP transport in mitochondria evidence for a metal-ion involved in transport catalysis by use of metallochromic indicators

This study introduces a new class of active-site directed probes with respect to ADP and ATP transport catalysis in rat liver mitochondria. The anionic monoazo dyes, e.g., $\text{p-(2-}\text{hydroxy}\text{-1-}\text{naphthylazo}\text{)}\text{naphtholsulfonic acid}$, are competitive inhibitors of carrier-mediated ADP uptake ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ 20–30 μM). The azo dyes also can displace the same amount of carrier-specific bound ADP as does carboxyatractyloside. Two essential substructures could be derived from a structure-activity study. Firstly, a sulfonic acid group in the para position relative to the azo bridge which becomes neutralized upon binding by a specifically located positive charge of the carrier protein. This electrostatic binding component, which presumably is represented by a strategic arginyl residue, seems to be essential for substrate binding as well as inhibitor binding. The second structural requirement for effective inhibition was found to be the $\text{o-}\text{hydroxy}$ or $\text{o,o′-}\text{dihydroxyazo}$ system, which is known to form stable complexes with metal ions by chelation. Experiments on prevention and reversal of dye-mediated inhibition revealed that the metal-chelating properties are responsible for the effects observed. In addition, using bovine serum albumin or the synthetic polymer Kollidone, inhibition could be prevented as well as abolished. It is postulated that a metal ion, possibly $\text{Mg}{}^{\mathrm{2+}}$, which is bound to the carrier protein plays an essential role for transport catalysis. The metal ion is assumed to form a functional ternary complex, i.e., a metal bridge complex between the carrier protein and its substrate.     

Role of the deinhibitor protein in the interconversion of the ATP,Mg-dependent protein phosphatase

The small molecular weight (± 9,000) heat stable deinhibitor protein, isolated from dog liver, not only protects the multisubstrate protein phosphatase from inhibition by inhibitor-1 and the modulator protein. It prevents the conversion of the active enzyme to the ATP,Mg-dependent enzyme form brought about by the modulator protein, and also affects the activation of the ATP,Mg-dependent protein phosphatase, probably by stabilizing the enzyme in its active conformation during the reversible activation by protein kinase F_A. Therefore the deinhibitor protein could be an important factor in the process of glycogen synthesis, which requires glycogen synthase and phosphorylase as dephosphorylated enzymes.     

Rapid isolation of bovine interphotoreceptor retinol-binding protein

A large retinol-binding protein, interphotoreceptor retinol-binding protein, is found only in the interphotoreceptor matrix of the eye, and may function in vitamin A transport for the visual cycle. Interphotoreceptor retinol-binding protein is the major glycoprotein of this matrix, and can be isolated rapidly by affinity-adsorption onto concanavalin A-Sepharose. The yield is approx. 0.25 mg per bovine eye. Its apparent M_r is 250 000 by gel-filtration chromatography, and 225 000 by native polyacrylamide-gradient gel electrophoresis; this protein band displays endogenous retinol fluorescence on such gels. As measured by SDS-polyacrylamide gel electrophoresis, the apparent M_r is 140 000. In the interphotoreceptor matrix most vitamin A-binding sites on this retinol-binding protein are unoccupied; however, addition of exogenous all-trans-retinol can saturate these sites. The apparent dissociation constant for retinol is 10⁻⁶ M, as measured by fluorimetric titration.     

Role of calcium in serum-stimulation of hexose transport in muscle cells

Serum stimulates glucose uptake into several cells in culture. In intact muscle, an increase in cytosolic free Ca2+ has been proposed to mediate the activation of glucose uptake by hormones and other stimuli [Cell Calcium (1980) 1, 311-325]. We report that hexose (2-deoxy-D-glucose) uptake into L6 muscle cells in culture is enhanced several-fold by fetal calf serum. The increase in uptake is due to stimulation of transmembrane transport, since serum also stimulated uptake of the non-metabolizable hexose 3-O-methyl-D-glucose. The role of Ca2+ in this stimulation was assessed: (i) stimulation of transport by serum was independent of the presence of extracellular Ca2+ during the incubation with serum; (ii) the intracellular levels of free Ca2+, measured by the fluorescence of the novel Ca-indicator quin-2, were identical in serum-stimulated and control cells. It is concluded that hexose transport can increase in muscle cells without concomitant changes in cytoplasmic free Ca2+.     

Polyamine transport,accumulation, and release in brain

Cycling of polyamines (spermine and spermidine) in the brain was examined by measuring polyamine transport in synaptic vesicles, synaptosomes and glial cells, and the release of spermine from hippocampal slices. It was found that membrane potential-dependent polyamine transport systems exist in synaptosomes and glial cells, and a proton gradient-dependent polyamine transport system exists in synaptic vesicles. The glial cell transporter had high affinities for both spermine and spermidine, whereas the transporters in synaptosomes and synaptic vesicles had a much higher affinity for spermine than for spermidine. Polyamine transport by synaptosomes was inhibited by putrescine, agmatine, histidine, and histamine. Transport by glial cells was also inhibited by these four compounds and additionally by norepinephrine. On the other hand, polyamine transport by synaptic vesicles was inhibited only by putrescine and histamine. These results suggest that the polyamine transporters present in glial cells, neurons, and synaptic vesicles each have different properties and are, presumably, different molecular entities. Spermine was found to be accumulated in synaptic vesicles and was released from rat hippocampal slices by depolarization using a high concentration of KCl. Polyamines, in particular spermine, may function as neuromodulators in the brain.     

A giant liposome for single-molecule observation of conformational changes in membrane proteins

We present an experimental system that allows visualization of conformational changes in membrane proteins at the single-molecule level. The target membrane protein is reconstituted in a giant liposome for independent control of the aqueous environments on the two sides of the membrane. For direct observation of conformational changes, an extra-liposomal site(s) of the target protein is bound to a glass surface, and a probe that is easily visible under a microscope, such as a micron-sized plastic bead, is attached to another site on the intra-liposomal side. A conformational change, or an angular motion in the tiny protein molecule, would manifest as a visible motion of the probe. The attachment of the protein on the glass surface also immobilizes the liposome, greatly facilitating its manipulation such as the probe injection. As a model system, we reconstituted ATP synthase (F_OF₁) in liposomes tens of μm in size, attached the protein specifically to a glass surface, and demonstrated its ATP-driven rotation in the membrane through the motion of a submicron bead.     

聚合高分子电解质分离纯化蛋白质

聚合高分子电解质含有大量阳离子或阴离子,通过静电作用结合带相反电荷的蛋白质,生成聚合高分子电解质-蛋白质复合物,电解质-蛋白质复合物通过桥连作用或疏水作用形成沉淀颗粒;聚合高分子电解质的选择性沉淀作用受电解质的分子量、添加剂量、溶液离子强度和pH的影响。用高分子电解质从大规模的低浓度溶液中选择性地沉淀目的蛋白质,为生物工程的下游处理开辟了一条新途径。     

Presence and regulation of ATP:citrate lyase from the citric acid producing fungus Aspergillus niger

ATP:citrate lyase (EC 4.1.3.8) has been identified in cell-free extracts from the filamentous fungus Aspergillus niger. The enzyme was located in the cytosol. It exhibits an activity at least ten times that of acetate-CoA-kinase (EC 6.2.1.1) during growth on carbohydrates as carbon sources, and is thus considered responsible for acetyl-CoA formation under these conditions. It is formed constitutively and its biosynthesis does not appear to be controlled by changes in the nitrogen or carbon source or type. ATP:citrate-lyase appears to be very labile during conventional purification procedures; a method involving fast protein liquid anion exchange chromatography was thus developed in order to obtain enzyme preparations sufficiently free of enzymes which could interfere with kinetic investigations. This preparation displays commonly known characteristics of ATP:citrate lyase with respect to substrate affinities and cofactor requirements, with the exception that the affinity for citrate is rather low (2.5 mM). No activator was found. The enzyme is inhibited by nucleoside diphosphates, nucleoside monophosphates and palmitoyl-CoA. Regulation of ATP:citrate lyase be the energy charge of the cytosol in relation to lipid or citric acid accumulation is discussed in view of these findings. Present address: Institut für Allgemeine Biochemie, Universität Wien, Währingerstrasse 38, A-1090 Wien, Austria     

Identification of a SNARE protein required for vacuolar protein transport in Schizosaccharomyces pombe

Intracellular vesicle trafficking is mediated by a set of SNARE proteins in eukaryotic cells. Several SNARE proteins are required for vacuolar protein transport and vacuolar biogenesis in Saccharomyces cerevisiae. A search of the Schizosaccharomyces pombe genome database revealed a total of 17 SNARE-related genes. Although no homologs of Vam3p, Nyv1p, and Vam7p have been found in S. pombe, we identified one SNARE-like protein that is homologous to S. cerevisiae Pep12p. However, the disruptants transport vacuolar hydrolase CPY (SpCPY) to the vacuole normally, suggesting that the Pep12 homolog is not required for vacuolar protein transport in S. pombe cells. To identify the SNARE protein(s) involved in Golgi-to-vacuole protein transport, we have deleted four SNARE homolog genes in S. pombe. SpCPY was significantly missorted to the cell surface on deletion of one of the SNARE proteins, Fsv1p (SPAC6F12.03c), with no apparent S. cerevisiae ortholog. In addition, sporulation, endocytosis, and in vivo vacuolar fusion appear to be normal in fsv1Delta cells. These results showed that Fsv1p is mainly involved in vesicle-mediated protein transport between the Golgi and vacuole in S. pombe cells.     

Calculation of thermodynamic properties of protein in Escherichia coli K-12 grown on succinic acid, energy changes accompanying protein anabolism, and energetic role of ATP in protein synthesis

Using an average of the results from three methods of calculation, estimations are made of the thermodynamic properties of a unit carbon formula weight (UCFW) of Escherichia coli K-12 protein. These resulted in values fro DeltaG(f) of -38.09 kJ (-9.10 kcal)/ UCFW, for DeltaH(f) of -68.18 kJ (-16.29 kcal)/UCFW, and for DeltaS(f) of -94.2 J (-22.5 cal)/UCFW deg. The absolute entropy of one UCFW of E. coli K-12 protein is calculated to be 73.8 J/UCFW deg. Using these values, the corresponding changes in thermodynamic properties accompanying the anabolism of protein by this microorganism to from one UCFW of protein by this microorganism to from one UCFW of protein are calculated to be 1.97 kJ (0.47 kcal)/UCFW for DeltaG, 0.75 kJ (0.18 kcal)/UCFW for DeltaH, and -4.09 J (-0.98 cal)/UCFW deg for DeltaS. All these values are sufficiently close to zero that they may be considered to be so. The question is raised as to the quantity of ATP energy conserved within the substance of the protein as it is synthesized from succinic acid. It is calculated that only 3.8% of the total free energy available from ATP that is required during protein anabolism can have been conserved within the substance of the protein, there being a net conversion of the remaninder into heat and entropy.     

Expression of the pCloDF13 encoded bacteriocin release protein or its stable signal peptide causes early effects on protein biosynthesis and Mg2+ transport

The effect of the pCloDF13 encoded bacteriocin release protein (BRP) onEscherichia coli cell lethality was studied. Induction of the BRP resulted in a strong inhibition of the incorporation of radioactive labeled amino acids and affected the transport of Mg²⁺ ions. Similar effects were obtained when the BRP stable signal peptide was expressed as a separate entity. Kinetic studies revealed that these effects occurred prior to quasi-lysis and release of cloacin DF13. The results indicated that the BRP induced cell lethality is caused by early effects on protein synthesis and Mg²⁺ transport, due to the accumulation of stable BRP signal peptides in the cytoplasmic membrane.