Cupric ion-mediated active transport of amino acids in membrane vesicles of Mycobacterium phlei.

In the absence of substrate oxidation, membrane vesicles of Mycobacterium phlei were shown to accumulate proline, glutamine, and glutamic acid mediated by Cu”+. The Cu’+- mediated uptake of amino acids was found to be an active process and required the presence of amino acid binding protein(s). Thus, membrane vesicles which lack active transport of a particular amino acid because of the loss of specific binding protein(s) do not accumulate these amino acids even in the presence of Cu+. The Cu’+-mediated uptake of proline exhibited a specific requirement for Na+. The Cu”--mediated uptake process was not inhibited by anaerobiosis and respiratory inhibitors, such as sodium azide and m-chlorocarbonylcyanide phenylhydrazone. How- ever, atebrin and o-phenanthroline were found to inhibit the uptake process. The results with various sulfhydryl blocking agents suggests that oxidation of available sulfhy- dry1 residues of the membrane protein(s) is required for the Cu”+-mediated uptake process. The uptake of amino acid was inhibited by p-chloromercuribenzene sulfonic acid or X-ethylmaleimide when added prior to the addition of Cu’+. In addition, efflux of amino acids, previously accumulated to the steady state level occurred when sulfhydryl reducing agents such as glutathione, cysteine, or dithiothreitol were added. Evidence is presented that disulfide configuration of the carrier or membrane protein(s) favors the transport of amino acids against the concentration gradient, whereas the sulfhydryl state of these proteins induces the efflux process. Studies show that there was 1:l stoichiometry for the oxidation of available sulfhydryl residues in the mem- brane to the reduction of Cu”, and the proton gradient generated as a result of the oxidation of sulfhydryl groups is presumably the driving force for the uptake of these amino acids. Numerous studies have been carried out to delineate the * This work was supported by grants from the National Science Foundation (BM572-018961, the National Institutes of Health, United States Public Health Service (AI 056371, the Hastings Foun- dation at the University of Southern California, School of Medicine and Bioscience Laboratories fellowship to A. J. J. The costs of publication of this article were defrayed in

[Y!lglutamine, and 1'4Clglutamic Acid Transport-The method for measuring transport of proline, glutamine, and glutamic acid in the membrane vesicles was essentially the same as described in earlier publications (13,14). The 1.5-ml assay system contained 25 mM potassium/4-(2-hydroxyethylj-l-piperazineethanesulfonic acid buffer (pH 7.0), 5 mM MgCl,, 10 mM NaCl, and 10 PM '%-labeled amino acids. Assay of Transport under Anaerobic Conditions-To study the cupric ion-stimulated transport of proline, glutamine, and glutamic acid under anaerobic conditions, Thunberg tubes were used. Oxygen was removed from the reaction system with a vacuum pump and the sealed environment was saturated with argon gas. This was repeated three times to ensure complete anaerobiosis. The reaction was initiated by the addition of the indicated amount of 14C-labeled amino acids from the side arm. The tubes were incubated in a shaking water bath at 30" for various time intervals, and at the end of the indicated time interval, the incubation mixture was diluted 10 times with 0.05 M potassium phosphate buffer, which was deoxygenated (pH 7.01, and filtered through a millipore filter, as described previously. Kinetics Vesicles-The uptake of proline, glutamine, and glutamic acid in the membrane vesicles of M. phlei has been shown to require substrate oxidation and to proceed against a concentration gradient (14). Of the various substrates used, ascorbate-TPD was found most effective in supporting the uptake of these amino acids, following in decreasing order of effectiveness were exogenous NADH, generated NADH, and succinate. However, no correlation between the rate of substrate oxidation and the level of amino . . acid accumulation has been observed (13, 14). As shown in Fig. 1, the addition of Cu'+ in the absence of exogenous substrate stimulated the uptake of proline, glutamine, and glutamic acid in the membrane vesicles of M. phlei. The Cut+-stimulated uptake of these amino acids was found to be an active process since there was a 6-to lo-fold concentration gradient established. The intravesicular space of membrane vesicles used for the calculation of concentration gradient was 1.7 pl/mg of membrane protein as described earlier (11). The amino acids accumulated in membrane vesicles were found not to be chemically modified or incorporated into proteins. Cupric sulfate and cupric chloride were found to stimulate the uptake of amino acids to the same extent with maximum effect at 150 FM. Cupric gluconate showed maximum effect at 105 PM. However, cuprous salts were ineffective.
Cupric ion-mediated uptake of amino acids other than glutamine, glutamic acid, and proline was also examined in these membrane vesicles. It was observed that the other amino acids, e.g. leucine, were not actively accumulated into the membrane vesicles by cupric ions (Fig. 1 The uptake of proline, glutamine, and glutamic acid mediated by Cr.?+ was found to be insensitive to various inhibitors such as azide and arsenate (Table I). The uptake was sensitive to cyanide (10 mM), but this inhibition was probably due to the known chemical interaction of cupric ions with cyanide (21). Irradiation of membrane vesicles at 360 nm has earlier been shown to inactivate the natural quinone MK-9 (II-H) and a light-sensitive component of the respiratory chain (25). The irradiated membrane vesicles are unable to oxidize succinate and NAD+-linked substrates (25). As shown in Table I, the cupric ion-stimulated uptake of proline, glutamine, and glutamic acid remained unaffected in irradiated membrane vesicles. However, the uptake of these amino acids was inhibited by atebrin or o-phenanthroline (Table I), which have been shown to block electron flow at the non-heme iron level in the respiratory chain of M. phlei (26). At the steady state levels of accumulated amino acids, the addition of atebrin or ophenanthroline elicited a rapid efIlux of these amino acids (Fig. 3). This may suggest that the effect of CL?++ occurs before the naphthoquinone (MK-9(11-H)) level, but after or at the flavoprotein-non-heme iron level of the respiratory chain. It is conceivable that o-phenanthroline may form a complex with CL?+; however, studies have shown that the Cu'+.ophenanthroline complex formation does not occur to any significant extent at pH 7.4 which was used in these studies. The Cu*++o-phenanthroline complex requires a high pH and can be detected by spectral shift. Moreover, the Cu2+.o-phenanthroline complex formation was observed only at a high concentration of CL?+ (3 mM). This concentration of Cu*+ is higher than that used in these studies.
Effect of Anaerobiosis on Transport -Studies with various respiratory inhibitors showed that the complete respiratory chain was not required for the cupric ion-stimulated uptake of amino acids. It was, therefore, of interest to determine whether oxygen itself was an obligatory requirement for the Cuz+-stimulated uptake. As shown in Fig. 1, the cupric mediated uptake of proline, glutamine, and glutamic acid was insensitive to anaerobiosis. It should be pointed out, however, that the presence of Cu"+ did inhibit succinate and NAD+linked oxidation in these membrane vesicles. Effect of Sulfhydryl Inhibitors -CL?+ is a known oxidizing agent of the sulfhydryl groups of various proteins. The possibility that the oxidation of sulfhydryl groups of membrane proteins was associated with the stimulated uptake of proline, glutamine, and glutamic acid by CL?+ was ascertained by using various sulthydryl blocking reagents. Membrane vesicles were incubated with p-CMBS or NEM prior to the addition of Cu'+ in order to block the available sulfhydryl groups; the transport of proline, glutamine, and glutamic acid in the presence of cupric chloride was then assayed. As shown in Fig. 2, sulfhydryl reagents inhibited the uptake of Cu*+-stimulated transport of proline (75%), glutamine (80%), and glutamic acid (85%). The inhibition by sulfhydryl reagents lowered the level of transport to about the level observed in the absence of substrate or Cu2+.
Effect of Cupric Ion on Sulfiydryl Residues -Studies were carried out to determine the total number of accessible sulfhy-dry1 groups in the membrane vesicles in the presence and x-x, amino acid transport in the presence of 150 PM CuClz and 100 PM NEM; 0. . . . 0, amino acid transport in the presence of 150 pM CuCl, and 79 pM p-CMBS. absence of Cu"+. It was observed that the total number of DTNB-reactive sulfhydryl residues was reduced by 50% in the presence of Cu2+ (Table II) under both aerobic and anaerobic conditions. Moreover, the number of DTNB-reactive sulfhydryl groups was increased (35%) ( Table II) when ophenanthroline was added at the steady state level of accumulation of amino acids, which has been shown to result in the efflux of amino acids (Fig. 3).
Since the Cu'+-stimulated uptake of proline, glutamine, and glutamic acid in membrane vesicles was seen to be associated with the oxidation of sulfhydryl groups of carrier or membrane protein(s), it was of interest to determine the effect of sulfhydryl reducing agents. Accumulated amino acids at steady state levels in membrane vesicles were efIluxed by reducing agents in the following order: dithiothreitol > glutathione > cysteine (Fig. 3).

Kinetics of Cupric
Ion-stimulated Transport -The concen- The sulfhydryl content of membrane vesicles and trichloroacetic acid-treated membrane vesicles in the presence and absence of Cu*+ was determined as described in the legend to 10.5 acid-treated membrane vesicles tration of Cu2+ ions necessary for maximal initial rate and the steady state levels of transport was found to be 150 pM. Higher concentrations of Cu*+ ions were found to inhibit amino acid uptake. Varying the concentration of Cu*+ did not affect the K,, value for proline, glutamine, and glutamic acid, but it did change the V,,, values ( Fig. 4 A to C). It is pertinent to mention that amino acids form a complex with Cu2+ (27) with a high stability constant (Cu*+-proline p2 = 16.58; Cu*+-glutamine pz = 14.20; Cu*+-glutamic acid fiz = 16.401, yet the K, values for the uptake of these amino acids mediated by Cu*+ remained unaltered. This may suggest that the Cu*+-mediated transport of these amino acids may occur as the Cu*+ . amino acids complex forms. Nevertheless, it should be noted that other amino acids with similar /$ constants, such as leucine 15.60, histidine 18.53, and lysine 13.90, are not transported in these vesicles by Cu2+. The K, values of proline (4 PM), glutamine (3.85 PM), and glutamic acid (4.25 PM) were similar to the K,,, values observed for these amino acids in the presence of succinate and NAD+linked substrates (13, 14).

Requirement
of Na+ for Proline Transport Mediated by C&+-The uptake of proline, in contrast to glutamine and glutamic acid, in membrane vesicles mediated by succinate and NAD+-linked substrates has been shown to have an absolute requirement for Na+ (12). It was observed that Cu2+mediated uptake of proline as compared to glutamine and glutamic acid also exhibited a requirement for Na+. Varying the concentration of Na+ did not affect the K,,, value for proline (4.2 rt 0.2), but it did change the V,,, value (Fig. 5). Effect of Temperature on Cu '+-stimulated Transport -The rate of enzymatic reactions is characteristically dependent upon temperature.
Hence, the rate and steady state levels of proline, glutamine, and glutamic acid were examined as a function of temperature.
The studies were performed with membrane vesicles at 4", lo", 20", 30", 40", and 50" in the presence of Cu2+. Both the rate of uptake and the steady state levels of transport increased from O-40" (data not shown); above 40" transport activity was slightly decreased. The temperature coefficient Q10 from 4-40" for the uptake of proline, glutamine, and glutamic acid mediated by Cu2+ was found to be in the range of 1.6 to 2.0, suggesting that the process is due to enzymatic catalysis (Table III).

Rate of Cupric
Ion Reduction by Membrane Vesicles -Since the sulfhydryl contents of membrane proteins decreased upon the addition of Cu2+, it was of interest to determine whether there was an accumulation of cuprous ion during this oxida-