Epidermal growth factor: Characteristics of specific binding in membranes from liver,placenta, and other target tissues

Epidermal growth factor, a 6,400-dalton polypeptide from the mouse submaxillary gland, binds specifically to cells and membranes derived from a variety of human, rat, mouse, and bovine tissues. Liver, placenta, skin, cornea, and cultured chondrocytes, Hela cells, and Chang liver cells bind large amounts of epidermal growth factor, whereas fat cells, resting and lectin-stimulated human peripheral lymphocytes, mouse thymocytes, cultured rat hepatoma cells, and mammary cells from virgin and pregnant mice bind little or no epidermal growth factor. The binding site for epidermal growth factor is distinct from receptors for other anabolic peptides such as insulin, nerve growth factor, and growth hormone. The binding of epidermal growth factor is rapid and reversible. The rate constant of association is approximately 10⁶ mole^?1 sec^?1, the rate constant of dissociation is about 6 × 10^?4 sec^?1, and the apparent equilibrium dissociation constant is about 10^?9m. Trypsin at low concentrations (50–200 μg/ml) destroys the receptor site for epidermal growth factor. The binding of epidermal growth factor by membranes is not accompanied by appreciable degradation of the peptide present in the medium or of that bound to the membranes. Use can be made of the high affinity and specificity of membranes for epidermal growth factor to measure by a competitive binding assay as little as 200 pg of EGF per ml (3 × 10^?11m).     

Double labeling of chromatin proteins,in vivo and in vitro,and their two-dimensional electrophoretic resolution

A modification of the two-dimensional electrophoretic method that involves nonequilibrium pH gradients has been adapted for high resolution of chromatin proteins from sea urchin embryos. A simple method of labeling the protein, in vitro, by reductive methylation with boro[³H]hydride to a specific activity of 100,000 cpm/μg of protein is detailed. Chromatin protein may be labeled, in vivo with ¹⁴C-amino acids, and newly synthesized (³H and ¹⁴C-labeled) and preexistent proteins (only ³H labeled) may be distinguished. The method reveals that sea urchin embryo chromatin contains over 200 proteins.     

Wilson and Wilson's comprehensive analytical chemistry: Vol. XII,Thermal Analysis,Part A,Simultaneous thermoanalytical examinations by means of the Derivatograph. By J. Paulik and F. Paulik,Elsevier, Amsterdam/New York, 1981. 278 pp., $83.00

The reductive methylation procedure of G.E. Means and R. E. Feeney (1968)Biochemistry7, 2192–2201) was adapted for ³H-labeling of membrane proteins using pigeon erythrocyte membrane. Usably high ³H incorporation into protein was obtained, e.g., 28 μCi/mg protein with 83 nmol (input) H₂CO/mg protein, B³H₄^? at 10 Ci/mmol, and a B³H₄^?/H₂CO ratio of 0.34. With this low H₂CO/protein ratio, methylation did not perturb ATP-dependent ⁴⁵Ca²⁺ uptake, Na⁺-dependent [¹⁴C]glycine uptake, membrane vesicle sealing, or isoelectric focusing patterns of methylated membrane proteins. The labeled membrane proteins were shown to be good tracers for the unlabeled proteins by using two-dimensional isoelectric focusing x sodium dodecyl sulfate gel electrophoresis.     

Chromatin proteins of sea urchin embryos: Dual origin from an oogenetic reservoir and new synthesis

The chromatin proteins of different embryonic stages, ranging from 16 cell to gastrula, of the sea urchin Strongylocentrotus purpuratus were labeled, in vivo, with ¹⁴C and were labeled, in vitro, with ³H. The proteins thus labeled were separated by high resolution two-dimensional electrophoresis. The extent of possible cytoplasmic contamination has been examined with reconstruction experiments. Gastrula chromatin contains over 200 separable nonhistone proteins, and about 90% of them are also detected at the 60-cell stage; cleavage stages have over all protein gel patterns displaying numerous differences with the pattern shown by chromatin from later stages. Differences in the proportion of histone to nonhistone proteins that are synthesized are observable at the different embryonic stages, with histones predominating in midcleavage. About half of the nonhistone proteins of the developing embryo that can be labeled with ³H, in vitro, are not labeled with ¹⁴C, in vivo, and hence, must originate from a reservoir of nonhistone proteins assembled during oogenesis.     

High affinity specific [3H] (+)PN 200-110 binding to dihydropyridine receptors associated with calcium channels in rat cerebral cortex and heart

The binding properties of the 1,4-dihydropyridine calcium channel antagonist, [³H](⁺)PN 200-110, were studied in rat cerebral cortical and cardiac homogenates (37°C, Krebs phosphate buffer). Specific binding of [³H](⁺)PN 200-110 was saturable, reversible, and of high affinity (K_d values are 35 and 64 pM for the cerebral cortex and heart, respectively). In parallel studies with [³H](⁺)PN 200-110, the dissociation constant of [³H]nitrendipine was 10–12 times higher. Substituted dihydropyridine calcium channel antagonists and agonists competitively inhibited specific [³H](⁺)PN 200-110 binding, but d-cis diltiazem enhanced and verapamil incompletely inhibited [³H](⁺)PN 200-110 binding in both the cerebral cortex and the heart. The effects of diltiazem and verapamil on [³H](⁺)PN 200-110 binding were due mainly to alterations in the dissociation constant (K_d), without alterations in the binding density (B_max). The new [³H](⁺)PN 200-110 receptor binding assay is remarkable for its low degree of nonspecific binding as compared to [³H]nitrendipine at physiological temperatures. [³H(⁺)PN 200-110 is a useful ligand for the further analysis of the dihydropyridine binding sites associated with calcium channels.     

APPLIED ISSUES The effect of ammonium nitrate on the feeding and development of larvae of the smooth newt, Triturus vulgaris (L.), and on the behaviour of its food source, Daphnia

1. Larvae of the smooth newt, Triturus vulgaris, were exposed to four concentrations of ammonium nitrate in artificial pond water (50, 100, 200 and 500 mg 1-^?1) under controlled laboratory conditions. 2. Larvae exposed to a 50 mg 1-^?1 solution of ammonium nitrate showed no significant difference to control larvae in feeding rate, mass at metamorphosis or time to metamorphosis. However, larvae exposed to 200 or 500 mg 1-^?1 ammonium nitrate, for a single period of 24, 48 or 72 h, were significantly smaller than controls at metamorphosis. Larvae exposed to 100 mg 1-^?1 had a significantly higher feeding rate than those reared under control conditions or those exposed to 200 or 500 mg 1-^?1 ammonium nitrate, but this was not reflected in their size at metamorphosis, which was not significantly different from the controls. Larval survival remained high in all trials. 3. In separate trials, Daphnia, used as a food source for the newt larvae, were exposed to the same series of ammonium nitrate concentrations. Daphnia behaviour was affected by exposure to 200 and 500 mg 1-^?1 ammonium nitrate, and in both cases animals were significantly more likely to be found at the top of the water column than those exposed to 50 or 100 mg 1-^?1 ammonium nitrate or the control medium.     

Subcellular distribution and isolation of the Ca2+ antagonist receptor associated with the voltage regulated Ca2+ channel from rabbit heart muscle

The Ca²⁺ antagonist binding sites associated with the voltage dependent calcium channel in rabbit myocardium were found to distribute with the sarcolemmal Na^– + K⁺ ATPase and adenylate cyclase activities during subcellular fractionation on sucrose-density gradients. The equilibrium dissociation constants (K_D) for the binding of [³H]nitrendipine and [³H]verapamil were 0.31 ± 0.04 nM and 4.1 ± 0.5 nM respectively, and displayed an average density of 0.55 ± 0.05 pmol/mg and 0.4 ± 0.03 pmol/mg protein respectively for the most enriched membrane fraction. The Ca²⁺2 antagonist binding sites were solubilized from the membranes with the detergent 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate, and specific binding sites for [³H]PN200-110, [³H]verapamil and [³H]diltiazem were isolated on a wheat-germ lectin column. The binding sites for [³H]PN200-110 were enriched about 2500 fold as compared with the original homogenate and displayed a density of 28.5 ± 8 pmole/mg protein in the isolated fraction. Sodium dodecyl sulfate gel electrophoresis of the isolated drug binding proteins indicated enrichment of proteins of Mr 170000, 140000, 130000, 100 000 and 53000. The isolated receptor contained an intrinsic kinase activity that phosphorylated glycoproteins of Mr 170 000 and 53000. Exogenously added cAMP-kinase stimulated phosphorylation of the 170000, 100000, 53 000 and 28000 Mr glycoproteins in the receptor fraction. The results of this study indicate that the binding sites for [³H]nitrendipine, [³H]PN200-110, [³H]verapamil and [³H]diltiazem residue on glycoprotein(s) which are of sarcolemmal origin, and co-purify together on wheat germ lectin columns. The polypeptide composition of the Ca²⁺ antagonist binding sites from cardiac muscle appears to be very similar to that of the dihydropyridine receptor in skeletal muscle.Abbreviations CHAPS 3-[-(3-cholamidopropyl) dimethylammonio]-propanesulphonate - SDS sodium dodecyl sulphate Scholar of the Ontario Heart and Stroke Foundation.     

Surface Proteins of Cultured Mouse Cerebellar Cells

Surface proteins of cultured monolayer cells from embryonic and early postnatal C57BL/6J mouse cerebella were identified by a lactoperoxidase-catalysed ¹³¹iodine labelling technique. Major iodinated polypeptides have molecular weights of approximately 200, 145, 120, 100, 85, 65, 50, and 30 X 10³ (P200, P145, ?) as estimated by sodium dodecylsulphate polyacrylamide gel electrophoresis. Membrane glycoproteins, of apparent molecular weights 200, 145, 100, 85, and 50 X 10³, are detected by biosynthetic labelling with [³H]fucose. The two major iodinated proteins are the glycoproteins P200 and P145. P145 is released from the cells into the medium together with other surface proteins. No changes in the patterns of labelled cerebellar cell surface proteins are detectable between embryonic day 17 and postnatal day 10. A pattern similar to the one seen with cerebellum is obtained with embryonic day 12 and 17 cerebral cortex. Cultured retinal cells from 2-day-old mice, skin fibroblasts, and l -cells display a distinctly different pattern, which does not contain P145 as a major iodinated component. In granule cell-enriched fractions of cerebellar cells the two glycoproteins P200 and P145 are proportionately increased, while three proteins, P100, P85, and P50, are more abundant in the glial cell-enriched fraction. These three polypeptides are also enriched in cells obtained from staggerer mutant mice. An antiserum against 4-day-old cerebellar cells (anti-NS-4) precipitates the 145 and 200 X 10³ molecular weight proteins, from lysates of both embryonic cerebral and postnatal cerebellar cells. From lysates of mouse retinal cells, anti-NS-4 antiserum precipitates two proteins with molecular weights of 140 and 210 X 10³. Rohrer H. and Schachner M. Surface proteins of cultured mouse cerebellar cells. J. Neurochem. 35, 792–803 (1980).     

Effect of L-DOPA pretreatment on invivo protein synthesis in various rat brain regions

A single dose of L-dihydroxyphenylalanine (L-DOPA, 500 mg/kg, i.p.) that caused massive disaggregation of brain polysomes also suppressed the incorporation of [³H] lysine into trichloracetic acid (TCA)-precipitable proteins of cortex, cerebellum, hypothalamus, brainstem and striatum. The magnitude of this inhibition of [³H] protein synthesis was similar in all brain regions studied, and was not related to changes in the specific activity of the precursor amino acid.     

Lindane degradation by root epiphytic bacterium Achromobacter sp. strain A3 from Acorus calamus and characterization of associated proteins

Abstract

Lindane degrading root epiphytic bacteria were isolated from wetland plant Acorus calamus. Bacterial strain A3 identified as Achromobacter sp. A3, showed maximum degradation potential of 88.7?±?1.24% for 50?mg?l^?1 lindane. Lindane biodegradation was followed by decrease in pH as well as increase in concentration of chloride ions in the culture medium. Lindane degradation potential of Achromobacter sp. A3 was also studied at different concentrations of lindane. Maximum degradation was at 10?mg l^?1 followed by 50?mg l^?1 and 100?mg l^?1 lindane. Also, lindane induced proteins were studied using SDS-PAGE. The induced proteins were identified as alpha/beta hydrolase fold-3 domain-containing protein, involved in lindane hydrolysis and extracellular solute-binding family protein having role in transmembrane transport of lindane for utilization of lindane by bacteria. The appearance of unique polypeptides in lane corresponding to media supplemented with lindane showed that the exposure of bacterial cells to lindane has resulted in regulative expression of certain proteins. So far as known, this is the first report to isolate and study lindane degrading root epiphytic bacteria from A. calamus.     

The effect of 7-oxa-13 prostynoic acid on the mechanism of action of bradykinin in human synovial fibroblasts

Bradykinin, a potent inflammatory mediator, induces an increment in intracellular cyclic AMP concentrations of human synovial fibroblasts and evokes the synthesis and release of ³H-arachidonic acid and ³H-E prostaglandins from these cells pre-labeled in their phospholipids. Fetal calf serum in the media also stimulates the synthesis and release of these labeled lipids from pre-labeled human synovial fibroblasts and potentiates the bradykinin-induced cyclic AMP response. The PGE₁ analogue, 7-oxa-13 prostynoic acid, completely abrogates both the bradkinin-induced cyclic AMP response and the bradykinin- and fetal calf serum-evoked release of labeled E-prostaglandins from pre-labeled cells. In serum-free media, the prostaglandin antagonist stimulated the release of ³H-arachidonic acid from pre-labeled human synovial fibroblasts and did not inhibit the bradykinin-induced release of this lipid.     

Evaluation of a Purification Procedure for the Muscarinic Receptor for the Purpose of Quantitative Receptor Assays of Anticholinergics

Abstract

The presented purification procedure for the muscarinic receptor from calf striatum includes the extraction of lipids with hexane in the first step and the removal of 39% of non-receptor proteins with 2 M NaCl in the second step. The simplicity of such an approach to the purification of the receptor warrants its use in the routine practice for quantitative purposes. The high affinity binding of tertiary ³H-dexetimide (³H-DEX) and quaternary ³H-N-methylscopolamine (³H-NMS) is preserved after the removal of irrelevant lipids and proteins from the P2-pellet.

The overall yield of receptors - 80%, when labelled with ³H-NMS, was satisfactory. Moreover, the final product, the NaCl-pellet, exerts a higher density of ³H-NMS binding sites per mg proteins by a factor of about 1.7. The overall yield of receptors and purification factor were lower, when measured with ³H-DEX. The total yield of ³H-DEX binding sites amounted to about 40% and the receptor density per mg protein decreased by a factor of 0.85.

We did not succeed in the improvement of the ratio specific/non-specific binding, neither for ³H-DEX nor for ³H-NMS for the purified receptor preparations. The use of ³H-NMS is preferable to ³H-DEX in plasma sample assays because of a negligible effect of plasma on ligand binding when compared with ³H-DEX.     

Assay for l-pipecolate oxidase activity in human liver: detection of enzyme deficiency in hyperpipecolic acidaemia

A direct assay method is described for l-pipecolate oxidase. The assay uses NaHSO₃ to trap the $\text{L}\text{-α-amino[}{}^3\text{H]adipate}$δ-semialdehyde (AAS) formed as a direct reaction product of l-pipecolate oxidase from l-[³H]pipecolic acid. The adduct so formed was separated from the substrate on Dowex 50 (H⁺) column. The product was identified as [³H]AAS by amino acid analysis after breaking down the adduct by boiling under acidic conditions. The assay is simpler and more specific than fluorometric methods; it is also more sensitive; requiring at most 16 μg of liver peroxisome-enriched protein per assay. We have used this assay procedure to detect l-pipecolate oxidase in skin fibroblasts obtained from a control subject and from patients of hyperpipecolic acidaemia and Zellweger syndrome and found that this enzyme activity is present in the control, but absent or decreased in the patients with the peroxisomal disorders.     

Enhancement of a pentacyclic tyrosine kinase inhibitor production in Cladosporium cf. cladosporioides by Cladosporol

The binaphthyl derivative cladosporol 3 was supplied from 60 to 200 mg l⁻¹ to shaken cultures of Cladosporium cf. cladosporioides. Compared to blank, fungal biomass was not affected by adding cladosporol till 100 mg l⁻¹: it rather increased at higher ratios between 150 and 200 mg l⁻¹. The production of the major pentacyclic metabolite 1, a cytokine production and tyrosine kinase inhibitor, was enhanced tenfold when cladosporol was supplied at the highest ratio (200 mg l⁻¹) to shaken growing cultures of the fungus. The bioconversion of cladosporol to cladosporol D through reductive cleavage of the epoxide group was also observed. Interest in this kind of metabolites lies in their potential activity vs DNA topoisomerase I.     

Decreased Benzodiazepine Receptor Density in Rat Cerebellum Following Neurotoxic Doses of Phenytoin

Abstract: The effect of acute and chronic administration of phenytoin on [³H]-flunitrazepam binding was examined in the rat cerebellum. There was no significant effect of phenytoin on [³H]flunitrazepam binding in the rat cerebellum 1 and 6 h after a single i.p. injection of 200 mg/kg of phenytoin. However, after 14 days and 28 days of chronic phenytoin administration, significant de-creases in [³H]flunitrazepam receptor density were observed, with no changes in apparent affinity constants in the rat cerebellum. This effect of phenytoin was dose-dependent, as lower doses of phenytoin (100 mg/kg/day) for 14 or 28 days produced no alterations in [³H]flunitrazepam binding in the rat cerebellum. Light-microscopic examination of the rat cerebellum treated with 200 mg/kg/day of phenytoin for 14 days showed degeneration of the Purkinje cells, with edematous Bergmann astrocytes. These data provide evidence for the neuronal localization of benzodiazepine receptors on cerebellar Purkinje cells.     

Identification of external membrane proteins of the T lymphoblast cell line CCRF-CEM

The 70 membrane proteins of the T lymphoblast cell line CCRF-CEM were characterized by

1. 1. [³⁵S]methionine internal radiolabeling;

2. 2. [¹²⁵I]iodine labeling by a lactoperoxidase-mediated method;

3. 3. [³H]fucose internal labeling;

4. 4. binding to a lentil lectin adsorbant column;

5. 5. susceptibility to digestion with limited amounts of papain.

Of the three methods of radiolabeling membrane proteins, [³⁵S]methionine best displayed all proteins although some individual proteins were heavily iodinated or fucosylated. Thirty proteins were externally exposed as defined by susceptibility to lactoperoxidase-mediated radio-iodination and to digestion with minute amounts of papain. Thirtyfive proteins were bound to a lentil lectin absorbant column. p44 (HLA-A and -B antigens) were iodinated, fucosylated, susceptible to papain digestion and bound to the lectin column. β₂-Microglobulin was iodinated and bound to the lectin column. The identifications and functions of other membrane proteins were not known. In general, proteins of high molecular weight (100 000 to 250 000 D) were more heavily radio-iodinated and fucosylated than were proteins of lower molecular weights. p95 was the most heavily fucosylated protein, p110, which had been identified only on T lymphoblasts, was fucosylated and was iodinated. p65, which was found only on the T lymphoblast line CCRF-CEM and could represent a lymphocyte subpopulation-specific molecule, was iodinated and fucosylated. p15 and p18 were equally and densely labeled with [³⁵S]methionine but only p18 was fucosylated and it was heavily radio-iodinated. These experiments help to define the external membrane proteins of a T lymphoblast cell line in part for the selection of proteins for isolation in order to raise antisera for immunodiagnostic and functional studies.     

Nuclear matrix as acceptor for cAMP-binding proteins

Using [³²P]-8-N₃-cAMP, a photoaffinity analog of cAMP, we have established that nuclear binding of cAMP is preferentially localized in the “nuclear matrix”. Two major radioactive bands corresponded to proteins of M_r 40 K and 50 K, and three minor bands to proteins of M_r 55, 150 and 200 K. Even though the molecular weight of the major nuclear binding proteins in the matrix are similar to those of the cytosolic cAMP binding proteins, the characteristics of the binding reaction in the nucleus were markedly different from those in the cytosol.     

Enrichment of Triadic and Terminal Cisternae Vesicles from Rabbit Skeletal Muscle

An enriched triad and terminal cisternae preparation was achieved from skeletal muscle through alterations of the differential centrifugation and muscle homogenization protocols. Both yield and specific activity (pmoles of radioligand binding per mg protein) were optimized for ³H-PN200-l10 (transverse tubule marker) and ³H-ryanodine (terminal cisternae marker) binding sites. By pelleting crude microsomes between 2,000 an 12,000 × g without any rehomogenizations, we improved both the yield and specific activity of transverse tubule and terminal cisternae markers in crude microsomes by approximately 4-fold to 1000–3000 pmoles binding sites (starting material: approximately 400 grams wet weight fast twitch skeletal muscle), with 10–15 pmoles/mg. Rehomogenization of the 1,000 × g pellet, which is typically discarded, allowed recovery of an additional 5000 pmoles PN200-110 binding sites and an additional 8000 pmoles ryanodine binding sites. Crude microsomes from the rehomogenized 1,000 × g pellets typically displayed specific activities of 20–25 pmoles binding/mg for both ³H-PN200-110 and ³H-ryanodine. Separation of crude microsomes on a sucrose gradient increased specific activity up to a maximum of 50 pmoles/mg in a specific fraction, a five- to ten-fold increase over standard triadic or terminal cisternae preparations. The mean specific activity for enriched triads was 30–40 pmoles/mg for both PN200-110 and ryanodine in pooled fractions, while pooled fractions of enriched terminal cisternae displayed low ³H-PN200-110 binding (3–5 pmoles/mg) and high ³H-ryanodine-specific activity (30–40 pmoles/mg).     

Effects of the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate on newly synthesized proteins in mouse epidermis

We have analyzed the effects of treatment of mouse epidermis with the potent tumor promoter TPA on the profile of newly synthesized proteins. TPA was applied to the skin of the intact mouse, and either 3 or 24 hr later skin fragments were pulse-labeled in vitro with ³⁵S-methionine for 4 hr. The epidermal proteins were extracted and separated by two-dimensional gel electrophoresis. Over 200 individual proteins were resolved in acidic gels. At least 10 of these showed major (by a factor of 5 or more) increases or decreases in response to TPA; eight of these appear to be keratin proteins. Two-dimensional gel profiles of basic proteins synthesized by mouse epidermis resolved over 100 individual proteins. Only one of these showed a significant change in response to TPA. This 41 kd protein increased more than 100-fold within 24 hr after the application of TPA. Treatment of mouse skin with mezerein, a plant diterpene structurally related to TPA, produces an almost identical change in the pattern of proteins produced. Four agents that induce hyperplasia but are not potent tumor promoters, ethylphenylpropiolate, acetic acid, turpentine oil and the Ca⁺⁺ ionophore A23187, modulate the synthesis of only three of the keratin proteins. Thus the changes in protein profiles induced by TPA and mezerein are not simply the consequence of hyperplasia. In addition, application to mouse skin of a glucocorticoid that is a potent inhibitor of tumor promotion inhibits most of the changes in protein profiles induced by TPA. Taken together, these results indicate that TPA and mezerein induce early and marked changes in the profile of specific epidermal proteins. It seems likely that some of these changes are directly related to the process of tumor promotion.     

Retention of Ca2+ by rat liver and rat heart mitochondria: Effect of phosphate,Mg2+, and NAD(P) redox state

Parallel measurements of Ca²⁺ uptake, oxygen consumption, endogenous Mg²⁺ efflux, and swelling in rotenone-poisoned rat liver and rat heart mitochondria showed that heart mitochondria is much more resistant to uncoupling by Ca²⁺ in the presence of phosphate than rat liver mitochondria. The extent of Mg²⁺ efflux and swelling induced by Ca²⁺ accumulation are much less pronounced in heart mitochondria. Uncoupling and swelling in liver mitochondria seem to result from the loss of membrane-bound Mg²⁺ as a consequence of Ca²⁺ recycling across the membrane as induced by phosphate. Exogenous Mg²⁺ protects liver mitochondria against the deleterious effects of Ca²⁺ by inhibiting a ruthenium red-insensitive Ca²⁺ efflux induced by phosphate. Phosphate does not induce recycling of Ca²⁺ in heart mitochondria. On the other hand, heart mitochondria respiring on NAD-linked substrates or with succinate in the absence of rotenone behave like liver mitochondria with respect to the alterations caused by Ca²⁺ recycling. In heart mitochondria the recycling of Ca²⁺ is related to the redox state of pyridine nucleotides, which suggests that the ruthenium red-insensitive efflux of Ca²⁺ is subject to metabolic control. In addition it has been observed that Sr²⁺does not undergo cyclic movements across the membrane. The data indicate that the efflux pathway is more specific for Ca²⁺ than the ruthenium red-sensitive influx transporter.