Ehrlich Cell Plasma Membranes Research Articles

Putrescine and chlorpheniramine inhibit Ehrlich ascites tumor cell plasma membrane ferricyanide reductase activity

The presence of putrescine or chlorpheniramine in the incubation medium of Ehrlich ascites tumor cells starved for 1 h significantly inhibits the rate of ferricyanide reduction by their plasma membrane redox system. Freshly harvested cells, without depletion of their intracellular pools of polyamines, and cells preincubated under conditions arranged to increase ornithine decarboxylase activity also reduced externally added ferricyanide at a lower rate than those cells starved for 1 h. All these data seems to indicate that the presence of putrescine is enough to significantly inhibit Ehrlich cell plasma membrane redox system activity.

Enhancement of the functional stability of solubilized nucleoside transporters by substrates and inhibitors

Purification of functional nucleoside transporters has been hampered by the instability of detergent-solublized proteins. The present study was undertaken to determine if the presence of specific transporter ligands in the solubilization medium could enhance the functional stability of the isolated proteins. Ehrlich cell plasma membranes were solubilized with 1% (w/v) octylglucoside (± transporter ligands) and reconstituted into liposomal membranes either immediately after solubilization or after storage for 48 hr at 6 °. Storage resulted in a parallel loss (≈60%) of [ 3H]nitrobenzylthioinosine (NBMPR) binding and reconstituted [ 3H]uridine uptake activities. Furthermore, upon storage, the relative amount of NBMPR-resistant [ 3H]uridine uptake by the reconstituted system dropped from 19 ± 2 to 8 ± 1% of the total mediated influx. The inclusion of high concentrations (>10 mM) of adenosine in the solubilization medium completely prevented the storage-induced loss of both [ 3H]NBMPR binding and [ 3H]uridine influx activity, and prevented the shift in NBMPR sensitivity. In addition, inclusion of adenosine in the solublization procedure increased the relative amount of NBMPR-resistant [ 3H]uridine uptake to 33 ± 2% of the total influx in proteoliposomes prepared immediately after the proteins were extracted from the plasma membrane (i.e. no storage). A partial protection of [ 3H]NBMPR binding activity was also obtained using 2′-deoxyadenosine, 2-chloroadenosine, uridine, and non-radiolabelled NBMPR, but not with cytidine, inosine, diazepam, dipyridamole, or dilazep. These results suggest that both NBMPR sensitivity and transporter stability are dependent upon the conformational state of the protein. The protective effects of adenosine analogues and other nucleosides are likely due to their binding to the substrate translocation site, thereby effectively “locking” the transporter in a stable conformation.

Identification of a Zn2 ‐sensitive component of Ehrlich cell plasma membrane redox system by CHAPS‐agarose‐polyacrylamide electrophoresis and in situ staining of activity

A procedure based on CHAPS-agarose-polyacrylamide electrophoresis and in situ staining of activity was used to detect a Zn(2+)-sensitive component of Ehrlich cell plasma membrane redox system. The procedure is so powerful that it allows to use crude plasma membrane fractions and can be easily adapted for use in an electrophoretic approach to the purification of this protein.

Effect of Detergents on Ligand Binding and Translocation Activities of Solubilized/Reconstituted Nucleoside Transporters

Purification of equilibrative nucleoside transporters has been hampered by the functional instability of the detergent-solubilized proteins. A variety of nonionic detergents were compared with octylglucoside (the most commonly used detergent in this regard) for their abilities to solubilize functionally stable nucleoside transporter proteins from Ehrlich cell plasma membranes. Transporter stability was assessed through the binding of the specific probe [3H]nitrobenzylthi- oinosine to freshly solubilized and stored (48 h/6°C) preparations. The most promising detergents were decylmaltoside and cyclohexylbutylmaltoside, both of which, like octylglucoside, solubilized over 70% of the transporters from the membrane. Decylmaltoside- and cyclohexylbutylmaltoside-solubilized transport proteins retained 61 and 83%, respectively, of their [3H]nitrobenzylthioinosine binding activity upon storage, compared to about 30% using octylglucoside. Decylmaltoside was also superior to octylglucoside in its capacity to solubilize the transporter in a state that retained its high affinity for the transport inhibitors dilazep (Ki= 11 nM, vs 75 nMin octylglucoside) and dipyridamole (Ki= 260 nM, vs 12 μMin octylglucoside). Reconstitution studies indicated that both the de- cylmaltoside- and cyclohexylbutylmaltoside-solubilized transporters were capable of mediating the uptake of [3H]uridine. Decylmaltoside was superior to cyclohexylbutylmaltoside, however, in both the enhanced transport activity of the resulting proteoliposomes (Vi= 21 pmol/mg/s vs 13 pmol/mg/s, respectively) and the lower nonmediated uptake observed in the decylmaltoside-derived vesicles (27% of total uptake at 4 min incubation). Nevertheless, cyclohexylbutylmaltoside may be useful in initial solubilization procedures due to its ability to selectively solubilize the nucleoside transporter from the plasma membrane. The rational use of these detergents will enable a more extensive purification of functional nucleoside transporters.

Effects of protein kinase C and phosphoprotein phosphatase modulators on Ehrlich cell plasma membrane redox system activity

Diacyl glycerols and phorbol esters, which activate protein kinases C, stimulated Ehrlich ascites tumor cell ferricyanide reductase activity. On the contrary, selective inhibition of active protein kinases C with bis-indolyl maleimide did not change the rate of ferricyanide reduction by Ehrlich cells. Selective inhibitors of phosphoprotein phosphatases, okadaic acid and cyclosporin A, also stimulated plasma membrane redox system. Taking all these data together, protein kinases or phosphoprotein phosphatases seemed to be involved in the multiple and complex regulation of Ehrlich cell plasma membrane redox system.

Cytoplasmic pH modulates the activity of the plasma membrane electron transport system in Ehrlich ascites tumour cells.

We report evidence for a modulation of Ehrlich cell plasma membrane redox activity by cytoplasmic pH. When Ehrlich cells were submitted to treatments leading to a slight decrease of cytoplasmic pH, there was a significant inhibition of plasma membrane ferricyanide reductase activity. However, those treatments which produced a slight alkalinization of the cytoplasm enhanced the activity of the plasma membrane redox system. Since it has been shown previously that plasma membrane redox activity controls cytoplasmic pH, it seems that plasma membrane redox activity and cytoplasmic pH mutually modulate each other.

Ehrlich cell plasma membrane redox system is modulated through signal transduction pathways involving cGMP and Ca2+ as second messengers.

Ehrlich cell plasma membrane ferricyanide reductase activity increased in the presence of mastoparan, a generic activator of G proteins, using either whole cells or isolated plasma membrane-fractions. Agents that increase intracellular cAMP also increased the rate of ferricyanide reduction by Ehrlich cells. For the first time, evidence is shown on a modulation of plasma membrane redox system by cGMP. In fact, permeant analogs of cGMP, dibutyryl cGMP, and 8-bromo-cGMP increased the rate of ferricyanide reduction by the Ehrlich cell plasma membrane redox system. Furthermore, specific inhibition of cGMP-phosphodiesterases by dipyridamole was also accompanied by an enhancement in the rate of ferricyanide reduction. On the other hand, treatments expected to increase cytoplasmic Ca2+ concentrations were accompanied by a remarkable stimulation of the reductase activity. Taking all these data together, it seems that the Ehrlich cell plasma membrane redox system is under a multiple and complex regulation by different signal transduction pathways involving G proteins, cyclic nucleotides, and Ca2+ ions.

Identification of the integrin alpha 3 beta 1 as a component of a partially purified A-system amino acid transporter from Ehrlich cell plasma membranes.

We have previously reported [McCormick and Johnstone (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7877-7881] the partial purification of the Na(+)-dependent A-system amino acid transporter from Ehrlich cell plasma membranes and have suggested that a 120-130 kDa peptide, a major component of the purified fraction [octyl glucoside (OG) extract], is involved in Na(+)-dependent amino acid transport. In the present study, N-terminal sequence analysis of the 120-130 kDa peptide revealed a sequence similar to that of the alpha 3 subunit of the integrin alpha 3 beta 1. The presence of alpha 3 beta 1 was confirmed by Western blots of the OG extract probed with anti-alpha 3 or -beta 1 antibodies. Western blots also showed that an antibody originally raised against the 120-130 kDa peptide crossreacts with both the alpha 3 and beta 1 integrin subunits. Co-purification of alpha 3 beta 1 and Na(+)-dependent transport activity suggested that the two activities might be associated. Evidence that alpha 3 plays a role in transport is shown by the fact that an antibody against human alpha 3, but not beta 1, removed transport activity (approximately 25% loss) from cholate-solubilized Ehrlich membranes. Further purification of OG extracts using concanavalin A and wheat-germ lectin columns resulted in the separation of transport activity from the bulk (but not all) of alpha 3 beta 1 integrin without loss of the transport activity. These results indicate that the integrin itself is not essential for amino acid transport. Reconstitution of a purified alpha 3 beta 1-depleted protein fraction showed high levels of Na(+)-dependent, alpha-methylaminoisobutyric-acid-inhibitable amino acid transport in proteoliposomes, whereas reconstituted integrin alone showed little transport activity. However, in the integrin-depleted fractions, high amino acid uptake occurred in K+ which compromised the accurate measurement of the Na(+)-dependent component of uptake. The data suggest that alpha 3 may be associated with the A-system transporter and may modulate the activity of this carrier. Moreover, transfection of K562 and RD cells with human alpha 3 and alpha 2 cDNA showed that the former but not the latter increased A-system transport, thus providing more direct evidence that alpha 3 may modulate A-system transport activity.

Involvement of essential histidine residue(s) in the activity of Ehrlich cell plasma membrane NADH-ferricyanide oxidoreductase

The existence of histidine residue(s) implicated in the active site of NADH-ferricyanide oxidoreductase in plasma membrane vesicles isolated from Ehrlich ascites tumour cells is investigated. The shape of the pH-dependence curve of the enzyme activity suggests that one or more histidine residues are located at (or near) the active site of the enzyme. This hypothesis is supported by the following experimental data: the loss of activity after treatment with diethyl pyrocarbonate (DEPC) or photooxidation by using Rose bengal, and the strong inhibition caused by Zn 2+ ions at micromolar concentrations. The combined arguments support the statement that histidine plays an essential role in the catalytic activity of NADH-ferricyanide oxidoreductase from Ehrlich ascites tumour cells.

Two phases of ferricyanide reductase activity in Ehrlich cell plasma membranes.

Ehrlich cell plasma membrane vesicles have a ferricyanide reductase activity that shows two phases. These two phases were kinetically characterized. Evidence is presented for a differential effect of trypsin on both phases.

Role of specific acidic lipids on the reconstitution of sodium-dependent amino acid transport in proteoliposomes derived from Ehrlich cell plasma membranes

The effect of acidic phospholipids on the activity of a Na(+)-dependent amino acid transporter (A system) from Ehrlich ascites cell plasma membranes was examined. Plasma membranes were solubilized in cholate/urea and reconstituted with Ba2(+)-precipitated asolectin (soybean phospholipid free of anionic phospholipids) replenished with different acidic phospholipids. In the absence of added acidic phospholipids, transport activity was very low. However, three acidic lipids [cardiolipin greater than phosphatidic acid (PA) greater than phosphatidylinositol] were capable of restoring transport activity (in the order given) to proteoliposomes made from Ba2(+)-precipitated asolectin, while other acidic phospholipids (phosphatidylserine and phosphatidylglycerol) were much less active in this respect. For restoration of optimal activity, PA containing at least one unsaturated fatty acyl moiety, particularly in the beta position, was required. PA containing only saturated fatty acids in the beta and gamma positions was largely inactive. No difference in restoration of function was observed on varying the saturated fatty acyl chain length in PA from 10 carbons to 18 carbons. The specific effects of PA on the A-system transporter were not shared by the Na(+)-independent amino acid exchange system (L system) or the glucose transport system. Treatment with poly(ethylene glycol) 8000 was shown to reduce the nonspecific permeability of the reconstituted proteoliposomes and to enhance Na(+)-dependent amino acid transport.

Evidence for an essential sulfhydryl group at the substrate binding site of the A-system transporter of Ehrlich cell plasma membranes

Plasma membrane suspensions of Ehrlich ascites cells solubilized with cholic acid were used to study the effects of sulfhydryl reagents on Na(+)-dependent amino acid transport. These suspensions were treated with the sulfhydryl binding agents p-chloromercuribenzenesulfonic acid or N-ethylmaleimide prior to reconstitution for the assay of transport activity. The proteoliposomes formed from dissolved membranes treated with p-chloromercuribenzenesulfonic acid showed no Na(+)-dependent alpha-aminoisobutyric acid transport, while N-ethylmaleimide pretreated membranes retained approximately 90% of the original activity. To avoid interference by the N-ethylmaleimide component, further studies were carried out with membranes pretreated with 200 microM N-ethylmaleimide prior to p-chloromercuribenzenesulfonic acid treatment. A concentration of 25 microM p-chloromercuribenzenesulfonic acid inhibited Na(+)-dependent alpha-aminoisobutyric acid transport by 50%. The degree of inhibition was dramatically reduced in the presence of substrates specific for the A transport system. Using an inhibition index to address the efficacy of inhibition in presence and absence of substrates, it could be shown that an index of 1.0 in presence of p-chloromercuribenzenesulfonic acid was reduced to 0.84 with (methylamino)isobutyric acid alone and 0.05 in the presence of 100 mM Na+ and 5 mM (methylamino)isobutyric acid. Na+ alone offered no protection. The results show that sulfhydryl group(s) on the amino acid carrier may be directly involved in substrate binding and that substrate binding sites are functional in the disaggregated membrane state. Furthermore, Na+ directly affects (methylamino)isobutyrate binding, since the degree of protection by the amino acid analogue against p-chloromercuribenzenesulfonic acid inhibition was influenced by the presence of Na+.(ABSTRACT TRUNCATED AT 250 WORDS)

Simple and effective purification of a Na+-dependent amino acid transport system from Ehrlich ascites cell plasma membrane.

A reconstitution assay was used to measure transport activity during purification of a Na+-dependent amino acid transporter from Ehrlich cell plasma membrane. Cholate/urea-solubilized membranes were fractionated on a Sepharose 6B column and transport activity was recovered in the column void volume. Centrifugation of the void volume fraction at 105,000 X g and reextraction of the pellet with 1% octyl glucoside led to recovery of an extract whose specific transport activity was nearly 30-fold higher than that of the original solubilized extract with a recovery of 38% of the original activity. The properties of amino acid uptake in the purified reconstituted transporter were identical to those in native plasma membrane vesicles. The major component present in the purified fraction had a molecular mass of 120-130 kDa. Strong evidence that this 120- to 130-kDa peptide contains a component of the amino acid transporter was obtained by immunoprecipitation of transport activity from solubilized membranes with an antibody against the 120- to 130-kDa peptide. This study tentatively identifies a component of the Na+-dependent amino acid transporter as a peptide with an apparent molecular mass of 120-130 kDa.

Volume enlargement and recovery of Na+-dependent amino acid transport in proteoliposomes derived from Ehrlich ascites cell membranes.

Na+-dependent amino acid transport can be reconstituted from solubilized Ehrlich cell plasma membranes by addition of asolectin vesicles, gel filtration, and a freeze-thaw cycle. Removal of phosphatidic acid (approximately 10% of the total lipid) by Ba2+ precipitation reduces the efficiency of reconstitution of Na+-dependent amino acid transport by approximately 73% and decreases intravesicular volume of the proteoliposomes by approximately 43%. The loss of transport activity is not due to exclusion of specific proteins during reconstitution. The phosphatidic acid-free liposomes are less permeable and require more time to attain an equilibrium distribution of solute. Transport activity and intravesicular volume can be restored to Ba2+-precipitated asolectin proteoliposomes by addition of egg-phosphatidic acid during reconstitution. The extent of recovery of transport activity is proportional to the change in intravesicular volume and depends on the amount of phosphatidic acid present. Replacement of phosphatidic acid with 20% phosphatidylserine or phosphatidylglycerol leads to increases in intravesicular volume with little or no increase in amino acid transport. Generation of phosphatidic acid in situ by treatment of Ba2+-precipitated proteoliposomes with phospholipase D also restored transport. The observed increase in transport activity (9-fold) is accompanied by a 46% increase in intravesicular volume, presumably caused by vesicle fusion. Phosphatidic acid is also required for successful reconstitution of Na+-dependent amino acid transport from pure phosphatidylcholine:phosphatidylethanolamine (1:1) mixtures with only a small change (approximately 16%) in intravesicular volume. The results provide evidence for both indirect and direct effects of phosphatidic acid on reconstitution of Na+-dependent amino acid transport. The indirect effects occur through enlargement of intravesicular volume, large vesicles showing higher rates of transport. However, there is also evidence to indicate a specific effect of phosphatidic acid on the Na+-dependent amino acid transporter, since other acidic lipids may change intravesicular volume without a commensurate change in transport activity.

Asymmetric reconstitution of the glucose transporter from Ehrlich ascites cell plasma membrane: Role of alkali cations

Gel chromatography of solubilized Ehrlich cell plasma membranes and preformed asolectin vesicles coupled to a freeze-thaw cycle results in the reconstitution of 3- O methyl- d-glucose transport. The transport activity of the liposomes formed is critically dependent on the cation present during reconstitution. Liposomes formed in K + show high levels of carrier-mediated 3- O-methyl- d-glucose uptake (495 pmol/min/mg protein) while those formed in Na + do not (33 pmol/min/mg protein). The inactivity in Na + is not due to a diminished incorporation of glucose transporter nor is it due to carrier molecules reconstituted with a different orientation from those in K + liposomes. Instead, the low glucose transport level in Na + liposomes is related to the small size of vesicles formed with Na +. A second freeze-thaw cycle in K + causes a two- to threefold increase in the available intravesicular volume of Na + liposomes and results in an eightfold increase in carrier-mediated 3- O-methyl- d-glucose uptake. K + liposomes, treated in an identical manner, show only a twofold increase in uptake. The glucose transporter was identified as a protein with a molecular mass range of 44.7 to 66.8 kDa, by the d-glucose inhibitable photoincorporation of [ 3H]cytochalasin B. The carrier protein is inserted in reconstituted vesicles in a nonrandom manner with at least 80% of the molecules oriented with the cytoplasmic domain accessible to the external medium. In contrast, the neutral Na +-dependent amino acid transport system appears to be randomly reconstituted.

A simple and efficient method for reconstitution of amino acid and glucose transport systems from Ehrlich ascites cells

Solubilized Ehrlich cell plasma membrane proteins were incorporated into lipid vesicles in the presence of added phospholipid, using Sephadex G-50 chromatography combined with a freeze-thaw step. Liposomes formed in K + exhibited high levels of Na +-dependent, α-aminoisobutyric acid uptake which was electrogenic and inhibited by other amino acids. The transport activity reconstituted was similar to that observed in native plasma membrane vesicles. In addition to transport by system A, leucine exchange activity (system L), Na +-dependent serine exchange activity (system ASC), and stereospecific glucose transport activity were also reconstituted. The latter was inhibited by d-glucose, d-galactose, cytochalasin B, and mercuric chloride. The medium used for reconstitution was critical for the recovery of Na +-dependent amino acid transport. The use of Na + in the reconstitution procedure led to formation of liposomes which displayed little Na +-dependent and gradient-stimulated amino acid uptake. In contrast, all transport activities studied were efficiently reconstituted in K + medium.

Occurrence of alpha-D-galactosyl-containing glycoproteins on Ehrlich tumor cell membranes.

Ehrlich ascites tumor cells were strongly agglutinated by 0.4 micrograms/mL Griffonia simplicifolia I-B4 isolectin (GS I-B4), indicating the presence of nonreducing terminal alpha-D-galactopyranose (alpha-D-Galp) residues on the cell surface. Incubation of the cells with GS I-B4 labeled with either fluorescein isothiocyanate (FITC-B4) or ferritin followed by examination with the light and electron microscope revealed a random distribution of alpha-D-Galp residues over the entire cell surface. Cell-binding studies with [3H]propionate-labeled GS I-B4 demonstrated a minimum of 18.1 X 10(6) alpha-D-Galp sites per Ehrlich cell. An enriched Ehrlich cell plasma membrane preparation subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with FITC-B4 revealed a number of alpha-D-galactosyl-containing glycoproteins ranging in molecular weight from 50 000 to over 200 000. The major plasma membrane glycoprotein of the Ehrlich cell (GP 130) was isolated in near homogeneous form by using nonionic detergent extraction, affinity chromatography over GS I-Sepharose 4B, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Injection of Ehrlich cells into the mouse peritoneal cavity stimulated the appearance of low levels of alpha-D-galactosyl-containing glycoproteins in the ascites fluid ranging in molecular weight from 34 000 to 260 000. These glycoproteins differed in molecular weight when compared to the alpha-D-galactosyl-containing glycoproteins observed in either ascites fluid induced with Freund's complete adjuvant or the glycoproteins in the Ehrlich cell plasma membrane.