The effects of N-ethylmaleimide on active amino acid transport in Escherichia coli

N-Ethylmaleimide (MalNEt) binds covalently and without specificity to accessible sulfhydryl residues in proteins. In some cases specificity has been imposed on this reaction by manipulating reaction conditions, yielding information concerning both enzyme mechanism and the identity of specific proteins (for example C.F. Fox and E.P. Kennedy (1965) Proc. Natl. Acad. Sci. U.S. 54, 891–899) and R.E. McCarty and J. Fagan (1973) Biochemistry 12, 1503–1507). We have examined the effects of MalNEt on the active accumulation of nine amino acids by Escherichia coli strains ML 308-225 and DL 54. Whole cells have been used in order that transport systems both dependent on and independent of periplasmic binding proteins could be studied under various conditions of energy supply for transport. Our results suggest that the systems transporting ornithine, phenylalanine and proline are those most likely to undergo inactivation by direct reaction of MalNEt with the transport apparatus, rather than merely via side effects such as interruption of their energy supply. The inhibition of proline transport is specifically enhanced by the presence of proline, competitive inhibitors of proline transport, or carbonylcyanide p-trifluoromethoxyphenylhydrazone during MalNEt treatment. The other eight systems tested showed no analogous effects.     

Proline porters effect the utilization of proline as nutrient or osmoprotectant for bacteria

Proline is utilized by all organisms as a protein constituent. It may also serve as a source of carbon, energy and nitrogen for growth or as an osmoprotectant. The molecular characteristics of the proline transport systems which mediate the multiple functions of proline in the Gram negative enteric bacteria, Escherichia coli and Salmonella typhimurium, are now becoming apparent. Recent research on those organisms has provided both protocols for the genetic and biochemical characterization of the enzymes mediating proline transport and molecular probes with which the degree of homology among the proline transport systems of archaebacteria, eubacteria and eukaryotes can be assessed. This review has provided a detailed summary of recent research on proline transport in E. coli and S. typhimurium; the properties of other organisms are cited primarily to illustrate the generality of those observations and to show where homologous proline transport systems might be expected to occur. The characteristics of proline transport in eukaryotic microorganisms have recently been reviewed (Horak, 1986).     

Location of the sites of reaction of N-ethylmaleimide in papain and chymotryptic fragments of the gizzard myosin heavy chain

The thiol of the gizzard myosin heavy chain, which reacts most rapidly with N-ethylmaleimide (MalNEt), has been located in the subfragment 2 region of myosin rod by fragmentation of [14C]-MalNEt-labeled myosin with papain and chymotrypsin. MalNEt reacts more slowly with thiols present in the 70- and 25-kilodalton (kDa) papain fragments of subfragment 1. The reaction of MalNEt with thiols present in these regions is increased on addition of ATP by factors of 2 and 10, respectively, when myosin is modified in 0.45 M NaCl where it is present in the extended, 6S conformation. The rate of increase of Mg2+-activated adenosinetriphosphatase (ATPase) activity, which reflects the loss of ability of myosin to assume the folded, 10S conformation, and the rate of loss of K+-EDTA-activated activity produced by MalNEt are both accelerated 5- to 10-fold on addition of ATP. The rates at which ATPase activities change agree closely to the reaction rates of MalNEt with the 25-kDa region of subfragment 1; therefore, the changes in these activities can be attributed to modification of a thiol of the 25-kDa segment. An increase in actin-activated ATPase activity produced by reaction of myosin with MalNEt in 0.45 M NaCl is accelerated by ATP by a factor of at least 4. Reaction with [14C]MalNEt in the presence of MgATP and 0.2 M NaCl, where myosin is in the 10S form, inhibits the incorporation of radioactive MalNEt into the 25-kDa papain fragment of subfragment 1. It also prevents the increase in actin-activated ATPase activity and preserves the ability of myosin to assume the 10S form.     

Identification of a PutP proline permease gene homolog from Staphylococcus aureus by expression cloning of the high-affinity proline transport system in Escherichia coli.

The important food-borne pathogen Staphylococcus aureus is distinguished by its ability to grow at low water activity values. Previous work in our laboratory and by others has revealed that proline accumulation via transport is an important osmoregulatory strategy employed by this bacterium. Furthermore, proline uptake by this bacterium has been shown to be mediated by two distinct transport systems: a high-affinity system and a low-affinity system (J.-H. Bae, and K. J. Miller, Appl. Environ. Microbiol. 58:471-475, 1992; D. E. Townsend and B. J. Wilkinson, J. Bacteriol. 174:2702-2710, 1992). In the present study, we report the cloning of the high-affinity proline transport system of S. aureus by functional expression in an Escherichia coli host. The sequence of the staphylococcal proline permease gene was predicted to encode a protein of 497 amino acids which shares 49% identity with the PutP high-affinity proline permease of E. coli. Analysis of hydropathy also indicated a common overall structure for these proteins.     

Membrane transport in Schistosoma mansoni: transport of amino acids by adult males.

The mechanisms by which adult male Schistosoma mansoni transport amino acids have been investigated using radioactive amino acids during 2-min incubation times. The transport constants (K_t) for mediated uptake of glycine, proline, methionine, arginine, glutamate, and tryptophan were calculated to be 0.60-1.05, 1.67-1.98, 2.0, 0.10-0.35, 0.30-0.50, and 0.5-1.0 mM, respectively. Maximal velocities (V_max) were 5.5–7.5, 25, 6.4, 1.5-2.0, 2.5, and 3.0–6.0 μmoles absorbed/g worm protein/2 min, respectively. Cysteine is taken up solely by diffusion. Proline uptake is unique in that no significant diffusion component was found. The other amino acids studied were absorbed by diffusion as well as by specific transport systems. In the 2-min incubation periods employed glycine, proline, glutamate, and methionine were not significantly metabolized indicating that the uptake studies using these substrates reflect transport. Metabolism of the other amino acids used in these studies was not examined. The specificity of the transport systems was studied by testing the inhibitory effects of various amino acids on the uptake of each of the amino acids studied. The results suggest the presence of at least five transport systems. There is a highly specific transport locus for proline, and one for acidic amino acids. There are probably at least two transport systems, each of broad and overlapping specificity, for most of the neutral amino acids. Basic amino acids also appear to be taken up by complex transport systems, at least one of which overlaps with the neutral sites. The results are discussed with respect to the nutrition of the parasite and the host-parasite relationship.     

Comparative study on effect of N-ethylmaleimide on the superoxide-generating system of guinea-pig eosinophils stimulated by soluble stimuli

1. The effect of N-ethylmaleimide (MalNEt) modification on O2- production by guinea-pig eosinophils mediated by different soluble stimuli was studied. 2. MalNEt pretreatment inhibited the O2- production stimulated by concanavalin A (Con A), cytochalasin E or digitonin, but not A23187 or sodium fluoride. 3. Particulate fractions from MalNEt-pretreated eosinophils before exposure to the stimulus showed the inhibition of the enhancement of NADPH-dependent O2- production induced by Con A, cytochalasin E or digitonin, but not A23187. 4. Treatment of eosinophils with MalNEt after stimulation had no effect on the NADPH oxidase activity. 5. These findings suggest that at least two pathways exist for the activation of the O2(-)-generating enzyme system, probably the NADPH oxidase system, in guinea-pig eosinophils.     

Sulfhydryl groups of the uncoupling protein of brown adipose tissue mitochondria. Distinction between sulfhydryl groups of the H+ channel and the nucleotide binding site

Mersalyl, 5,5'-dithio-bis(2-nitrobenzoate) (Nbs2) and fluorescent Thiolyte DB react with SH groups in the H+ channel (SHc) of the uncoupling protein of brown adipose tissue mitochondria, as inferred from their inhibition of H+ transport. Cl- transport by the uncoupling protein was unaffected. Using these modifiers and N-ethylmaleimide (MalNEt), distinct SH groups (SHB) in the purine nucleotide binding site were identified. Nbs2 reacts more readily with the SHB than with the SHc groups, but mersalyl and Thiolyte DB are more reactive with the SHc groups. MalNEt reacts exclusively with the SHB. GDP inhibition is fully prevented after sufficient modification of the SHB. Pretreatment with p-diazobenzenesulfonate (N2PhSO2) suppresses only 20-25% of fluorescence of Thiolyte-DB-labeled uncoupling protein on SDS/PAGE gels, while MalNEt suppresses 66% and Nbs2 80-90%. Since N2PhSO2 also affects the GDP binding site, these results demonstrate that the N2PhSO2-reactive residue is not identical with the SHB.     

Characteristics and osmoregulatory roles of uptake systems for proline and glycine betaine in Lactococcus lactis.

Lactococcus lactis subsp. lactis ML3 contains high pools of proline or betaine when grown under conditions of high osmotic strength. These pools are created by specific transport systems. A high-affinity uptake system for glycine betaine (betaine) with a Km of 1.5 microM is expressed constitutively. The activity of this system is not stimulated by high osmolarities of the growth or assay medium but varies strongly with the medium pH. A low-affinity proline uptake system (Km, > 5 mM) is expressed at high levels only in chemically defined medium (CDM) with high osmolarity. This transport system is also stimulated by high osmolarity. The expression of this proline uptake system is repressed in rich broth with low or high osmolarity and in CDM with low osmolarity. The accumulated proline can be exchanged for betaine. Proline uptake is also effectively inhibited by betaine (Ki of between 50 and 100 microM). The proline transport system therefore probably also transports betaine. The inhibition of proline transport by betaine results in low proline pools in cells grown in high-osmotic-strength, betaine-containing CDM. The energy and pH dependency and the influence of ionophores on the activity of both transport systems suggest that these systems are not proton motive force driven. At low osmolarities, proline uptake is low but significant. This low proline uptake is also inhibited by betaine, although to a lesser extent than in cells grown in high-osmotic-strength CDM. These data indicate that proline uptake in L. lactis is enzyme mediated and is not dependent on passive diffusion, as was previously believed.     

A novel type of coupling between proline and galactoside transport in Escherichia coli.

Exit of thiomethylgalactoside (TMG) from preloaded cells induced the accumulation of proline. Likewise, proline exit stimulated TMG accumulation. Since a proton ionophore (carbonylcyanide-m-chlorophenylhydrazone) abolished these effects, a protonmotive force was implicated as the "intermediate" in the coupling reaction. The evidence suggests that the exit of TMG resulted in proton exit, which produced either a membrane potential (inside negative or a pH gradient (outside acid) or both. This inwardly directed protonmotive force provided the energy for proline entry and accumulation. Thus the energy coupling was not via a common transport protein but by proton movements which coupled the two separate H+-dependent transport processes.     

Active transport of proline by Coxiella burnetii

The obligate intracellular rickettsia, Coxiella burnetii, was shown to possess an energy dependent proline transport system which displayed a high degree of specificity and was highly dependent on pH. Transport was maximal at pH 3.0 to 4.5, a pH range approximately that of the host cell phagolysosome where the agent replicates. Transport was inhibited by the uncouplers carbonyl cyanide m-chlorophenylhydrazone and dinitrophenol, but not by sodium arsenite. In the presence of glutamate, a preferred energy source, proline uptake was enhanced more than two-fold. This enhancement of proline uptake was greatly decreased in the presence of sodium arsenite. The addition of glutamate decreased the apparent Km for proline transport from 45 microM to 15 microM, with the Vmax increasing from 3.6 pmol s-1 (mg dry wt)-1 to 4.8 pmol s-1 (mg dry wt)-1. Two proline analogues, furoic acid and azetidine-2-carboxylic acid, were effective inhibitors of proline transport. D-Proline, 4-hydroxyproline, glycine and proline amide inhibited transport minimally, while no inhibition was seen with succinate, pyruvate or glutamate.     

Reactivity of the sulphydryl groups of the mitochondrial phosphate carrier

The rate of reaction of - SH groups of the mitochondrial phosphate carrier with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) and N-ethylmaleimide (MalNEt) was followed by measuring the inhibition of phosphate transport. The changes in the rate of reaction caused by alterations of the ionic composition of the matrix were compared with changes of the total intramitochondrial phosphate content, the intramitochondrial K+ content and the value of intramitochondrial pH. The ionic composition was manipulated by addition of valinomycin to non-respiring or to respiring mitochondria and by addition of inorganic phosphate to respiring and non-respiring mitochondria. From all these variables it was the changes of the intramitochondrial pH which correlated with the - SH group reactivity. Internal acidification decreased and internal alkalinization increased the rate of reaction of mitochondrial phosphate carrier with both Nbs2 and MalNEt. Nbs2 did not penetrate the inner mitochondrial membrane as assayed by determination of the acid-soluble thiol content of the matrix. From this fact it follows that the Nbs2-reactive SH groups of the carrier were accessible from the outer surface of the inner membrane in our experiments. It is concluded that intramitochondrial pH modifies the reactivity of the externally oriented - SH groups indirectly. A hypothesis is presented according to which protonation and deprotonation of the carrier molecule on the inner side could induce a conformational change of the whole protein altering also the microenvironment of the - SH groups near the opposite surface.     

Construction and properties of bifunctionally active membrane-bound fusion proteins. Escherichia coli proline carrier linked with beta-galactosidase

For construction of bifunctionally active membrane-bound fusion proteins, we designed plasmids encoding fusion proteins in which the carboxyl terminus of Escherichia coli proline carrier was joined to the amino terminus of E. coli beta-galactosidase directly or with a collagen linker inserted between the two. The expressions of these fusion proteins complemented deficiencies in both proline transport and beta-galactosidase activity in E. coli cells. The fusion proteins were stable and mostly localized in the cytoplasmic membrane. The proline transport activities of the fusion proteins were kinetically similar to that of the wild type proline carrier. The beta-galactosidase moiety of the collagen-linked fusion protein was liberated from membrane vesicles by collagenase treatment. The Km value of released beta-galactosidase for o-nitrophenyl beta-D-galactopyranoside hydrolysis was similar to that of membrane-bound beta-galactosidase in the fusion protein. These results indicated that the fusion proteins are bifunctionally active and exhibit normal proline transport and beta-galactosidase activities. The crypticity of the beta-galactosidase activity associated with the fusion proteins indicated that the carboxyl terminus of the proline carrier was located on the cytoplasmic side of the membrane.     

Stimulatory effect of lithium ion on proline transport by whole cells of Escherichia coli.

The effect of monovalent cations on proline transport in whole cells of Escherichia coli K-12 has been examined. Lithium ion added to the uptake medium stimulated proline transport severalfold and K+ and Na+ were slightly effective, whereas Rb+, Cs+, and NH4+ were completely without effect. The stimulatory effect of Li+ on proline transport was not due to an increase in osmolarity of the uptake medium, and d 5 mM p-chloromercuribenzene sulfonic acid completely blocked this effect of Li+ without having any effect on the basal rate of proline transport. The Arrhenius plots for Li+-stimulated transport showed a clear transition point at 35 degrees C in addition to 20 degrees C which was also detectable in the basal transport. Lithium ion stimulated proline transport synergistically in the presence of glucose and succinate as a carbon source. The addition of 2.5 mM KCN or 0.5 mM arsenate did not inhibit this synergistic effect, although the presence of these inhibitors inhibited completely the stimulation of proline transport induced by the addition of carbon source. Carbonylcyanide m-chlorophenylhydrazone and 2,4-dinitrophenol blocked both the basal and Li+-stimulated proline transport. When membrane potential of E. coli cells was measured by the dibenzyldimethylammonium uptake method, the incubation of Li+ with the cells did not affect the preexisting membrane potential. These results suggest that Li+ stimulates proline transport by intact cells of E. coli in a manner somewhat affecting membrane component(s) different from the transport carrier of proline. It is uncertain whether the effect of Li+ is directly involved in the mechanisms of energy coupling of proline transport.     

A Novel Type of Coupling Between Proline and Galactoside Transport in Escherichia coli

Exit of thiomethylgalactoside (TMG) from preloaded cells induced the accumulation of proline. Likewise, proline exit stimulated TMG accumulation. Since a proton ionophore (carbonyl-cyanide-m-chlorophenylhydrazone) abolished these effects, a proton-motive force was implicated as the “intermediate” in the coupling reaction. The evidence suggests that the exit of TMG resulted in proton exit, which produced either a membrane potential (inside negative) or a pH gradient (outside acid) or both. This inwardly directed protonmotive force provided the energy for proline entry and accumulation. Thus the energy coupling was not via a common transport protein but by proton movements which coupled the two separate H⁺-dependent transport processes.     

Mutational analysis of the Escherichia coli phosphate-specific transport system, a member of the traffic ATPase (or ABC) family of membrane transporters. A role for proline residues in transmembrane helices.

The Escherichia coli Pst system is a periplasmic phosphate permease. A mutational analysis of the requirement for function of specific charged residues or proline residues in the two hydrophobic subunits (PstC and PstA) has been carried out. No residues, among 19 charged residues altered, were found to be essential for phosphate uptake, although some alterations resulted in partial effects. Evidence was obtained that the 3 residues, R220 in the PstA protein and R237 and E241 in the PstC protein, previously shown to be required for phosphate transport (Cox, G. B., Webb, D., Godovac-Zimmermann, J., and Rosenberg, H. (1988) J. Bacteriol. 170, 2283-2286; Cox, G. B., Webb, D., and Rosenberg, H. (1989) J. Bacteriol. 171, 1531-1534), interact with each other. A feature of the proposed structures of the PstA and PstC proteins was 2 pairs of proline residues in putative transmembrane helices 3 and 4. While individual substitutions of these proline residues by leucine resulted in loss of phosphate transport activity substitution by alanine only had partial effects. However, if the proline to alanine changes were paired then, depending on the particular subunit, markedly different effects were obtained. The double mutation in the PstA protein resulted in a permanently "closed" system, whereas the double mutation in the PstC protein resulted in a permanently "open" transport system.     

Effect of maleimide derivatives on superoxide-generating system of guinea-pig neutrophils stimulated by different soluble stimuli

The effect of modification of maleimide derivatives on superoxide production by guinea-pig neutrophils induced by a variety of different soluble stimuli was studied. Pretreatment of neutrophils by showdomycin, a very slowly penetrating-SH reagent, did not affect superoxide production by all of the stimuli used, suggesting no exposure of sulfhydryl groups involved in superoxide-generating system on the cell surface. Pretreatment with N-ethylmaleimide (MalNEt), a considerably penetrating-SH reagent, markedly inhibited superoxide production stimulated by formyl-methionyl-leucyl-phenylalanine (HCO-Met-Leu-Phe), cytochalasin E or digitonin, but not superoxide production stimulated by the ionophore A23187 or sodium fluoride. The oxygen consumption stimulated by HCO-Met-Leu-Phe or cytochalasin E was inhibited by MalNEt pretreatment, whereas the oxygen consumption stimulated by A23187 was not inhibited by MalNEt. The inhibition by MalNEt of superoxide production did not appear to be due to the interference with binding of the affected stimuli, since MalNEt pretreatment did not inhibit the release of lysozyme, granule enzyme, induced by HCO-Met-Leu-Phe, cytochalasin E or digitonin. Particulate fractions from MalNEt-pretreated neutrophils before exposure to the stimulus exhibited the inhibition of the enhancement of NADPH-dependent superoxide production induced by HCO-Met-Leu-Phe, cytochalasin E or digitonin, but not A23187, whereas treatment of neutrophils with MalNEt after activation by these stimuli had no effect on the NADPH oxidase activity in particulate fractions. Direct exposure of particulate fractions from A23187-stimulated neutrophils to MalNEt showed no actual susceptibility of NADPH oxidase to MalNEt inhibition. These findings suggest that the inhibitory effect of MalNEt is caused by the modification of the process of the activation by the affected stimuli of the superoxide system, probably NADPH oxidase and that at least two mechanisms exist for activation of superoxide-generating system in guinea-pig neutrophils on the basis of the susceptibility to MalNEt inhibition.     

Proline uptake through the major transport system of Salmonella typhimurium is coupled to sodium ions.

Strains of Salmonella typhimurium deficient in one or more of the proline transport systems have been constructed and used to study the mechanism of energy coupling to transport. Proline uptake through the major proline permease (PP-I, putP) is shown to be absolutely coupled to Na+ ions and not to H+ ions as has previously been assumed. Transport through the minor proline permease (PP-II, proP), however, is unaffected by the presence or absence of Na+. The effect of Na+ on the kinetics of proline uptake shows that external Na+ increases the Vmax for transport. It seems probable that proline transport through PP-I is also coupled to Na+ ions in Escherichia coli.     

Developmental regulation of proline transport in Leishmania donovani

Leishmania donovani are the causative agents of kala azar in humans. These organisms cycle between the proline-rich environment of the sand fly vector (extracellular promastigotes) and the sugar-rich condition in the mammalian host (intracellular amastigotes). Parasites have adapted to these extreme changes in proline concentrations: promastigotes utilize proline as a carbon source, whereas amastigotes utilize sugars and fatty acids. Previous studies have suggested that promastigotes and amastigotes express distinct proline transporters. However, the information available on these transporters is limited. In this work, proline transport was investigated in axenic L. donovani cultures. Three transport systems were identified: cation-dependent and -independent proline transporters in promastigotes (systems A and B, respectively) and a single cation-independent transporter in amastigotes (system C). Systems A and C have broad specificity to almost all amino acids and obtain optimum activity at acidic pH ranges (pH 6 and 5, respectively). System B is more specific to proline, as it is inhibited by only five amino acids. Temperature response analyses indicated that the transporters of both promastigotes and amastigotes perform best at 37 degrees C. The activity of system A during parasite differentiation was assessed. The transport activity of system A disappeared 3 days after promastigotes were induced to differentiate into amastigotes. In these cells, elevated temperature and acidic pH each suppressed the activity of system A. When amastigotes were induced to differentiate back into promastigotes, system A resumed its activity 24 h after differentiation was initiated. In conclusion, L. donovani obtain proline transport systems that are stage specific, regulated by both pH and temperature. This paper constitutes the first investigation of amino acid transport in axenic L. donovani.     

The organic osmolytes betaine and proline are transported by a shared system in early preimplantation mouse embryos

Betaine and proline protect preimplantation mouse embryos against increased osmolarity and decreased cell volume, implying that they may function as organic osmolytes. However, the transport system(s) that mediates their accumulation in fertilized eggs and early embryos was unknown, and previously identified mammalian organic osmolyte transporters could not account for their transport. Here, we report that there is a single saturable transport component shared by betaine and proline in 1-cell mouse embryos. A series of inhibitors had nearly identical effects on both betaine and proline transport by this system. In addition, K(i) values for reciprocal inhibition of betaine and proline transport were approximately 100-300 microM, similar to K(m) values ( approximately 200-300 microM) for their transport, and both had similar maximal transport rates (V(max)). The K(i) values for inhibition of betaine and proline transport by dimethylglycine were similar ( approximately 2 mM), further supporting transport of both substrates by a single transport system. Finally, betaine and proline transport each required Na(+)- and Cl(-). These data were consistent with a single, Na(+)- and Cl(-)-requiring, betaine/proline transport system in 1-cell mouse embryos. While betaine was only transported by a single saturable system, we found an additional, less conspicuous proline transport route that was betaine-insensitive, Na(+)-sensitive, and inhibited by alanine, leucine, cysteine, and methionine. Furthermore, we showed that betaine, like proline, is present in the mouse oviduct and thus could serve as a physiological substrate. Finally, accumulation of both betaine and proline increased with increasing osmolarity, consistent with a possible role as organic osmolytes in early embryos.     

Mutant of Escherichia coli defective in response to colicin K and in active transport.

A mutant of Escherichia coli has been isolated that grows poorly on succinate and exhibits a markedly reduced sensitivity to colicin K. This mutant is also deficient in the respiration-linked transport of proline and thiomethyl-beta-D-galactoside but appears normal for the adenosine triphosphate-dependent transport of glutamine and arginine. A temperature-conditional revertant of the mutant grows on succinate and is sensitive to colicin K at 27 C, but fails to grow on succinate and is insensitive to colicin K at 42 C. Proline transport in the temperature-conditional revertant is reduced at 42 C when either glucose or succinate is used as energy source. Glutamine transport, on the other hand, is normal at 42 C with glucose as energy source, but is reduced with succinate, although not to the same extent as is proline transport. The lack of growth on succinate and the deficiencies in transport at 42 C are not due to a temperature-dependent lesion in either the electron transport chain or in Ca2+, Mg2+-activated adenosine triphosphatase activity. Membrane vesicles prepared from the temperature-conditional revertant are impaired in proline transport at both 27 and 42 C. These findings suggest the existence in the cytoplasmic membrane of E. coli cells of a component, presumably protein, that is required for colicin K action and that functions in respiration-linked and, to a lesser degree, in adenosine triphosphate-dependent active transport systems. This protein may serve as the primary target of colicin K action.