Modification of system A amino acid carrier by diethyl pyrocarbonate

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THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 2, Issue of January 15, pp. 798-802, 1991 Printed in U.S.A.

Modification of System A Amino Acid Carrier byDiethyl Pyrocarbonate” (Received for publication, September 11,1990)

Joan Bertran$, Angela Roca, Emilia Pola, XavierTestar, Antonio Zorzano, and Manuel Palacing From the Ilepartamentde Bioquimica i Fisiologia, Facultat deBiologia, Universitat de Barcelona, A d a . Diagonal 645, 08028 Barcelona, Spain

Sodium-dependentalaninetransportinplasma by high concentrations of 2-(methy1amino)isobutyricacid (3, membrane vesicles from rat liver was inactivated ina 8). time- and concentration-dependent fashion by prior There is a paucity of data regarding the molecular propertreatment of membranes with the acylating reagent ties of amino acid transport systems A and ASC. Very rediethyl pyrocarbonate (DEPC).Bothcomponents of cently, a 120-130-KDa peptide has beenidentified as a putaNa+/alaninecotransport(systems A and ASC) were tive component of system A carrier activityin Ehrlich ascites inhibited.Exposure of vesiclestop-bromophenacyl cell plasma membranes (9). There evidence is that the system bromide and methyl p-nitrobenzenesulfonate, which A carrier is a glycoprotein (10, 11); andlike the system ASC share withDEPC reactivity against histidine residues, carrier, it shows sensitivity to sulfhydryl-preferring reagents also led to inhibitionof alanine transport throughsys- (12-16). Besides differences in substrate specificity and modtems A and ASC. The presence of Na+ (100 mM NaCl) and L-alanine (10 mM) during exposure of vesicles to ulation of their activities, the only structural difference found different DEPC protected against inactivation of system A (but between system A and ASC carriers is related to the substrate requirements to protect both systems against inacnot system ASC) transport activity. This protective effect was specific and required the presence of L- tivation by sulfhydryl reagents (16). A striking functional differencebetween systems A and alanine since the presence of L-phenylalanine alone (10 mM) or L-phenylalanine plus Na+ (100 mM NaCl) ASC is the strong pH dependence of system A activity (12, 17, 18). Inhepaticplasmamembrane vesicles, sodium-dedid not cause any detectable protection. This overall and reaches pattern of protection is opposite to that previously pendent alanine transport is inactivated at6.0pH foundagainst specific sulfhydrylreagents (i.e. N- maximal activity aroundpH 8.0 (19). This pH dependence of ethylmaleimide), where protection of system ASC was system A activity points to the existence of amino acid resinearly maximal. The pH profile for DEPC-dependent due(s) in the carrier witha pK, of -7, suggesting the particiinhibition of system A transport activity suggests mod- pation of histidyl residue(s). To gain informationonthe ification of amino acid residue(s) with a pK,. of -7, structural basis of the pH dependence of system A activity most likely histidine(s), in close parallel with the pH and, ingeneral, onthestructures of system A and ASC dependence of system A transport activity. Our resultscarriers, we have investigated the sensitivity of both systems suggest the presence of critical histidine residues on to modification mediated by three different histidine-modithe system A carrier that may be responsible for the fying reagents. Protection obtained with substrates provides pH dependence of system A transport activity. direct evidence for binding of diethyl pyrocarbonate (DEPC)’ to the system A carrier. Furthermore, the clear pH dependence of DEPC-mediated system A carrier modification strongly suggests that residue(s) modified by DEPC, probably histiIn hepatocytes, neutral amino acids are taken up primarilydine, are involved in the pH dependenceof system A carrier through systems A, ASC, and L (1-3). Transport by system activity. A isNa+-dependentandtranslocatesshortpolar,straight chain amino acids, including the nonmetabolizable analog 2EXPERIMENTALPROCEDURES (methy1amino)isobutyric acid. System A transport activity is Membrane Vesicle Preparation-Plasma membrane vesicles were subjected to hormonal regulation, trans-inhibition, and adaptative regulation in a variety of cell types (4-7). System prepared from the livers of 200-300-g male Wistar rats fed ad libitum according to the method of van Amelsvoort et al. (20) modified as ASC also shows Na’ dependence, and it issubjected to trans- describedelsewhere (16). Liversfrom fed rats were minced in 5 stimulation (1, 8 ) ; this system carries neutral amino acids volumes of cold 0.25 M sucrose, 0.2 mM CaCb, 10 mM Hepes, pH 7.5 with small side chains, especially those with an -OH or -SH (buffer A). After homogenization and filtration through nylon cloth, group, excluding N-methylated amino acid derivatives. The homogenates were diluted 1:3 with 0.25 M sucrose, 0.2 mMCaCIZ, 1 sodium-dependent transport of alanine is due to the activity mM EDTA, 10 mM Hepes, pH 7.5 (buffer B). The homogenate was centrifuged at 30,000 X g for 20 min, and pellets were resuspended in of systems A and ASC, the former component being inhibited buffer B and centrifuged a t 700 X g for 10 min. The supernatant was * This work was supported in part by Research Grant P B 573-86 from the Direccibn General deInvestigacibnCientificay Tkcnica, Spain. The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore hereby be marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $Recipient of a predoctoral fellowship from the “Ministerio de Educacibn y Ciencia,” Spain. To whom correspondence shouldbe addressed.

collected, and the pelletwas resuspended in buffer B and centrifuged as before. The supernatants from the two 700 X g centrifugations were pooled (typically 48 ml), mixed with 6 ml of isotonic Percoll (90% Percoll, 0.25 M sucrose, pH 7.5), and centrifuged a t 30,000 X g The abbreviations used are: DEPC, diethyl pyrocarbonate; Hepes, N-2-hydroxyethylpiperazine-N’-2-etbanesulfonic acid; Pipes, piperazine-N,N”bis(2-ethanesulfonic acid); Hepps, N-2-hydroxyethylpiperazine-N’-3-propanesulfonicacid.


Binding of Diethyl Pyrocarbonate to System A


supplemented with histidine required block to the excess of unreacted histidine-modifying reagent. Vesicles were immediately centrifuged at 30,000 X g for 20 min, washed again in buffer B, and centrifuged again a t 30,000 X g for 10 min,which results in 1,000-fold dilution of chemicals present during the modification reactions. Finally, vesicles were resuspended in buffer A and immediately used for transport measurements. Transport Assay and DatuAnalysis-Vesicle aliquots andreaction mixtures were preincubated at 26 "C for 5 min. To assay total alanine transport, membrane vesicles (10 pl, 30-40 pg of protein) were incu0 05 1 10 Time (min) bated in a medium (final volume of 40 pl) containing 0.25 M sucrose, 0.2 mM CaC12, 10 mM MgC12, 100 mM NaSCN, 0.2 mM ~ - [ 2 , 3 - ~ H ] alanine (specific activity of 0.126 pCi.nmol"), 10 mM Hepes/KOH, pH 7.5. Alanine uptake was terminated by adding 1 ml of ice-cold 0.25 M sucrose, 0.2 mM CaC12, 100 mM NaCl, 10 mM Hepes, pH 7.5 (buffer C).The diluted membraneswere immediately filtered through nitrocellulose filters (pore size of 0.45 pm, diameter of 25 mm). Filters were washedonce with 4 ml of ice-cold buffer C and placed in scintillation vials containing 6 ml of scintillation mixture, andradioactivity was measuredin aliquid scintillationspectrometer.The nonspecific binding of radioactivity to the filters in the absence of vesicles was
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