Proc. Natl. Acad. Sci. USA Vol. 76, No. 5, pp. 2138-2142, May 1979 Biochemistry Neuroactive drugs inhibit trypsin and outer membrane protein processing in Escherichia coli K-12 (procaine/cocaine/neostigmine/atropine/DNA cloning) RANDALL C. GAYDA*, GORDON W. HENDERSON, AND ALVIN MARKOVITZt Department of Microbiology, University of Chicago, Chicago, Illinois 60637 Communicated by Josef Fried, January 29, 1979

ABSTRACT Previous studies demonstrated that a cloned It is now established that precursor proteins exist for a 2-megadalton (MDal) fragment of Escherichia coli DNA con- number of bacterial proteins that are located outside the inner tained the structural gene for major outer membrane protein membrane as periplasmic proteins (12, 13), proteins of the outer a (also known as 3b or M2 (40 kDal). The present study demon- strates that M2 is synthesized from a 42-kDal precursor that also membrane (5, 13, 14), or extracellular enzymes (15). Most of is present in the outer membrane. The conversion of the 42-kDal these precursors are approximately 2 kDal larger than the precursor to M2 is inhibited by a number of different local an- mature protein and, where it is known, the portion removed esthetics (procaine, piperocaine, lidocaine, cocaine), by the proteolytically is at the NH2 terminus. The best known example neuroactive drug atropine, and by the classical try in inhibitors is the precursor of the lipoprotein, which contains an additional Na-tosyllysine chloromethyl ketone (TLCK) and benzamidine. NH2-terminal sequence of 20 amino acids (5). The signal hy- Our kinetic studies demonstrate that the amidase action of pure trypsin is inhibited competitively by the local anesthetics tested pothesis was proposed by Blobel and Dobberstein (16) to explain (excluding lidocaine) as well as by atropine and neostigmine. the mechanism of transport of proteins through the cell mem- A mechanism of action for local anesthetics as well as atropine brane of eukaryotes and is currently applied to work with in E. coli may be to inhibit trypsinlike proteases, in a competi- bacteria. This hypothesis proposes that the extra NH2-terminal tive manner, in the region of the outer membrane. The mecha- peptide acts as a signal to provide a ribosome-membrane nism of action of these compounds in regulating nerve con- junction and thus provides a topology for translational as well duction in man may have certain features in common with the as unidirectional transport of a polypeptide chain through a mechanism proposed in E. coli. membrane. There is some question as to whether or not the One of the major outer membrane proteins of Escherchia coli signal sequence must be processed during transport. The in vivo K-12 is the 40-kilodalton (kDal) protein a (1, 2) [also known as detection of the precursor of arabinose-binding protein (13) and 3b (3) and M2 (4); see ref. 5 for review]. Protein a is detected a mutant prolipoprotein (14) suggests that processing can occur in the outer membrane of strains grown at 37°C but not at 30°C after transport is complete. (2, 6). A 2-MDal cloned fragment of E. coli K-12 DNA contains In this report we demonstrate that the 42-kDal polypeptide the structural gene for protein M2 (4, 7), and studies with Ml is the precursor of the 40-kDal outer membrane polypeptide plasmid mutants demonstrated that this protein is important M2. Furthermore, the precursor can be found in the outer in repressing the synthesis of the capsular polysaccharide (8) membrane fraction. In the course of these studies we examined in lon (capR) strains that overproduce the polysaccharide the effect of a number of local anesthetics and other neuroactive (unpublished data). The 2-MDal DNA fragment was originally drugs, as well as classical trypsin inhibitors, on the conversion isolated by using the cloning vehicle pSC101, the resultant of MI to M2 in minicells containing plasmid pMC44. Many of plasmid being designated pMC44. Plasmid pMC44-coded these compounds inhibited the processing and most were shown proteins were determined by transforming a minicell-pro- to be competitive inhibitors of trypsin. ducing mutant to tetracycline resistance with the DNA, iso- lating the minicells, and incubating them in [-5S]methionine. MATERIALS AND METHODS Polypeptides were separated in sodium dodecyl sulfate (Na- Materials were obtained as described (4) or from the following DodSO4) polyacrylamide gels by electrophoresis. A 42-kDal sources: cocaine HCl, a gift of C. R. Schuster; procaine HC1, polypeptide, designated Ml, was also produced by plasmid procainamide HC1, neostigmine bromide, atropine sulfate, pMC44-containing minicells. Several observations suggested carbamoylcholine chloride, Na-tosyllysine chloromethyl ketone that Ml is a precursor of the 40-kDal polypeptide M2: plasmid (TLCK), and benzamidine HC1, Sigma; lidocaine HCl, a gift mutants were obtained that specified neither Ml nor M2 (un- from H. G. Vassallo (Astra Pharmaceutical Products, Fram- published data); the amount of Ml varied from 0 to 10% of the ingham, MA); piperocaine HC1, Lilly. amount of M2; the amount of MI was influenced by the pres- The minicell-producing strain DS410 (17) was supplied by ence of tetracycline during [-5S]methionine-labeling as well as D. Mount. Strain LonMin was provided by H. Adler and was by the complexity of the medium in which the minicell-pro- prepared by conjugation between Hfr strain M6 [a capR6 (Ion) ducing strain was grown (4). In addition, when plasmid pMC44 mutant] (18) and minicell-producing strain X925 (19). The was in a minicell-producing strain that contained a Ion (capR) LonMin strain is mucoid, is UV sensitive [i.e., lon (capR)], and mutation that decreases proteolysis of nonsense and missense retains the ability to produce minicells. Plasmid pMC44 DNA protein fragments (9-11), the amount of Ml detected by was transferred to strains DS410 and LonMin by transformation [35S]methionine-labeling of minicells was approximately equal (7). All experiments were performed with minicells from strain to that of M2 (unpublished data). Abbreviations: Dal, daltons; NaDodSO4, sodium dodecyl sulfate; The publication costs of this article were defrayed in part by page TLCK, Na-tosyllysine chloromethyl ketone. charge payment. This article must therefore be hereby marked "ad- * Present address: Department of Bacteriology and Immunology, vertisement" in accordance with 18 U. S. C. §1734 solely to indicate University of California, Berkeley, CA 94720. this fact. t To whom reprint requests should be addressed. 2138 Downloaded by guest on September 26, 2021 Biochemistry: Gayda et al. Proc. Nati. Acad. Sci. USA 76 (1979) 2139 DS410 except where strain LonMin is specifically men- tioned. For purification of minicells and labeling of plasmid pMC44-specified proteins, bacteria were grown in minimal medium M9 supplemented with 0.5% Casamino acids (4). The procedures for the isolation and labeling of minicells have been 0 described (4). To pulse label the minicells, purified minicells x were resuspended to OD620 nm of 0.5 and incubated for 5 min at 370C; then, [35S~methionine [200 approximately 400 U) AtCi/ml; C Ci/mmol (1 Ci = 3.7 X 1010 becquerels)] was added. The .C minicells were incubated for the desired time and either 20 vol 0 of 00C "stop buffer" (10 mM Tris, pH 7.4/5 mM EDTA/0.1 211 mM phenylmethylsulfonyl fluoride/0.29 mM chloramphenicol) a) or 20 vol of prewarmed fresh labeling medium containing 100 C,, mM L-methionine was added; in the latter case the incubation was continued and finally terminated by the addition of 2 vol of 00C stop buffer. To ensure rapid chilling, the mixture was swirled in a dry ice/acetone bath for 30 sec. NaDodSO4/polyacrylamide gel electrophoresis and fluo- rography were as described (4). Exposed x-ray films (Kodak Time, min XR-5) were scanned with a Joyce-Loebl microdensitometer and FIc. 1. Effect of procaine on ["5Slmethionine incorporation into the peaks were quantitated by cutting them out and weighing minicells containing plasmid pMC44. Minicell suspensions were in- them, except where stated otherwise. cubated at 37°C in the indicated concentration of procaine (added For assay of trypsin and its inhibition, hydrolysis of N- 30 sec before the addition of [35Slmethionine). Duplicate samples were (Y-benzoyl-DL-arginine p-nitroanilide was measured by fol- taken at intervals and the radioactive protein was determined (4). lowing change in absorbance at 405 nm as described (20). It has been shown that the Ki calculated for a second competitive NaDodSO4 gel electrophoresis followed by fluorography and inhibitor is not affected by the fact that a DL mixture is used as densitometer tracing. Previous studies (4) had revealed that substrate and the D form is itself a competitive inhibitor (21). 40-50% of the [35S]methionine incorporation in pMC44-con- However, apparent Vmax = Vmax/(l + Km/Ki) and apparent taining minicells was accounted for by the major outer mem- Kni = K,1(1 + Kj/Ki) in which Ki is the dissociation constant brane protein M2. The results presented in Fig. 2 reveal that, for the D form of the substrate (21). The following modifications with increasing procaine concentrations, outer membrane of the assay (20) were used: buffer was 0.1 M Tris, pH 8/0.02 protein M2 is diminished to near zero and polypeptide Ml (42 M CaC12; assays were performed at 220C; and inhibitors were kDal) replaces M2 as the major peak. Another polypeptide dissolved in H20. Trypsin was trypsin-TPCK from Worth- specified by the 2-MDal DNA fragment of pMC44 (4), poly- ington. Ki was calculated from experiments in which two or peptide M5 (25 kDal), was eliminated by piocaine and there more inhibitor concentrations were used at each of three sub- was an increase in the relative amount of polypeptides between strate concentrations. A basic computer program was used to approximately 27 and 31 kDal. Several polypeptides that are fit initial rate data for variable substrate concentrations to a present in smaller quantities than M5 and are of lower molec- hyperbolic function by an iterative least squares method, as ular weight (4) did not vary in relative amounts as a function originally proposed by Wilkinson (22). The error function is of procaine concentration although their synthesis was inhibited assumed to yield a homogeneous envelope in a hyperbolic fit. by procaine (data not shown). The data of Fig. 2 are consistent The program and advice on its use were provided by J. Westley. with the hypothesis that Ml is the precursor of M2 and that The points in Fig. 4 were experimentally determined and the proteolytic processing is inhibited by procaine. slopes were determined with the aid of the computer pro- To test this hypothesis we performed pulse-chase experi- gram. ments in several different ways. The data indicate that pulse Staphylococcus aureus V8 protease (Miles) was used as de- labeling for 5 and 15 sec followed by a 1-min chase leads to scribed (13, 23). When two doses of protease were used, one was higher amounts of Ml than does steady-state labeling; chases added to the gel pocket with the gel slice (13, 23) and a second for 10 min resulted in most of the label appearing in M2 (Table aliquot was added to the pocket when the blue dye from the gel slice had just entered the stacking gel. a M2 d Ml RESULTS M5 Ml M5 M2f Effect of Procaine on Minicells Containing Plasmid pMC44. Procaine caused increased incorporation of [35S]me- b thionine into protein of pMC44-containing minicells (Fig. 1). The initial rate of incorporation was increased approximately 4-fold by 20 mM procaine. There was an inhibition of incor- poration at 30 min that cannot be the result of lack of [35S]_ -11 methionine (Fig. 1, 60 and 80 mM). The stimulation by pro- c f caine appears to be specific for plasmid pMC44 because no change in the rate of [35S]methionine incorporation was ob- served with the parental cloning vehicle pSC101 (results not shown). FIG. 2. Effect of procaine on [35S]methionine incorporation into The effect of procaine on the quality of the proteins syn- polypeptides synthesized in minicells containing plasmid pMC44. thesized was determined by separating the polypeptides on (a-f) Procaine at 0, 20, 40, 60, 80, and 100 mM, respectively. Downloaded by guest on September 26, 2021 2140 Biochemistry: G'ayda et al. Proc. Natl. Acad. Sci. UISA 76 (1979) 1). A 15-min pulse labeling in the presence of 10() mM procaine M a h a d e f g h yielded an MI/(M1 + M2) ratio of 0.73, and much of the ra- .< 10 dioactivity in MI could be chased into M2. The extent of 42-> am y a chasing was decreased blut not eliminated by chloramphenicol. When procaine was present at a high concentration during the / . 1-min chase period but not during the 5- or 15-sec pulse, the highest ratios of M1/(MI + M2) were observed. It is difficult 18 - -> to draw any conclusion other than that MI is being processed to M2. In the pulse--chase experiments containing procaine 14->- during the pulse we detected accumulation of polypeptides in 142> x the 27- to 31-kDal range (as in Fig. 2) but there was no evidence 12 that these were chased into M5 (data not shown). ->t i sS. The fact that we could increase the amount of MI present by adding procaine allowed us to purify adequate quantities FiG. 3. Proteolysis of polypeptide MI and outer membrane of MI to compare it with M2. Minicells containing pMC44 were protein M2 with S. aureus V8 protease. Lanes a and h, 135S]methio- labeled with a mixture of 14C-labeled amino acids in the pres- nine-labeled polypeptides from minicells containing plasmid pMC44; molecular weights were determined previously (ref. 4; unpublished ence of 40 mM procaine, Ml and M2 were separated on Na- results). For preparative purposes, MI was separated from M2 by a DodSO4/polyacrylamide gel electrophoresis, and the gels, larger distance than in a and h by increasing the time of electropho- stained with Coomassie blue, were dried and sul)jected to ra- resis. Other lanes: b, MI with trace of protease; c, M2 with trace of dioautography. The upper half of the band of Ml and the lower protease; d, MI with 0.5 pg of protease; e, M2 with 0.5 pg of protease; half of the f1ind of N42 were cut out, rehydrated, transferred f, MI with 5 pg of protease (two doses); g, M2 with 5 pg of protease to slots in another gel, and digested with varying concentrations (two doses). of S. aureus V8 protease (13, 23). The results indicate that, with the highest quantity of enzyme used, six major polypeptides that the isolated minicells contained large quantities of M2 but were produced from MI that had the same mobilities as six little Ml. All other digestion experiments with similar quantities produced from M2 (Fig. 3, f and g). Two polypeptides pro- as wvell as 0.05 Atg of VS protease yielded results consistent with duced from M2 were absent from MI (Fig. 3, peptides y and those of Fig. 3. The data demonstrate that both MI and M2 are z). Partial digestion revealed two peptides produced from Ml cleaved by VS protease into at least 11 peptide fragments that that were not produced from M2 (Fig. 3, peptides w and x). All have identical mobilities, 2 that are present in Ml (Fig. 3, w and of the other major partially digested peptides produced from x) but not in M2, and 2 that are present in M2 but not in MI M2 were also produced from Ml. It is important to note that, (Fig. 3, y and z). Several peptides seen in partially digested M2 although the amount of radioactivity in M2 used for digestion (between w and x) that are not apparent in partially digested in each lane was approximately 3 times that of Ml, the chemical MI (Fig. 3) are seen on longer exposure of the fluorogram. amount of M2 may have been 20 times higher due to the fact Polypeptide MI Is Associated with the Other Membrane. pMC44-containing minicells were labeled in the absence or 'Fable 1. Relative amounts of polypel)tides MI and M2 during presence of 30 mM procaine and the outer membranes were pulse-chase experiments purified by extraction with sodium lauryl sarcosinate (Sarkosyl) followed by centrifugation on an isopycnic sucrose gradient (4). Pulse, Chase,* Mit The mean buoyant density of these outer membranes was 1.22, sec mim Other conditions (Ml + M2) as expected (4). In both normal and procaine-treated outer 5 1 None 0.27 membranes, NIl was present and the ratio of MI to M2 was the 5 10 None 0.048 same as in whole minicells. Thus, Ml was transported to the 5 1 Procaine (100 mM) in chase 0.87 outer membrane without being processed to M2 in the absence, 5 1 Chloramphenicol (1 mM) in chase ND as well as in the presence, of procaine. Effect of Trypsin on Envelope Fraction (Inner and Outer 15 1 None 0.22 Membrane Fraction). Outer membrane polypeptide M2 is 15 10 None 0.042 resistant to trypsin (24). To determine whether Ml was also 15 1 Procaine in chase 0.86 resistant to trypsin, envelope fractions from minicells containing 15 1 Chloramphenicol in chase ND procaine-amplified Ml were treated with three concentrations of trypsin (10, 50, and 100 Ag/ml) for 1 hr at 370C. Ml was 900 0 None 0.14 completely digested and a prominent 17-kDal peptide fragment 900 0 Procaine (100 mM) during pulse 0.73 appeared but Ml was not processed to M2. The quantity of 900 60 Procaine (100 mM) during pulse; 0.37 polypeptide M2 appeared to be unchanged. procaine (5 mM) in chase Effect of Temperature on Ml Processing. Protein M2 is not 900 60 Procaine (100 mM) in pulse and 0.76 foumid in the outer membrane of E. coli K-12 grown at 30'C (6). chase Why? We labeled PMC44-containing minicells (from strains 900 60 Procaine (100 mM) during pulse; 0.47 LonMin and X984) at 320C and found that, compared to la- 5 mM procaine + 1 mM beling at 37°C, synthesis of M2 was greatly decreased, little or chloramphenicol in chase no Ml was apparent, and there was a large increase in radio- * Chase always included 100 mM L,-methionine. activity in a 20.5-kDal polypeptide. However, when procaine t Polypeptides containing a total of approximately 1000 cpm of was added to pMC44-containing minicells from the LonMin a5S-labeled protein were separated by NaDodSO4/polyacrylamide strain, MI accumulated at 320C and there-was no radioactivity gel electrophoreses, detected by exposure of fluorograms for 1 week, in the 20.5-kDal The results are consistent with and quantitated. Exposure of a 5-sec-pulse sample with no chase polypeptide. (containing 700 cpm of 35S-labeled protein) for 6 weeks yielded the the interpretation that Ml is synthesized at 320C but is rapidly usual pattern of 35Slaheled polypeptides but no 35Shabeled Ml or processed to a 20.5-kDal polypeptide and possibly other smaller M2. Similar results were obtained with 5- and 15-sec-pulse samples fragments unless procaine is present to prevent this alternate chased in chloramphenicol. ND, not detected. route for processing Ml. Downloaded by guest on September 26, 2021 Biochemistry Gayda et al. Proc. Natl. Acad. Sci. USA 76 (1979) 2141

600 A 600o B Table 2. Effect of neuroactive drugs on polypeptide synthesis in 500 -33.2 mM 500 33.2 / plasmid pMC44-containing minicells and on try sin mM/ 400 400 M\ /16.6 mM Conc. 24.9 mM tested, Mit Inhibition I No inhibitor 300 16.6 mM 300 Compound* mM (MI + M2) of trypsint No inhibitor 200 200 Nople - 0.05-0.1 -2C 100 100 OKi = 42.3 mM K; = 22 mM Procaine 30 0.5 42.3 i 0.95 Procainamide 20 0.2 22.2 ± 1.2 0 ) 5 10 5 10 E Piperocaine 10 0.9 29.2 + 1.6 :I 600 C 6010-D Cocaine 25 0.6 137 + 16 500 5010 50 0.9 16.6 mM 16.6 mM Lidocaine 15 0.6 No inhibition 4010 30 0.9 400 K, 8.3 mM 3010 8.3 mM 23.3 ± 2.7 2No inhibitor Atropine 10 0.6 20 -No 20 0.9 Neostigmine 15-30 0.05 33.1 ± 3.2 100 K= 10 137 0 = 33. lmM Carbamoylcholine 3--30 0.05 Not done 20 0.5 0.0166§ 0 5 10 0 5 10 Benzamidine 1/[S], mM-, TLCK 4 0.9 1 * Minicells of strain DS410 containing plasmid pMC44 were labeled Flc. 4. Competitive inhibition of trypsin by procaine (A), pro- cainamide (B), cocaine (C), and neostigmine (D). V is in Mrmol/min. with [KS]methionine for 20 min at 370C in the presence of drug. t The ratio M1/(M1 + M2) was determined by visual inspection of [S] is mM. appropriately exposed fluorograms. In many instances, both higher and lower concentrations of drug were tested and yielded results consistent with those reported. Effect of Anesthetics and Other Neuroactive Drugs on Dissociation constants (Ki in mM) indicated (±SEM) were deter- Proteolytic Processing of MI to M2 and on Trypsin. Several mined from data of Fig. 4 and similar experiments. Intercepts were local anesthetics, including cocaine, procainamide, piperocaine, identical, within the SEM for all concentrations of the inhibitors and lidocaine, inhibited conversion of Ml to M2 (Table 2). tested. Atropine also inhibited conversion of MI to M2 but neither § From ref. 25. TLCK is an active site titrant of trypsin (25). neostigmine nor carbamoylcholine was inhibitory. The well- known trypsin inhibitors benzamidine and TLCK were also active. When we considered the ester or amide bond present in the local anesthetic molecules, it appeared that they might There is little polypeptide with the electrophoretic mobility be active not only because they were lipophilic and perturbed of MI normally found in outer membrane preparations of membranes but also because they might be specific inhibitors whole cells of E. coli K-12 (unpublished results). However, of a trypsin-like activity. The results of Fig. 4 show that pro- when isolated minicells containing plasmid pMC44 were la- caine, procainamide, cocaine, and neostigmine are indeed beled with [""SIniethionine at 370(C, in the presence or absence competitive inhibitors of trypsin. Table 2 presents the calculated of 30 mM procaine, both radioactive MI and M2 were found dissociation constant (Ki) for the compounds tested in Fig. 4 as in the purified outer membrane preparations. The ratio of Ml well as other compounds that are also competitive inhibitors to M2 in the outer membrane fraction was approximately the of trypsin. The only local anesthetic that was not an inhibitor same as in the whole mninicells. Thus, MI was translocated and of trypsin but inhibited conversion of MI to M2 was lido- assembled into the outer membrane of minicells without nec- caine. essarily being processed at 370C. Our pulse-chase studies with and without procaine (Table 1) indicate that most of the MI can be chased into NM2 but similar experiments with purified outer DISCUSSION membrane wvould be required to prove that those molecules of We have presented the following evidence that polypeptide MI in the outer membrane are in fact chased into M2 in the Ml (42 kDal) is the precursor of outer membrane protein M2 outer membrane. However, the fact that the M1/M2 ratio (40 kDal). Polypeptide MI, as well as polypeptide M2, is found synthlesize(I is the same in the purified outer membranes as in in purified preparations of outer membrane. Pulse-chase ex- the whole minicells is consistent with the expectation that MI periments with minicells containing the structural genes for MI is first translocated to the outer membrane and is then processed and M2 are difficult to interpret in any way other than that MI to M2. is converted to M2 (Table 1). Inhibitors of proteolysis including Protein M2 was not found in the outer membrane of E. coli procaine, benzamidine, and TLCK prevented the appearance K-12 grown at 30'C (6). Why not? Ion (capR) strains are de- of M2 and caused MI to accumulate. Purified M2 and MI were fective in degrading bo-th nonsense and missense polypeptides both cleaved by S. aureus V8 protease to a series of polypeptides (9-11). In unpublished studies we had determined that with identical mobilities, and intermediate digestion revealed pMC44-containing minicells from a rninicell strain that contains two polypeptides unique to MI as well as two unique to M2 a Ion (capR) mutation (strain LonMin) incorporates equal (Fig. 3). The location of the extra 2-kDal segment in MI was amounts of radioactivity into MI anid N12 after labeling at 370C. riot determined. The localization of MI to the outer membrane When such minicells were labeled at 300C, little M2 and a trace as well as the knowledge that the precursor of an outer mem- of M I were (Ietecte(1 together with large amounts of a 20.5-kDal brane protein, the lipoprotein, contains the extra peptide on the polypeptide; when procaine was added, Ml accumulated and NH2 terminus (5) leads us to believe that the extra peptide of none of the 20.5-kDal polypeptide appeared. These results are MI is at the NH2 terminus and may be a signal sequence (16). consistent with the imlterlpretation that at 30'C MI is rapidly Only amino acid sequence analysis of the proteins will prove processed to a 20.5-kDal polypeptide unless procaine is present this point. to Iblock this alternate route for processing Ml. Therefore, it Downloaded by guest on September 26, 2021 2142 Biochemistry: Gayda et al. Proc. Natl. Acad. Sci. USA 76 (1979) appears that M2 is not detected in E. coli K-12 grown at 30'C We appreciate the advice of J. Westley and F. J. Kezdy on assays of (6) because its precursor, Ml, is processed not to M2 but to other trypsin and its inhibition. We thank J. Westley, F. J. Kezdy, L. S. product(s), possibly a 20.5-kDal polypeptide as well as other Seiden, and H. Nikaido for suggestions concerning the manuscript. The polypeptides. inspiration of Diane C. Markovitz is acknowledged. This research was to seek a that prevented conversion supported by U.S. Public Health Service Grant AI-06966 from the Our studies compound National Institute of Allergy and Infectious Diseases (to A.M.) and of MI to M2 led to the discovery that procaine was effective. American Cancer Society Grants VC116 (to A.M.) and PF1296 (to Furthermore, total incorporation of [35S]methionine was R.C.G.). stimulated by procaine in minicells containing plasmid pMC44 (Fig. 1), and most of this incorporation was into MI and M2 1. Lugtenberg, B., Meyers, J., Peters, R., van der Hoek, R. & van (Fig. 2). If the stimulation were due simply to increased trans- Alphen, L. (1975) FEBS Lett. 58, 254-258. port of [35Sjmethionine, we would have expected procaine to 2. Lugtenberg, B., Peters, R., Bernheimer, H. & Berendser, W. stimulate incorporation in minicells containing other plasmids (1976) Mol. Gen. Cenet. 147, 251-263. 3. Bassford, P. J., Diedrich, D. L., Schnaitman, C. L. & Reeves, P. such as the cloning vehicle used to construct pMC44 (i.e., (1977) J. Bacteriol. 131, 608-622. pSC101); procaine did not do this. We have found that, in 4. Gayda, R. C. & Markovitz, A. (1978) J. Bacteriol. 136, 369- minicells, functional mRNA for M1/M2 synthesis appears to 380. be more stable in the presence of rifampicin than mRNA for 5. DiRienzo, J. M., Nakamura, K. & Inouye, M. (1978) Annu. Rev. polyppptides coded for by the pSC101 portion of pMC44 (4). Biochem. 47, 481-532. These results lead us to suggest the possibility of a rifampicin- 6. Manning, P. A. & Reeves, P. (1977) FEMS (Fed. Eur. Microbiol. excluding "compartment" for Ml/M2 mRNA, perhaps asso- Soc.) Microbiol. Lett. 1, 275-278. ciated with membrane-bound polysomes (13). The apparent 7. Berg, P. E., Gayda, R., Avni, H., Zehnbauer, B. & Markovitz, A. delay in synthesis of Ml and M2 during short pulses of (1976) Proc. Natl. Acad. Sci. USA 73, 697-701. [35S]_ 8. Markovitz, A. (1977) in Surface Carbohydrates of the Procaryotic methionine compared to other plasmid-coded polypeptides Cell, ed. Sutherland, I. (Academic, New York), pp. 415-462. (Table 1) supports the idea of a compartment. Such a com- 9. Shineberg, J. B. & Zipser, D. (1973) J. Bacteriol. 116, 1469- prtment might be made more accessible to [V5Slmethionine 1471. by procaine and could account for the increased specific in- 10. Kowit, J. D. & Goldberg, A. L. (1977) J. Biol. Chem. 252, corporation. Further studies are required to determine the basis 8350-8357. of the separate effect of stimulation of [35S]methionine incor- 11. Gottesman, S. & Zipser, D. (1978) J. Bacteriol. 133, 844-851. poration by procaine. 12. Inouye, H. I. & Beckwith, J. (1977) Proc. Natl. Acad. Sci. USA Procaine was previously shown to prevent the formation of 74, 1440-1444. active alkaline a periplasmic en- 13. Randall, L. R., Hardy, S. J. S. & Josefsson, L. (1978) Proc. Natl. epzymatically phosphatase, Acad. Sci. USA 75, 1209-1212. zyme in E. coli, but not the formation of inactive monomers 14. Lin, J. J., Kanazawa, H., Ozols, J. & Wu, H. C. (1978) Proc. Natl. (26). In view of subsequent work indicating that alkaline Acad. Sci. USA 75, 4891-4895. pbosphatase monomer is synthesized as a precursor protein (12) 15. Traficante, L. J. & Lampen, J. 0. (1977) J. Bacteriol. 129, apd our results, it seems likely that procaine was preventing 184-190. processing of alkaline phosphatase precursor. 16. Blobel, G. & Dobberstein, B. (1975) J. Cell Biol. 67, 835-851. Perhaps the most significant portion of the present work is 17. Reeve, J. N. (1977) Mol. Gen. Genet. 158, 73-79. the discovery that a number of neuroactive compounds that 18. Markovitz, A. & Rosenbaum, N. (1965) Proc. NatI. Acad. Sci. have lipophilic moieties as well as ester or amide linkages inhibit USA 54, 1084-1091. proteolytic conversion of MI to M2 in pMC44-containing 19. Frazer, A. C. & Curtiss, R., III (1976) in Current Topics in Mi- crobiology and Immnunolgoy, eds. Arber, W., et al. (Springer, minicells and that these same compounds, with the exception New York), Vol. 69, pp. 1-84. of lidocaine, are competitive inhibitors of trypsin. These com- 20. Kassell, B. (1970) Methods Enzymol. 19, 844-852. pounds may be useful in studies of protein processing in eu- 21. Mares-Guia, M. & Shaw, E. (1965) J. Biol. Chem. 240, 1579- karyotic cells; they have been selected for their low toxicity in 1585. comparison with literally thousands of compounds (27). There 22. Wilkinson, G. N. (1961) Biochenm. J. 80, 324-332. are reports that local anesthetics inhibit lipid breakdown in fat 23. Cleveland, D. W., Fischer, S. G., Kirschner, NI. W. & Laemmli, ells of humans (28). Local anesthetics have also been shown U. K. (1977) J. Biol. Chem. 252, 1102-1106. to affect the mobility and distribution of cell surface receptors, 24. Nakamura, K. & Mizushima, S. (1976) J. Biochem. (Tokyo) 80, via their effects on microtubules and microfilaments 1411-1422. po)ssibly 25. Walsh, K. A. (1970) Methods Enzymol. 19, 41-63. (29). Perhaps a proteolytic reaction is retrospectively implicated 26. Tribhuwan. R. C. & Pradhan, D. S. (1977) J. Bacteriol. 131, in some of these reactions on the basis of our results. It now 431-437. appears that serious consideration should be given to the pos- 27. Goodman, L. S. & Gilman, A. (1975) The Pharmacological Basis sibility that the mechanism(s) of molecular action of these of Therapeutics (Macmillan, New York). neuroactive compounds in animals may involve inhibition of 28. Arner, P., ArnerO. & Ostman, J. (1973) Life Sci. 13,161-169. a hypothetical trypsin-like reaction at cell surfaces or mem- 29. Poste, G., Papahadjopoulos, D. & Nicolson, C. L. (1975) Proc. branes. Nadt. Acad. Sci. USA 72, 4430-4434. Downloaded by guest on September 26, 2021