Amino Group for the Transport of Peptides in Escherichia Coli

Biochem. J. (1971) 123, 245-253 245 Printed in Great Britain

Tke Requirement for the Protonated a-Amino Group for the Transport of Peptides in Escherichia coli

By J. W. PAYNE Microbiological Research Establishment, Porton, Salisbury, Wilts., U.K. (Received 15 February 1971)

Many glycine peptides support growth of a glycine auxotroph of E8cherichia coli. If the a-amino group of these peptides is methylated, the products are still utilized for growth, and also retain comparable ability with the unsubstituted peptides to compete with natural peptides for transport into the cell. In contrast, glycine peptides devoid of an a-amino group, or that have the a-amino group substituted by one of a number of acyl groups are not utilized, although E. coli possesses intracellular enzymic activity able tor release glycine from such compounds; further, these derivatives do not compete with natural peptides for transport into the cell.

A number of studies have been carried out over acetylation of the a-amino group of lysine and the last few years into the process of peptide trans- arginine homopeptides effectively destroyed their port in bacteria (Payne & Gilvarg, 1971). In nutritional capacity. However, acetylation not general, these studies have sought to establish only substitutes the a-amino group but also re- which structural features of a peptide play a role moves its positive charge. It appears of some in the transport process. The uptake of a peptide importance therefore, before any mechanism of into a bacterium can be established from the ability peptide transport can be proposed, to assess the of a peptide to support the growth of an appropriate importance or otherwise of this positive charge on amino acid auxotroph (Gilvarg & Katchalski, the oc-amino group. 1965). It was demonstrated that lysine and In the present study, we describe the relative arginine homopeptides, for example, could support abilities of glycine oligopeptides and a-N-methyl- the growth of the respective Escherichia coli auxo- glycine oligopeptides (sarcosine peptides) to enter trophs, but that nutritional effectiveness ceased E. coli; N-methylation leaves intact the positive abruptly beyond the tetrapeptides (Gilvarg & charge on the amino group. Further, to sub- Katchalski, 1965). With other homologous oligo- stantiate the generality of the earlier conclusions peptides, analogous observations were made that based on the a-N-acetylated lysine and arginine nutritional effectiveness was lost by higher oligopeptides we have prepared a variety of a-N- mers (Gilvarg & Katchalski, 1965; Payne & acylglycine peptides and have tested them for their Gilvarg, 1968a; Payne, 1968). The structural para- ability to enter E. coli. meter responsible for the loss of nutritional efficiency was shown to be the overall volume of the peptide (Payne & Gilvarg, 1968b). Further studies MATERIALS have shown that separate transport systems exist Oligopeptides. Lysine and ornithine homo-oligopeptides for the uptake of dipeptides and of oligopeptides were prepared by the controlled polymerization of (Payne & Gilvarg, 1968a; Payne, 1968), which c- N- benzyloxycarbonyl-lysine - N- carboxylic anhydride differ with respect to their terminal carboxyl-group (Bergman, Zervas & Ross, 1935) and S-N-benzyloxy- requirements. Whereas a dipeptide must possess carbonylornithine-N-carboxylic anhydride in a manner an unsubstituted terminal carboxyl group to analogous to that described by Payne & Gilvarg (1968a). be transported, oligopeptides with the terminal The individual oligomers were selectively eluted from a carboxyl group substituted or even completely column of CM-cellulose (Na+ form) with a gradient of absent are still readily taken up (Payne & exponentially increasing salt concentration by the procedure of Stewart & Stahmann Di- and tri- In the presence of an (1962). Gilvarg, 1968a). contrast, glycine were obtained from British Drug Houses Ltd., unsubstituted a-amino group is important for the Poole, Dorset, U.K. All other peptides were purchased transport of both di- and oligo-peptides (Gilvarg & from Cyclo Chemical Corp., through Baxter Laboratories, Katchalski, 1965; Losick & Gilvarg, 1966). This Thetford, Norfolk, U.K., or from Sigma (London) conclusion was based on the observation that Chemical Co. Ltd., London S.W.6, U.K. 246 J. W. PAYNE 1971 Table 1. Characterization of N-acyl derivatives Electrophoretic mobilities are relative to aspartic acid (1.0) at pH 6.5, and serine (1.0) at pH 2.1, measured from the neutral band. The net charge is that expected from the nature of each particular acyl substituent' Molecular weights are estimated on the basis of the electrophoretic mobility and net charge by the method of Offord (1966). Abbreviations are as follows: Suc, succinyl; Glt, glutaryl; Mal, maleyl; Cit, citraconyl; Ac, acetyl; Sarc, sarcosyl; n.d., not determined. Electrophoretic mobility Estimated Theoretical Net charge molecular molecular Peptide at pH6.5 pH6.5 pH2.1 weight weight Suc-Gly -2 1.49 n.d. 189 185 Suc-Gly2 -2 1.27 n.d. 240 242 Suc-Gly3 -2 1.15 n.d. 285 299 Glt-Gly -2 1.40 n.d. 202 199 Glt-Gly2 -2 1.24 n.d. 250 256 Glt-Gly3 -2 1.12 n.d. 293 313 Mal-Gly2 -2 1.30 n.d. 232 258 Mal-Gly3 -2 1.17 n.d. 275 315 Cit-Gly2 -2 1.23 n.d. 254 272 Cit-Gly3 -2 1.11 n.d. 300 329 Ac-Gly2 -1 0.89 n.d. 160 174 Ac-Gly3 -1 0.74 n.d. 212 231 Sarc-GlY2 0 0.00 1.33 138 146 Sarc-Gly3 0 0.00 1.15 190 203 Sarc-Ala 0 0.00 1.24 160 160 Saro-Ser 0 0.00 1.15 189 176 Gly-Sare 0 0.00 1.27 153 146 Gly2 0 0.00 1.40 120 132 Gly3 0 0.00 1.20 176 189

N-Substituted glycine oligopeptides. Standard proce- N-acyl derivatives were characterized by high-voltage dures are usually employed for the acylation of proteins paper electrophoresis by using a Shandon flat-plate by using several common acid anhydrides (Riordan & apparatus with a buffer (pH 6.5) containing pyridine Vallee, 1967; Klotz, 1967). However, for the acylation of (8%, v/v) and acetic acid (0.4%, v/v). At this pH value simple glycine peptides the lower pKaof the o-amino group the glycine substrates are neutral, whereas the N-acyl (about 7.2) in the glycine oligopeptides compared with derivatives each possesses an overall negative charge. that of an c-amino group (about 10.3) allows the reaction Electrophoretograms were developed with cadmium- to be readily carried out at neutral pH, thus decreasing ninhydrin reagent (Heilmann, Barrolier & Watzke, 1957), the spontaneous hydrolysis of the acid anhydride (Klotz, followed by chlorine-tolidine (Von Aux & Neher, 1963). 1967). The oc-N-acyl derivatives of glycine oligopeptides The initial colour reaction with cadmium-ninhydrin were prepared as follows. The glycine substrate was showed the complete absence from the neutral band of any treated with the appropriate anhydride at pH 7.1 by using of the glycine starting materials, except for the citraconyl a Radiometer Titrimeter (Copenhagen, Denmark) in its derivatives, which contained a few per cent of the free pH-stat mode. Glycine (160mg, 2.13mmol), diglycine peptide; the citraconyl group is the most acid-labile of the (300mg, 2.28 mmol) or triglycine (400mg, 2.10 mmol) was acyl derivatives and the free peptides probably arose dissolved in 1M-NaHCO3 (5.Oml) in the titration cup of through deacylation (Dixon & Perham, 1968). The acetyl, the titrimeter and the pH was adjusted to 7.1. The succinyl and glutaryl derivatives are all ninhydrin- anhydride (2.5mmol) was added in a single amount to the negative, a fact that is used as a test for complete acylation stirred solution at room temperature. The pH was main- (Klotz, 1967). The maleyl, and in particular the citraconyl, tained at 7.1 by the automatic addition of lOm-NaOH. derivatives gave a yellow colour with cadmium-ninhydrin, The reaction was rapid and in all cases was judged to be characteristic of peptides with unsubstituted N-terminal complete (addition of base ceased) after about 20min. glycine. However, we believe that this may be attributed The pH of the final reaction mixture was adjusted to to deacylation ofthese acid-labile substituents during run- 7.5 before storage at -10°C. These solutions were diluted ning and subsequent drying of the electrophoretograms, as required and were used directly for bacterial growth for the ninhydrin colour response was considerably greater studies. The anhydrides used were acetic anhydride, if heat was used. All the N-acyl derivatives were readily maleic anhydride, 2-methylmaleic anhydride (citraconyl detected as single spots on the electrophoretograms by anhydride) obtained from British Drug Houses Ltd., chlorine-tolidine. By the method of Offord (1966), it is and succinic anhydride and glutaric anhydride purchased possible to estimate a value for the molecular weight of from Ralph N. Emanuel Ltd., London S.E.1, U.K. each species from the observed mobilities of the N-acyl Glutaric anhydride was recrystallized before use. The derivatives and from the net charge on each derivative. Vol. 123 Vc-AMINO GROUP IN PEPTIDE TRANSPORT 247 The results of such calculations are shown in Table 1 and a consequence of the cleavage of the peptide bond, as a confirm that the observed products are indeed the ex- preliminary procedure to assess rapidly the peptidase pected N-acyl derivatives. Glycine and sarcosine peptides activity in any incubation mixture. Extinction changes were characterized by high-voltage electrophoresis at were automatically recorded with a Gilford model 2000 pH2.1 in acetic acid (8%, v/v) and formic acid (2%, v/v). recording spectrophotometer. This assay is extremely The a-N-methyl compounds are ninhydrin-positive, but simple, and with appropriate controls, lends itself readily yield a purple colour which for full development takes to determination of the usual parameters of an enzymic somewhat longer at 1050C than for the unsubstituted reaction. Samples of this same incubation mixture may glycine peptides, which stain yellow. Each gave a single subsequently be used for standard colorimetrio assays. spot with cadmium-ninhydrin with the expected electro- In confirmation of peptidase action the products of these phoretic mobility. reactions were characterized by high-voltage electrophoresis as described above. METHODS RESULTS Bacterial 8train8. The following strains of E. coli W (A.T.C.C. 9637) were used. Strain M-26-26 is a lysine The N-acyl derivatives of glycine were syn- auxotroph that lacks the decarboxylase that converts thesized to assess the importance of the ac-amino me8o-diaminopimelic acid into L-lysine (Davis, 1952; group of a peptide to the bacterial utilization of Dewey & Work, 1952). Strain M-26-26.R is a spontaneous peptides. Some of these compounds can be con- non-auxotrophic revertant of strain M-26-26 correspond- sidered to be either N-acyl peptide derivatives or ing to the wild-type strain. Strain M-123 is a glycine- serine auxotroph derived from strain M-22-93, originally peptides that lack an a-amino group. Thus, an obtained from Dr W. Maas (Payne, 1968). The bacterial a-acetyl substituted amino acid or peptide may cultures were grown in the medium A of Davis & Mingioli equally well be regarded as a glycyl peptide devoid (1950). Bacterial growth was followed by measuring the of its a-amino group. Similarly, a-propionyl, ac- E560 in a Bausch and Lomb Spectronic 20 instrument. succinyl and oc-glutaryl derivatives respectively Inocula (0.1-0.2 ml) were removed from a culture growing may be considered as alanyl, aspartyl and glutamyl exponentially on minimal medium and added to the test peptides devoid of a-amino groups. In interpreting solutions (6.Oml) in culture tubes (20mm diam.). The the results of the bacterial utilization of these N- culture tubes were incubated at 370C with shaking. it is to con- E. coli K12, strain AS013, a lysine auxotroph derived acyl compounds occasionally helpful from strain 2001c, F-, Thr-, Leu-, thiamin-, was kindly sider that some N-acyl derivatives are peptides provided by Dr C. Gilvarg. This strain was grown in without their a-amino groups. minimal medium M-56 (Weismeyer & Cohn, 1960) supplemented with 0.5% glucose; the required amino acids were Growth response of glycine auxotroph to free and N- provided at 50mg/litre and thiamin at 5mg/litre. Peptidase assay. To prepare cell-free extracts, bacteria 8ub8tituted glycine peptides were grown in minimal medium, harvested in exponential In earlier studies it was shown that the homo- phase, washed once with minimal medium A and then oligopeptides up to and including hexaglycine could disrupted for 10min in a Braun cell disintegrator. The act as a source of glycine for the glycine auxotroph extract was separated from cell debris by centrifugation at 15000g for 50min. Protein concentration was deter- M-123 (Payne, 1968). However, in the present study mined by the method of Warburg & Christian (1941). when the N-acyl compounds were tested at con- Peptide cleavage was measured in an incubation mixture centrations up to 3.0 ,umol/ml, that is about six containing 50mM-potassium phosphate buffer, pH7.1, times the concentration at which glycine oligopep- crude extract corresponding to 0.5-1.Omg of protein, and tides can sustain exponential growth, no growth peptide or peptides in a total volume of 1.2ml. When was observed. These N-acyl derivatives do not required, Co2+, Zn2+, Mg2+ and Mn2+ were each added to inhibit growth, for at 3.0,umol/ml they are without give a final concentration of 0.5mm. Reaction mixtures effect on the growth of the wild-type strain or on were incubated at 37°C, samples were removed periodic- M-123 on media ally, and the peptidase action was stopped by delivering the growth of auxotroph supple- into HCI with lysine peptides, or by cooling in ice-water, mented with glycine. Slow linear growth was with other peptides. Cleavage of lysine peptides was observed with the citraconyl derivatives, but this followed by the ninhydrin method of Shimura & Vogel was compatible with that expected from their (1966), which is specific for lysine and gives no colour content of deacylated contaminants. However, with lysine peptides. Cleavage of glycine peptides was a-glutamylglycine and a-aspartylglycine each at assayed by the liberation of ninhydrin-positive material 0.8,umol/ml supported exponential growth of strain (ac-amino groups) by using the procedure of Moore & M-123, whereas neither of the corresponding Stein (1948). By this procedure, the absorption at zero peptides that lack the a-amino group, that is, N- time was identical for the N-acyl derivatives tested and for the control containing protein alone, confirming that glutaryl- and N-succinyl-glycine, was utilized. these N-acyl peptides are not contaminated with the Similarly, a-N-acetyldiglycine and a-N-acetyltri- unsubstituted parent peptides. glycine did not support growth although they are We occasionally made use of the decrease in E220 that is the analogues of tri- and tetra-glycine respectively, 248 J. W. PAYNE 1971 both of which allow exponential growth (Payne, section. Crude extracts contain enzymic activity 1968). able to cleave all peptides tested, with the exception When sarcosylglycine and sarcosyldiglycine were of glycylsarcosine, for which we could detect no tested at concentrations up to 5.0,umol/ml very cleavage (Table 2). Thus, since the cell contains slow growth ofauxotroph M- 123 was observed, with enzymes able to release glycine from the non- a minimum generation time of 120 min; the rate of utilizable N-acyl peptides as easily as from the growth was not faster with higher concentrations of utilizable unsubstituted peptides, it implies that these peptides. With sarcosylalanine and glycyl- the N-acyl derivatives are unable to enter the cell. sarcosine no growth was observed after incubation On the other hand, the very slow growth obtained for 8h. None of these compounds was inhibitory with sarcosylglycine and sarcosyldiglycine may at this concentration to the growth of the wild type stem from their lower rates of cleavage (about or to the growth of auxotroph M-123 in media one-third that of the unsubstituted peptides) which supplemented with glycine. Sarcosine itself may supply glycine at a rate less than the critical (1.5,mol/ml) is unable to support growth of the rate required to sustain exponential growth. At glycine auxotroph; it is also without inhibitory first sight the fact that sarcosylserine is also cleaved effect, although certain other a-N-methyl-amino at a slow rate but nevertheless supports exponential acids are inhibitory to E. coli (Fowden, Lewis & growth of auxotroph M-123 appears to invalidate Tristram, 1967). However, sarcosylserine was the above argument, but it should be remembered found to support exponential growth of auxotroph that auxotroph M-123 requires either glycine M-123, from which it must be concluded that E. coli or serine for growth, and serine appears to be possesses the means to concentrate and to cleave nutritionally superior; indeed the rate of growth on ac-N-methyl-peptides. glycylserine is considerably faster than that of The results of the above growth tests indicate glycylglycine. Thus, the slow cleavage of sarco- that peptides that either lack an a-amino group, or sylserine may nevertheless provide a superior that have this group substituted in certain ways nutrient at a rate adequate for exponential growth. cannot be utilized by E. coli. The question that In view of the importance of cations to the activity remains is whether this arises because these com- of a number of peptidases (Simmonds, 1970; pounds are immune to intracellular peptidase Sussman & Gilvarg, 1971), the assays were repeated action, or because they are unable to enter the cell in the presence ofvarious cations (Table 2). Ofthose via the peptide transport systems. To decide tested, Co2+ had the greatest effect, producing between these alternatives we have carried out the about a tenfold stimulation of cleavage of the following tests. unsubstituted and the sarcosyl peptides but no stimulation with glycylsarcosine or the N-acyl Enzymic cleavage ofglycinepeptidesandN-substituted derivatives. glycinepeptides Cell extracts were prepared and peptidase assays Competitive effects ofN-sub8titutedpeptides were carried out by using glycine and N-sub- Competition that overcomes growth inhibition. We stituted glycine peptides as described in the Methods tested the ability of the various peptide derivatives

Table 2. Cleavage of glycine peptides by crude extracts and by cation-supplemented extracts of E. coli W Cell extracts were prepared, and peptidase assays were carried out as described in the Methods section. Results are expressed as ,umol of oc-amino groups (glycine) released/min per mg of protein per ml. For abbreviations see Table 1. Cleavage of peptide Crude extract supplemented with Crude extract Peptide alone Co2 + Mn2+ Zn2 + Mg2+ Gly2 0.021 0.121 0.008 0.003 0.026 Gly3 0.015 0.171 0.044 0.016 0.017 Sarc-Gly 0.006 0.134 0.004 0.003 0.005 Sarc-Gly2 0.008 0.115 0.010 0.010 0.004 Sarc-Ser 0.005 0.200 0.009 n.d. n.d. Gly-Sarc 0.000 0.000 0.000 n.d. n.d. Ac-Gly, 0.021 0.014 n.d. n.d. n.d. Ac-Gly3 0.018 0.012 n.d. n.d. n.d. Glt-Gly3 0.021 0.013 n.d. n.d. n.d. Suc-Gly3 0.014 n.d. n.d. n.d. n.d. Vol. 123 1a-AMINO GROUP IN PEPTIDE TRANSPORT 249 At comparable concentrations neither the free amino acid nor diglycine are effective, a result to be expected from the distinct modes of uptake employed by amino acids, dipeptides and oligopeptides (Payne & Gilvarg, 1971; Payne, 1968). In further studies it was shown that the N-acyl peptides afforded no protection against inhibition even at a concentration 100 times that at which N-methyltriglycine was effective. Norleucine and norvaline both inhibit the growth of E. coli and peptides containing these amino acids 0.4- are inhibitory if they can enter the cell and be cleaved. The peptides norleucylnorvaline, glycyl- norvaline, alanylnorvaline, glycylnorleucine, glycyl-

0.2- glycylnorleucine and norvalylglycylglycine were all inhibitory to various extents. In confirmation of the fact that these oligopeptides containing un- natural amino acids use the peptide transport 0 2 4 6 8 system to enter the cell we found that a mutant Time (h) unable to transport oligopeptides (Payne, 1968), is not inhibited by these tripeptides. It was there- Fig. 1. Ability of glycine peptides to overcome the fore possible, in a manner analogous to the tri- inhibitory effect oftriornithine on the growth of wild-type ornithine experiments, to test whether the N- E. coli W. Except where indicated, triornithine was substituted glycine peptides are able to use the present at a concentration of 0.04pmol/ml. Glycine, transport system by measuring their ability to diglycine and N-methyldiglycine were tested at con- the centrations up to 0.5,umol/ml. N-Succinyl- and N- overcome the inhibitory effect of norvaline and glutaryl-glycine, N-acetyl-, N-succinyl- and N- norleucine peptides. The results (Fig. 2) were glutaryl-diglycine, N-acetyl-, N-succinyl-, N-glutaryl- analogous to those observed with triornithine. and N-maleyl-triglycine were tested at concentrations up Triglycine and N- methyltriglycine are almost to 1.4,umol/ml. o, No triornithine; *, + triglycine equally efficient in restoring growth whereas the (0.033,umol/ml); *, + N-methyltriglycine (0.033,umol/ N-acyl derivatives, even at much higher con- ml); A, + N-methyltriglycine (0.016,umol/ml); A, centrations, afford no protection. Similarly, glycine + triornithine alone or + N-acylglycine peptides, N- is devoid of competitive ability. A noteworthy case methyldiglycine, diglycine or glycine. of competition of a type distinct from that con- cerned with transport is possible in this system, for to use the peptide transport systems by measuring it should be recalled that norleucine toxicity arises their competitive efficiency in limiting the entry from its interference with the functions of methio- of various unsubstituted peptides. Triornithine has nine (Fowden et al. 1967), and that its toxic effect been shown to be extremely toxic to E. coli (Payne, can be overcome by the addition of methionine to 1968). The exact mechanism of its toxicity is still the growth medium. We confirmed that the unclear, but it results from the build-up of a high inhibitory effect of glycylnorleucine and glycyl- intracellular concentration caused by the absence glycylnorleucine (each at 1.0 ,umol/ml) can be of peptidases able to cleave the tripeptide (Payne, reversed by adding methionine (1.5,umol/ml) to the 1968; Sussman & Gilvarg, 1970). Thus, if tri- media. Further, the inhibitory effect of norleucine ornithine can be prevented from entering the cell, itself can be reversed non-specifically by leucine, growth may be achieved. presumably because the two amino acids share a Fig. 1 shows that an approximately equimolar common permease and leucine can therefore com- concentration of triglycine can virtually overcome petitively prevent the uptake of norleucine. How- the inhibitory effect of triornithine, and a family of ever, with glycylnorleucine or glycylglycylnorleu- growth curves may be obtained by using graded cine (each at 1.0,umol/ml), leucine (4.0,umol/ml) concentrations of triglycine, 0.15,umol/ml com- lacked any ability to reverse inhibition, in accord pletely reversing the inhibition caused by 0.04 ,umol with the distinct transport systems used by amino of triornithine/ml. N-Methyltriglycine also over- acids and peptides. comes triornithine inhibition (Fig. 1), suggesting It is noteworthy that the inhibition observed that the transport system does not distinguish with glycylglycylnorleucine at high concentrations between a peptide with a free ac-amino group and one (above 0.25 ,umol/ml) cannot be completely reversed with a methylated ac-amino group. The reversal of by increased concentrations of peptide com- inhibition is not attributable to the glycine moiety. petitors. The result accords with an earlier finding 250 J. W. PAYNE 1971

0.4- 0.4

0.2 -

0.2- 0 2 4 6 8 Time (h)

Fig. 2. Ability of glycine peptides to overcome the 0 2 4 6 8 10 12 inhibitory effect of glycylglycylnorleucine in auxotroph Time (h) M-26-26. Except where indicated, glycylglycylnorleucine was present at a concentration of0.082,umol/ml. Glycine, Fig. 3. Ability of glycine peptides to reverse the inhibi- diglycine and N-methyldiglycine were tested at concen- tory effect of valine peptides on E. coli K12 strain ASO13. trations up to 2.0,umol/ml. N-Succinyl- and N-glutaryl- To preserve clarity, tests with the N-acyl peptides are not glycine, N-acetyl-, N-succinyl- and N-glutaryl-diglycine, shown, but these were performed as follows: N-succinyl- and N-acetyl-, N-succinyl- and N-maleyl-triglycine were and N-glutaryl-glycine, and N-acetyl-, N-succinyl- and tested at concentrations up to 3.Otmol/ml. o, No glycyl- N-glutaryldiglycine were used at concentrations up to glycylnorleucine; *, + triglycine (0.82,umol/ml); L, 20,umol/ml in the presence of glycylvaline. N-Acetyl-, + N-methyltriglycine (0.82,mol/ml); A, + N-methyltri- N-succinyl-, N-glutaryl- and N-maleyl-triglycine were glycine (0.33,umol/ml); *, glycylglycylnorleucine alone or tested at concentrations up to 20,umol/ml in the presence + N-acylglycine peptides, N-methyldiglycine, diglycine ofvalyldiglycine. The results in each case paralleled those or glycine. obtained with the valine peptides alone. o, No glycylvaline or valyldiglycine; *, valyldiglycine (0.4,umol/ml) + triglycine (20 umol/ml) or + N-methyltriglycine (20 tmol/ with peptide-transport-deficient mutants, that in ml); *, + glycylvaline (0.4,umol/ml), + diglycine (l,0umol/ a ml) or + N-methyldiglycine (10jumol/ml); A, + glycyl- addition to entry via specific peptide-transport valine (0.4,tmol/ml) or valyldiglycine (0.4,tmol/ml). system, neutral peptides can enter E. coli by a concentration-dependent mode that is insensitive to competition and is perhaps a diffusive process. Presumably, at high concentrations glycylglycyl. In an effort to obtain further evidence for the norleucine can enter the cell by this secondary importance of the positively charged oc-amino group process at a rate sufficient to provide an inhibitory another strain of E. coli was examined. E. coli internal concentration of norleucine. K12 possesses an unusual sensitivity to valine Diglycine did afford slight protection at very high (Leavitt & Umbarger, 1962) and to valyl peptides molar ratios relative to glycylglycylnorleucine, in (A. J. Sussman & C. Gilvarg, personal communica- agreement with the previous findings that dipep- tion). The following valyl compounds completely tides possess limited ability to use the oligopeptide inhibited growth of strain AS013 in our standard transport system (Payne & Gilvarg, 1971). We test culture, valine (0.4,umol/ml), glycylvaline attempted to use this test system to measure the (0.6,umol/ml), valylglycine (0.4,umol/ml), trivaline ability ofglycine dipeptides to overcome the inhibi- (0.1 ,umol/ml), valylglycylglycine (0.5,umol/ml). tion caused by glycylnorleucine and by norleucyl- The effects of peptides on the inhibition of growth norvaline, but in neither case could we demonstrate by valine peptides were therefore examined. Di- any effect at competitor/inhibitor molar ratios up glycine and sarcosylglycine are indistinguishable in to 10: 1. However, this may simply reflect a more their ability to overcome glycylvaline inhibition, complicated process of dipeptide uptake that may and similarly triglycine and sarcosyldiglycine are involve several systems. equally efficient in reversing valylglycylglycine Vol. 123 Vc-AMINO GROUP IN PEPTIDE TRANSPORT 251 inhibition (Fig. 3). Further, the observations are specific for these combinations, for at these same I1.0 concentrations, neither diglycine nor sarcosylglycine affect inhibition by valylglycylglycine, and triglycine and sarcosyldiglycine do not affect the 0.8 inhibition by glycylvaline. The competitors do not relieve the inhibition by valine itself. In accord with all other studies we found the N-acyl peptides to be devoid of competitive activity in this test system. 0.6 0 Competition that lead8 to growth inhibition. In unI contrast with the above situation in which competition between oligopeptides for uptake can 0.4 r curtail the entry of toxic peptides and thus enhance growth, it should be possible by competitively inhibiting the transport of a peptide containing a 0.2- required amino acid to prevent growth of an auxotrophic strain. The growth of the lysine auxotroph on trilysine can be competitively inhibited by the addition of 0 2 4 6 8 triglycine or N-methyltriglycine to the growth media (Fig. 4). However, trilysine/triglycine molar Time (h) ratios of about 1: 50 are required before this inhibi- Fig. 4. Ability of glycine peptides to inhibit growth of tion is detectable. In contrast, no specific inhibition lysine auxotroph M-26-26 on trilysine. Except where of growth on trilysine was observed with any of the indicated trilysine was present at a concentration N-acyl compounds when tested at molar ratios of of 0.04,umol/ml. Glycine, diglycine and N-methyldiglycine were tested at concentrations up to 8.5 jtmol/ml. up to 300: 1 relative to trilysine. Further, as N-Acetyl-, N-suceinyl-, N-glutaryl- and N-maleyl expected, neither glycine itself nor dipeptides of derivatives of diglycine and triglycine were tested at glycine inhibited growth on trilysine. concentrations up to 14,umol/ml. 0, Trilysine alone, Ab8ence of competitive peptida8e activity. A or + glycine, diglycine, N-methyldiglycine, or N-acyl possible objection to the interpretation of the above peptides; o, + N-methyltriglycine (1.6,umol/ml); *, competition studies is that certain of the peptides + N-methyltriglycine (3.2,umol/ml); A, + triglycine may compete with one another for cleavage by (3.2p,mollml); 0, no trilysine. intracellular peptidases. The negative results with the N-acyl peptides may simply reflect the absence of a competitive effect on peptidases unlike the that is the toxic moiety. Similarly, tests in which unsubstituted and the N-methylated peptides. peptide-dependent auxotrophic growth is inhibited However, to accommodate this explanation, the (Fig. 4) demand that the competitors inhibit cleav- varied nature of the above tests requires that the the a of effects age of trilysine. Earlier studies indicated that active competitors be capable of variety cleavage of trilysine by crude extracts of E. coli is on intracellular peptidases. Thus, to overcome tri- and unaffected by relative concentrations of triglycine ornithine inhibition, triglycine N-methyltri- ten times that which on glycine must activate or induce peptidase activity inhibits growth trilysine There is considerable evi- (Payne, 1968). We have now extended these studies, to cleave triornithine. and by using the Shimura & Vogel (1966) assay for dence to indicate that peptidases are constitutive lysine, find that the cleavage of trilysine by crude and are not induced by growth in the presence of & extracts is unaffected by either triglycine or by N- peptides (Sussman Gilvarg, 1970, 1971; Vogt, at molar ratios that cause inhibi- 1970), and further, it has been shown that the methyltriglycine tri- tion of growth on trilysine. Cleavage of dilysine is peptidase(s) activity responsible for cleaving of or ornithine is distinct from that which cleaves glycine similarly unaffected by the presence diglycine peptides (Payne, 1968; Sussman & Gilvarg, 1970). N-methyldiglycine in the incubation mixture. In addition, triglycine neither activates nor inhibits the peptidase activity that cleaves triornithine in a DISCUSSION crude extract of E. coli. In contrast with the situation with triornithine, The experiments reported above were designed to the effects observed with the peptides containing investigate the function of the a-amino group in the norleucine or valine (Figs. 2 and 3) require that the process of peptide transport in E. coli. Previous peptide competitors inhibit the cleavage ofthe toxic studies showed that a-acetylation of lysine and peptides, for in each case it is the free amnino acid arginine oligopeptides rendered them nutritionally 252 J. W. PAYNE 1971 ineffective for lysine and arginine auxotrophs growing bacterium the results could explain the (Gilvarg & Katchalski, 1965; Losick & Gilvarg, lack of growth of the N-acyl derivatives (although 1966). In this case, the auxotrophs possessed presenting difficulties of interpretation for the N- peptidases able to cleave the acetylated peptides methyl derivatives). However, exponential- and and the discrimination against these compounds stationary-phase cells frequently differ in peptidase was considered to arise from their inability to enter activity and it is not possible to decide from the cell. Simmonds and co-workers showed that measurements of the peptidase activity of broken the a-benzyloxycarbonyl derivatives of glycyl- cells whether or not a peptide might be expected to phenylalanine, glycyltyrosine and prolylproline support growth (Simmonds, 1970). Certainly, the were much inferior to the free peptides in supporting simple observation that a cell extract contains the growth of phenylalanine, tyrosine (Simmonds, activity able to cleave a peptide does not mean that Tatum & Fruton, 1947), and proline auxotrophs the same peptide will support growth of an appro- (Simmonds & Fruton, 1948) respectively. The priate auxotroph. proline mutant was also unable to utilize a-N- C02+ caused no stimulation in the cleavage of the acetylphenylalanylproline for growth (Simmonds & N-acyl derivatives or of glycylsarcosine, and Fruton, 1948). Dunn & Dittmer (1951) reported electrophoretic studies on the initial cleavage pro- that a number of peptides containing ,-2-thienyl- ducts of sarcosyldiglycine in the presence of C02+ alanine possessed inhibitory properties for E. coli revealed glycine but not diglycine, suggesting that equal to the free amino acid, but that the corre- the enzyme is unlikely to be an aminopeptidase. sponding a-N-benzyloxycarbonyl peptides were Other Co2+-activated peptidases have beenreported about 1000 times less inhibitory. Unfortunately, in (Sussman & Gilvarg, 1970, 1971). most of the above studies it was not demonstrated In spite of the lack of growth in certain cases, whether the cell could cleave the a-N-acyl peptides there can be little doubt that N-methyl peptides with intracellular peptidases and it is impossible can use the peptide transport system, for in every to decide whether the distinction arises at the level competitive test they were comparable in activity of uptake or cleavage. with the corresponding unsubstituted peptide. In an attempt to gain further insight into the The diverse nature of the growth-competition ex- function of the a-amino group we prepared a series periments, and the direct tests that indicated the of substituted peptides with different amino sub- absence of any effects on peptidase activity, leave stituents. Thus, a-N-acetyl-, cc-N-succinyl- and inhibition of peptide uptake as the most probable a-N-glutaryl-glycine peptides may be regarded as explanation for the results of the competitive tests. N-glycyl, N-aspartyl and N-glutamyl peptides In contrast with peptides, the N-methylation of devoid of a-amino groups; free glycine peptides and certain amino acids prevents their uptake by N-methylglycine peptides both possess a positively bacteria (Britten & McClure, 1962), although charged a-amino group; a-N-acetylglycine peptides several of the amino acid transport systems in have no positive charge, and in a-N-maleyl-, c-N- mammalian cells can tolerate ac-N-methylation citraconyl-, ax-N-succinyl- and a-N-glutaryl-gly- (Christensen & Handlogten, 1968). The fact that cine peptides the substituted ac-amino group has none of the N-acyl derivatives permits growth of acquired an overall negative charge. the glycine auxotroph although the cells possess Since sarcosylserine supports exponential growth the enzymic machinery able to release glycine from of a glycine-serine auxotroph, and cells lack extra- these compounds suggests that they cannot enter cellular peptidase activity (Sussman & Gilvarg, the cell. The complete absence ofcompetitive action 1971) an cc-N-methylated peptide can evidently be with these compounds accords with this conclusion. accumulated and broken down by E. coli. The It therefore appears that the peptide transport other N-methyl derivatives may be unable to sup- systems of E. coli can tolerate substitution of a port growth because E. coli lacks peptidase activity peptide a-amino group provided that this can be able to split these compounds at a sufficient rate. achieved with retention of the positive charge. For As crude extracts readily split the N-acyl deriva- this interpretation, it is necessarily assumed that tives, the nutritional discrimination against these neither steric effects nor overall negative charge are compounds may result from their limited uptake. responsible for the inactivity of the N-acyl deriva- The effect of cations on peptidase activity was note- tives studied here but further studies are needed to worthy. Co2+ activated cleavage of the free pep- substantiate this point, the most pertinent perhaps, tides and the N-methyl compounds by peptidases; being the utilizability of peptides with various no other cations tested enhanced peptidase action, cc-N-alkyl substituents. and in certain cases inhibition was observed, although optimum conditions of pH and cation I am most grateful to Dr C. Gilvarg for discussions on concentration were not explored. Therefore, if the the possible mechanisms of peptide transport and for Co2+-sensitive peptidase(s) are fully active in the permission to quote material on bacterial peptidases be- Vol. 123 a-AMINO GROUP IN PEPTIDE TRANSPORT 253 fore its publication. I also thank Dr H. E. Wade for Moore, S. & Stein, W. H. (1948). J. biol. Chem. 176, 367. critically reading the manuscript and Miss Vanessa Taylor Offord, R. E. (1966). Nature, Lond., 211, 591. and Mr Richard Blake for their excellent technical assis- Payne, J. W. (1968). J. biol. Chem. 243, 3395. tance. Payne, J. W. & Gilvarg, C. (1968a). J. biol. Chem. 243,335. Payne, J. W. & Gilvarg, C. (1968b). J. biol. Chem. 243, 6291. REFERENCES Payne, J. W. & Gilvarg, C. (1971). Adv. Enzymol. (in the Bergman, M., Zervas, L. & Ross, W. F. (1935). J. biol. Press). Chem. 111, 245. Riordan, J. F. & Vallee, B. L. (1967). In Method8 in Britten, R. J. & McClure, F. T. (1962). Bact. Rev. 26, 292. Enzymology, vol. 11, p. 565. Ed. by Hirs, C. H. W. Christensen, H. N. & Handlogten, M. E. (1968). J. biol. New York: Academic Press Inc. Chem. 243, 5428. Shimura, Y. & Vogel, H. J. (1966). Biochim. biophys. Acta, Davis, B. D. (1952). Nature, Lond., 169, 534. 118, 396. Davis, B. D. & Mingioli, E. S. (1950). J. Bact. 60, 17. Simmonds, S. (1970). Biochemi8try, Easton, 9, 1. Dewey, D. & Work, E. (1952). Nature, Lond., 169, 533. Simmonds, S. & Fruton, J. S. (1948). J. biol. Chem. 174, Dixon, H. B. F. & Perham, R. N. (1968). Biochem. J. 705. 109, 312. Simmonds, S., Tatum, E. L. & Fruton, J. S. (1947). J. biol. Dunn, F. W. & Dittmer, K. (1951). J. biol. Chem. 188, Chem. 170, 483. 263. Stewart, J. W. & Stahmann, M. A. (1962). J. Chromat. Fowden, L., Lewis, D. & Tristram, H. (1967). Adv. 9, 233. Enzymol. 29, 89. Sussman, A. J. & Gilvarg, C. (1970). J. biol. Chem. 245, Gilvarg, C. & Katchalski, E. (1965). J. biol. Chem. 240, 6518. 3093. Sussman, A. J. & Gilvarg, C. (1971). A. Rev. Biochem. Heilmann, J., Barrolier, J. & Watzke, E. (1957). Hoppe- (in the Press). Seyler's Z. phy8iol. Chem. 309, 219. Vogt, V. M. (1970). J. biol. Chem. 245, 4760. Klotz, I. M. (1967). In Methods in Enzymology, vol. 11, Von Aux, E. & Neher, R. (1963). J. Chromat. 12, 329. p. 576. Ed. by Hirs, C. H. W. New York: Academic Warburg, 0. & Christian, W. (1941). Biochem. Z. 310, Press Inc. 384. Leavitt, R. I. & Umbarger, H. E. (1962). J. Bact. 83, 624. Weismeyer, H. & Cohn, M. (1960). Biochim. biophy8. Losick, R. & Gilvarg, C. (1966). J. biol. Chem. 241, 2340. Acta, 39, 417.