Proc. Natl. Acad. Sci. USAVol. 76, No. 5, pp. 2138-2142, May 1979Biochemistry

Neuroactive drugs inhibit trypsin and outer membrane proteinprocessing in Escherichia coli K-12

(procaine/cocaine/neostigmine/atropine/DNA cloning)

RANDALL C. GAYDA*, GORDON W. HENDERSON, AND ALVIN MARKOVITZtDepartment of Microbiology, University of Chicago, Chicago, Illinois 60637

Communicated by Josef Fried, January 29, 1979

ABSTRACT Previous studies demonstrated that a cloned2-megadalton (MDal) fragment of Escherichia coli DNA con-tained the structural gene for major outer membrane proteina (also known as 3b or M2 (40 kDal). The present study demon-strates that M2 is synthesized from a 42-kDal precursor that alsois present in the outer membrane. The conversion of the 42-kDalprecursor to M2 is inhibited by a number of different local an-esthetics (procaine, piperocaine, lidocaine, cocaine), by theneuroactive drug atropine, and by the classical try in inhibitorsNa-tosyllysine chloromethyl ketone (TLCK) and benzamidine.Our kinetic studies demonstrate that the amidase action of puretrypsin is inhibited competitively by the local anesthetics tested(excluding lidocaine) as well as by atropine and neostigmine.A mechanism of action for local anesthetics as well as atropinein E. coli may be to inhibit trypsinlike proteases, in a competi-tive manner, in the region of the outer membrane. The mecha-nism of action of these compounds in regulating nerve con-duction in man may have certain features in common with themechanism proposed in E. coli.

One of the major outer membrane proteins of Escherchia coliK-12 is the 40-kilodalton (kDal) protein a (1, 2) [also known as3b (3) and M2 (4); see ref. 5 for review]. Protein a is detectedin the outer membrane of strains grown at 37°C but not at 30°C(2, 6). A 2-MDal cloned fragment of E. coli K-12 DNA containsthe structural gene for protein M2 (4, 7), and studies withplasmid mutants demonstrated that this protein is importantin repressing the synthesis of the capsular polysaccharide (8)in lon (capR) strains that overproduce the polysaccharide(unpublished data). The 2-MDal DNA fragment was originallyisolated by using the cloning vehicle pSC101, the resultantplasmid being designated pMC44. Plasmid pMC44-codedproteins were determined by transforming a minicell-pro-ducing mutant to tetracycline resistance with the DNA, iso-lating the minicells, and incubating them in [-5S]methionine.Polypeptides were separated in sodium dodecyl sulfate (Na-DodSO4) polyacrylamide gels by electrophoresis. A 42-kDalpolypeptide, designated Ml, was also produced by plasmidpMC44-containing minicells. Several observations suggestedthat Ml is a precursor of the 40-kDal polypeptide M2: plasmidmutants were obtained that specified neither Ml nor M2 (un-published data); the amount of Ml varied from 0 to 10% of theamount of M2; the amount of MI was influenced by the pres-ence of tetracycline during [-5S]methionine-labeling as well asby the complexity of the medium in which the minicell-pro-ducing strain was grown (4). In addition, when plasmid pMC44was in a minicell-producing strain that contained a Ion (capR)mutation that decreases proteolysis of nonsense and missenseprotein fragments (9-11), the amount of Ml detected by[35S]methionine-labeling of minicells was approximately equalto that of M2 (unpublished data).

It is now established that precursor proteins exist for anumber of bacterial proteins that are located outside the innermembrane as periplasmic proteins (12, 13), proteins of the outermembrane (5, 13, 14), or extracellular enzymes (15). Most ofthese precursors are approximately 2 kDal larger than themature protein and, where it is known, the portion removedproteolytically is at the NH2 terminus. The best known exampleis the precursor of the lipoprotein, which contains an additionalNH2-terminal sequence of 20 amino acids (5). The signal hy-pothesis was proposed by Blobel and Dobberstein (16) to explainthe mechanism of transport of proteins through the cell mem-brane of eukaryotes and is currently applied to work withbacteria. This hypothesis proposes that the extra NH2-terminalpeptide acts as a signal to provide a ribosome-membranejunction and thus provides a topology for translational as wellas unidirectional transport of a polypeptide chain through amembrane. There is some question as to whether or not thesignal sequence must be processed during transport. The in vivodetection of the precursor of arabinose-binding protein (13) anda mutant prolipoprotein (14) suggests that processing can occurafter transport is complete.

In this report we demonstrate that the 42-kDal polypeptideMl is the precursor of the 40-kDal outer membrane polypeptideM2. Furthermore, the precursor can be found in the outermembrane fraction. In the course of these studies we examinedthe effect of a number of local anesthetics and other neuroactivedrugs, as well as classical trypsin inhibitors, on the conversionof MI to M2 in minicells containing plasmid pMC44. Many ofthese compounds inhibited the processing and most were shownto be competitive inhibitors of trypsin.

MATERIALS AND METHODSMaterials were obtained as described (4) or from the followingsources: cocaine HCl, a gift of C. R. Schuster; procaine HC1,procainamide HC1, neostigmine bromide, atropine sulfate,carbamoylcholine chloride, Na-tosyllysine chloromethyl ketone(TLCK), and benzamidine HC1, Sigma; lidocaine HCl, a giftfrom H. G. Vassallo (Astra Pharmaceutical Products, Fram-ingham, MA); piperocaine HC1, Lilly.The minicell-producing strain DS410 (17) was supplied by

D. Mount. Strain LonMin was provided by H. Adler and wasprepared by conjugation between Hfr strain M6 [a capR6 (Ion)mutant] (18) and minicell-producing strain X925 (19). TheLonMin strain is mucoid, is UV sensitive [i.e., lon (capR)], andretains the ability to produce minicells. Plasmid pMC44 DNAwas transferred to strains DS410 and LonMin by transformation(7). All experiments were performed with minicells from strain

Abbreviations: Dal, daltons; NaDodSO4, sodium dodecyl sulfate;TLCK, Na-tosyllysine chloromethyl ketone.* Present address: Department of Bacteriology and Immunology,University of California, Berkeley, CA 94720.

t To whom reprint requests should be addressed.

2138

The publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be hereby marked "ad-vertisement" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

Proc. Nati. Acad. Sci. USA 76 (1979) 2139

DS410 except where strain LonMin is specifically men-tioned.

For purification of minicells and labeling of plasmidpMC44-specified proteins, bacteria were grown in minimalmedium M9 supplemented with 0.5% Casamino acids (4). Theprocedures for the isolation and labeling of minicells have beendescribed (4). To pulse label the minicells, purified minicellswere resuspended to OD620 nm of 0.5 and incubated for 5 minat 370C; then, [35S~methionine [200 AtCi/ml; approximately 400Ci/mmol (1 Ci = 3.7 X 1010 becquerels)] was added. Theminicells were incubated for the desired time and either 20 volof 00C "stop buffer" (10 mM Tris, pH 7.4/5 mM EDTA/0.1mM phenylmethylsulfonyl fluoride/0.29 mM chloramphenicol)or 20 vol of prewarmed fresh labeling medium containing 100mM L-methionine was added; in the latter case the incubationwas continued and finally terminated by the addition of 2 volof 00C stop buffer. To ensure rapid chilling, the mixture wasswirled in a dry ice/acetone bath for 30 sec.

NaDodSO4/polyacrylamide gel electrophoresis and fluo-rography were as described (4). Exposed x-ray films (KodakXR-5) were scanned with a Joyce-Loebl microdensitometer andthe peaks were quantitated by cutting them out and weighingthem, except where stated otherwise.

For assay of trypsin and its inhibition, hydrolysis of N-(Y-benzoyl-DL-arginine p-nitroanilide was measured by fol-lowing change in absorbance at 405 nm as described (20). It hasbeen shown that the Ki calculated for a second competitiveinhibitor is not affected by the fact that a DL mixture is used assubstrate and the D form is itself a competitive inhibitor (21).However, apparent Vmax = Vmax/(l + Km/Ki) and apparentKni = K,1(1 + Kj/Ki) in which Ki is the dissociation constantfor the D form of the substrate (21). The following modificationsof the assay (20) were used: buffer was 0.1 M Tris, pH 8/0.02M CaC12; assays were performed at 220C; and inhibitors weredissolved in H20. Trypsin was trypsin-TPCK from Worth-ington. Ki was calculated from experiments in which two ormore inhibitor concentrations were used at each of three sub-strate concentrations. A basic computer program was used tofit initial rate data for variable substrate concentrations to ahyperbolic function by an iterative least squares method, asoriginally proposed by Wilkinson (22). The error function isassumed to yield a homogeneous envelope in a hyperbolic fit.The program and advice on its use were provided by J. Westley.The points in Fig. 4 were experimentally determined and theslopes were determined with the aid of the computer pro-gram.

Staphylococcus aureus V8 protease (Miles) was used as de-scribed (13, 23). When two doses of protease were used, one wasadded to the gel pocket with the gel slice (13, 23) and a secondaliquot was added to the pocket when the blue dye from the gelslice had just entered the stacking gel.

RESULTSEffect of Procaine on Minicells Containing Plasmid

pMC44. Procaine caused increased incorporation of [35S]me-thionine into protein of pMC44-containing minicells (Fig. 1).The initial rate of incorporation was increased approximately4-fold by 20 mM procaine. There was an inhibition of incor-poration at 30 min that cannot be the result of lack of [35S]_methionine (Fig. 1, 60 and 80 mM). The stimulation by pro-caine appears to be specific for plasmid pMC44 because nochange in the rate of [35S]methionine incorporation was ob-served with the parental cloning vehicle pSC101 (results notshown).The effect of procaine on the quality of the proteins syn-

thesized was determined by separating the polypeptides on

0

x

U)C.C0211

a)C,,

Time, min

FIc. 1. Effect of procaine on ["5Slmethionine incorporation intominicells containing plasmid pMC44. Minicell suspensions were in-cubated at 37°C in the indicated concentration of procaine (added30 sec before the addition of [35Slmethionine). Duplicate samples weretaken at intervals and the radioactive protein was determined (4).

NaDodSO4 gel electrophoresis followed by fluorography anddensitometer tracing. Previous studies (4) had revealed that40-50% of the [35S]methionine incorporation in pMC44-con-taining minicells was accounted for by the major outer mem-brane protein M2. The results presented in Fig. 2 reveal that,with increasing procaine concentrations, outer membraneprotein M2 is diminished to near zero and polypeptide Ml (42kDal) replaces M2 as the major peak. Another polypeptidespecified by the 2-MDal DNA fragment of pMC44 (4), poly-peptide M5 (25 kDal), was eliminated by piocaine and therewas an increase in the relative amount of polypeptides betweenapproximately 27 and 31 kDal. Several polypeptides that arepresent in smaller quantities than M5 and are of lower molec-ular weight (4) did not vary in relative amounts as a functionof procaine concentration although their synthesis was inhibitedby procaine (data not shown). The data of Fig. 2 are consistentwith the hypothesis that Ml is the precursor of M2 and thatproteolytic processing is inhibited by procaine.To test this hypothesis we performed pulse-chase experi-

ments in several different ways. The data indicate that pulselabeling for 5 and 15 sec followed by a 1-min chase leads tohigher amounts of Ml than does steady-state labeling; chasesfor 10 min resulted in most of the label appearing in M2 (Table

a M2 d

M5 Ml

b

Ml

M5 M2f

c

FIG. 2. Effect of procaine on [35S]methionine incorporation intopolypeptides synthesized in minicells containing plasmid pMC44.(a-f) Procaine at 0, 20, 40, 60, 80, and 100 mM, respectively.

Biochemistry: Gayda et al.

-11

f

Proc. Natl. Acad. Sci. UISA 76 (1979)

1). A 15-min pulse labeling in the presence of 10() mM procaineyielded an MI/(M1 + M2) ratio of 0.73, and much of the ra-dioactivity in MI could be chased into M2. The extent ofchasing was decreased blut not eliminated by chloramphenicol.When procaine was present at a high concentration during the1-min chase period but not during the 5- or 15-sec pulse, thehighest ratios of M1/(MI + M2) were observed. It is difficultto draw any conclusion other than that MI is being processedto M2. In the pulse--chase experiments containing procaineduring the pulse we detected accumulation of polypeptides inthe 27- to 31-kDal range (as in Fig. 2) but there was no evidencethat these were chased into M5 (data not shown).The fact that we could increase the amount of MI present

by adding procaine allowed us to purify adequate quantitiesof MI to compare it with M2. Minicells containing pMC44 werelabeled with a mixture of 14C-labeled amino acids in the pres-ence of 40 mM procaine, Ml and M2 were separated on Na-DodSO4/polyacrylamide gel electrophoresis, and the gels,stained with Coomassie blue, were dried and sul)jected to ra-dioautography. The upper half of the band of Ml and the lowerhalf of the f1ind of N42 were cut out, rehydrated, transferredto slots in another gel, and digested with varying concentrationsof S. aureus V8 protease (13, 23). The results indicate that, withthe highest quantity of enzyme used, six major polypeptideswere produced from MI that had the same mobilities as sixproduced from M2 (Fig. 3, f and g). Two polypeptides pro-duced from M2 were absent from MI (Fig. 3, peptides y andz). Partial digestion revealed two peptides produced from Mlthat were not produced from M2 (Fig. 3, peptides w and x). Allof the other major partially digested peptides produced fromM2 were also produced from Ml. It is important to note that,although the amount of radioactivity in M2 used for digestionin each lane was approximately 3 times that of Ml, the chemicalamount of M2 may have been 20 times higher due to the fact

'Fable 1. Relative amounts of polypel)tides MI and M2 duringpulse-chase experiments

Pulse, Chase,* Mitsec mim Other conditions (Ml + M2)

5 1 None 0.275 10 None 0.0485 1 Procaine (100 mM) in chase 0.875 1 Chloramphenicol (1 mM) in chase ND

15 1 None 0.2215 10 None 0.04215 1 Procaine in chase 0.8615 1 Chloramphenicol in chase ND

900 0 None 0.14900 0 Procaine (100 mM) during pulse 0.73900 60 Procaine (100 mM) during pulse; 0.37

procaine (5 mM) in chase900 60 Procaine (100 mM) in pulse and 0.76

chase900 60 Procaine (100 mM) during pulse; 0.47

5 mM procaine + 1 mMchloramphenicol in chase

* Chase always included 100 mM L,-methionine.t Polypeptides containing a total of approximately 1000 cpm of

a5S-labeled protein were separated by NaDodSO4/polyacrylamidegel electrophoreses, detected by exposure of fluorograms for 1 week,and quantitated. Exposure of a 5-sec-pulse sample with no chase(containing 700 cpm of 35S-labeled protein) for 6 weeks yielded theusual pattern of 35Slaheled polypeptides but no 35Shabeled Ml orM2. Similar results were obtained with 5- and 15-sec-pulse sampleschased in chloramphenicol. ND, not detected.

M a h a d e f g h.< 1042-> am y a

/ .

18 - ->

14->-142> x

12 ->t i sS.

FiG. 3. Proteolysis of polypeptide MI and outer membraneprotein M2 with S. aureus V8 protease. Lanes a and h, 135S]methio-nine-labeled polypeptides from minicells containing plasmid pMC44;molecular weights were determined previously (ref. 4; unpublishedresults). For preparative purposes, MI was separated from M2 by alarger distance than in a and h by increasing the time of electropho-resis. Other lanes: b, MI with trace of protease; c, M2 with trace ofprotease; d, MI with 0.5 pg of protease; e, M2 with 0.5 pg of protease;f, MI with 5 pg of protease (two doses); g, M2 with 5 pg of protease(two doses).

that the isolated minicells contained large quantities of M2 butlittle Ml. All other digestion experiments with similar quantitiesas wvell as 0.05 Atg of VS protease yielded results consistent withthose of Fig. 3. The data demonstrate that both MI and M2 arecleaved by VS protease into at least 11 peptide fragments thathave identical mobilities, 2 that are present in Ml (Fig. 3, w andx) but not in M2, and 2 that are present in M2 but not in MI(Fig. 3, y and z). Several peptides seen in partially digested M2(between w and x) that are not apparent in partially digestedMI (Fig. 3) are seen on longer exposure of the fluorogram.

Polypeptide MI Is Associated with the Other Membrane.pMC44-containing minicells were labeled in the absence orpresence of 30 mM procaine and the outer membranes werepurified by extraction with sodium lauryl sarcosinate (Sarkosyl)followed by centrifugation on an isopycnic sucrose gradient (4).The mean buoyant density of these outer membranes was 1.22,as expected (4). In both normal and procaine-treated outermembranes, NIl was present and the ratio of MI to M2 was thesame as in whole minicells. Thus, Ml was transported to theouter membrane without being processed to M2 in the absence,as well as in the presence, of procaine.

Effect of Trypsin on Envelope Fraction (Inner and OuterMembrane Fraction). Outer membrane polypeptide M2 isresistant to trypsin (24). To determine whether Ml was alsoresistant to trypsin, envelope fractions from minicells containingprocaine-amplified Ml were treated with three concentrationsof trypsin (10, 50, and 100 Ag/ml) for 1 hr at 370C. Ml wascompletely digested and a prominent 17-kDal peptide fragmentappeared but Ml was not processed to M2. The quantity ofpolypeptide M2 appeared to be unchanged.

Effect of Temperature on Ml Processing. Protein M2 is notfoumid in the outer membrane of E. coli K-12 grown at 30'C (6).Why? We labeled PMC44-containing minicells (from strainsLonMin and X984) at 320C and found that, compared to la-beling at 37°C, synthesis of M2 was greatly decreased, little orno Ml was apparent, and there was a large increase in radio-activity in a 20.5-kDal polypeptide. However, when procainewas added to pMC44-containing minicells from the LonMinstrain, MI accumulated at 320C and there-was no radioactivityin the 20.5-kDal polypeptide. The results are consistent withthe interpretation that Ml is synthesized at 320C but is rapidlyprocessed to a 20.5-kDal polypeptide and possibly other smallerfragments unless procaine is present to prevent this alternateroute for processing Ml.

2140 Biochemistry: G'ayda et al.

Proc. Natl. Acad. Sci. USA 76 (1979) 2141

600o BA

-33.2 mM

24.9 mM16.6 mM

No inhibitor

OKi = 42.3 mM

) 5 10

600 C

50016.6 mM

400 K, 8.3 mM

2No inhibitor

100 K= 137

0 5 10

500

400

300

200

100

60

50

40

30

20

10

33.2 /mM/M\ /16.6 mMI No inhibitor

K; = 22 mM

5 10

10-D

1016.6 mM10

10 8.3 mM

-No

0 = 33.lmM

0 5 101/[S], mM-,

Flc. 4. Competitive inhibition of trypsin by procaine (A), pro-

cainamide (B), cocaine (C), and neostigmine (D). V is in Mrmol/min.[S] is mM.

Effect of Anesthetics and Other Neuroactive Drugs on

Proteolytic Processing of MI to M2 and on Trypsin. Severallocal anesthetics, including cocaine, procainamide, piperocaine,and lidocaine, inhibited conversion of Ml to M2 (Table 2).Atropine also inhibited conversion of MI to M2 but neitherneostigmine nor carbamoylcholine was inhibitory. The well-known trypsin inhibitors benzamidine and TLCK were alsoactive. When we considered the ester or amide bond presentin the local anesthetic molecules, it appeared that they mightbe active not only because they were lipophilic and perturbedmembranes but also because they might be specific inhibitorsof a trypsin-like activity. The results of Fig. 4 show that pro-

caine, procainamide, cocaine, and neostigmine are indeedcompetitive inhibitors of trypsin. Table 2 presents the calculateddissociation constant (Ki) for the compounds tested in Fig. 4 as

well as other compounds that are also competitive inhibitorsof trypsin. The only local anesthetic that was not an inhibitorof trypsin but inhibited conversion of MI to M2 was lido-caine.

DISCUSSIONWe have presented the following evidence that polypeptideMl (42 kDal) is the precursor of outer membrane protein M2(40 kDal). Polypeptide MI, as well as polypeptide M2, is foundin purified preparations of outer membrane. Pulse-chase ex-

periments with minicells containing the structural genes for MIand M2 are difficult to interpret in any way other than that MIis converted to M2 (Table 1). Inhibitors of proteolysis includingprocaine, benzamidine, and TLCK prevented the appearanceof M2 and caused MI to accumulate. Purified M2 and MI were

both cleaved by S. aureus V8 protease to a series of polypeptideswith identical mobilities, and intermediate digestion revealedtwo polypeptides unique to MI as well as two unique to M2(Fig. 3). The location of the extra 2-kDal segment in MI was

riot determined. The localization of MI to the outer membraneas well as the knowledge that the precursor of an outer mem-brane protein, the lipoprotein, contains the extra peptide on theNH2 terminus (5) leads us to believe that the extra peptide ofMI is at the NH2 terminus and may be a signal sequence (16).Only amino acid sequence analysis of the proteins will prove

this point.

Table 2. Effect of neuroactive drugs on polypeptide synthesis inplasmid pMC44-containing minicells and on try sin

Conc.tested, Mit Inhibition

Compound* mM (MI + M2) of trypsint

Nople - 0.05-0.1Procaine 30 0.5 42.3 i 0.95Procainamide 20 0.2 22.2 ± 1.2Piperocaine 10 0.9 29.2 + 1.6Cocaine 25 0.6 137 + 16

50 0.9Lidocaine 15 0.6 No inhibition

30 0.9Atropine 10 0.6 23.3 ± 2.7

20 0.9Neostigmine 15-30 0.05 33.1 ± 3.2Carbamoylcholine 3--30 0.05 Not doneBenzamidine 20 0.5 0.0166§TLCK 4 0.9 1

* Minicells of strain DS410 containing plasmid pMC44 were labeledwith [KS]methionine for 20 min at 370C in the presence of drug.

t The ratio M1/(M1 + M2) was determined by visual inspection ofappropriately exposed fluorograms. In many instances, both higherand lower concentrations of drug were tested and yielded resultsconsistent with those reported.Dissociation constants (Ki in mM) indicated (±SEM) were deter-mined from data of Fig. 4 and similar experiments. Intercepts wereidentical, within the SEM for all concentrations of the inhibitorstested.

§ From ref. 25.TLCK is an active site titrant of trypsin (25).

There is little polypeptide with the electrophoretic mobilityof MI normally found in outer membrane preparations ofwhole cells of E. coli K-12 (unpublished results). However,when isolated minicells containing plasmid pMC44 were la-beled with [""SIniethionine at 370(C, in the presence or absenceof 30 mM procaine, both radioactive MI and M2 were foundin the purified outer membrane preparations. The ratio of Mlto M2 in the outer membrane fraction was approximately thesame as in the whole mninicells. Thus, MI was translocated andassembled into the outer membrane of minicells without nec-essarily being processed at 370C. Our pulse-chase studies withand without procaine (Table 1) indicate that most of the MI canbe chased into NM2 but similar experiments with purified outermembrane wvould be required to prove that those molecules ofMI in the outer membrane are in fact chased into M2 in theouter membrane. However, the fact that the M1/M2 ratiosynthlesize(I is the same in the purified outer membranes as inthe whole minicells is consistent with the expectation that MIis first translocated to the outer membrane and is then processedto M2.

Protein M2 was not found in the outer membrane of E. coliK-12 grown at 30'C (6). Why not? Ion (capR) strains are de-fective in degrading bo-th nonsense and missense polypeptides(9-11). In unpublished studies we had determined thatpMC44-containing minicells from a rninicell strain that containsa Ion (capR) mutation (strain LonMin) incorporates equalamounts of radioactivity into MI anid N12 after labeling at 370C.When such minicells were labeled at 300C, little M2 and a traceof M I were (Ietecte(1 together with large amounts of a 20.5-kDalpolypeptide; when procaine was added, Ml accumulated andnone of the 20.5-kDal polypeptide appeared. These results areconsistent with the imlterlpretation that at 30'C MI is rapidlyprocessed to a 20.5-kDal polypeptide unless procaine is presentto Iblock this alternate route for processing Ml. Therefore, it

600

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400

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0E:I

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(6) because its precursor, Ml, is processed not to M2 but to otherproduct(s), possibly a 20.5-kDal polypeptide as well as otherpolypeptides.Our studies to seek a compound that prevented conversion

of MI to M2 led to the discovery that procaine was effective.Furthermore, total incorporation of [35S]methionine wasstimulated by procaine in minicells containing plasmid pMC44(Fig. 1), and most of this incorporation was into MI and M2(Fig. 2). If the stimulation were due simply to increased trans-port of [35Sjmethionine, we would have expected procaine tostimulate incorporation in minicells containing other plasmidssuch as the cloning vehicle used to construct pMC44 (i.e.,pSC101); procaine did not do this. We have found that, inminicells, functional mRNA for M1/M2 synthesis appears tobe more stable in the presence of rifampicin than mRNA forpolyppptides coded for by the pSC101 portion of pMC44 (4).These results lead us to suggest the possibility of a rifampicin-excluding "compartment" for Ml/M2 mRNA, perhaps asso-ciated with membrane-bound polysomes (13). The apparentdelay in synthesis of Ml and M2 during short pulses of [35S]_methionine compared to other plasmid-coded polypeptides(Table 1) supports the idea of a compartment. Such a com-prtment might be made more accessible to [V5Slmethionineby procaine and could account for the increased specific in-corporation. Further studies are required to determine the basisof the separate effect of stimulation of [35S]methionine incor-poration by procaine.

Procaine was previously shown to prevent the formation ofepzymatically active alkaline phosphatase, a periplasmic en-zyme in E. coli, but not the formation of inactive monomers(26). In view of subsequent work indicating that alkalinepbosphatase monomer is synthesized as a precursor protein (12)apd our results, it seems likely that procaine was preventingprocessing of alkaline phosphatase precursor.

Perhaps the most significant portion of the present work isthe discovery that a number of neuroactive compounds thathave lipophilic moieties as well as ester or amide linkages inhibitproteolytic conversion of MI to M2 in pMC44-containingminicells and that these same compounds, with the exceptionof lidocaine, are competitive inhibitors of trypsin. These com-pounds may be useful in studies of protein processing in eu-karyotic cells; they have been selected for their low toxicity incomparison with literally thousands of compounds (27). Thereare reports that local anesthetics inhibit lipid breakdown in fatells of humans (28). Local anesthetics have also been shown

to affect the mobility and distribution of cell surface receptors,po)ssibly via their effects on microtubules and microfilaments(29). Perhaps a proteolytic reaction is retrospectively implicatedin some of these reactions on the basis of our results. It nowappears that serious consideration should be given to the pos-sibility that the mechanism(s) of molecular action of theseneuroactive compounds in animals may involve inhibition ofa hypothetical trypsin-like reaction at cell surfaces or mem-branes.

We appreciate the advice of J. Westley and F. J. Kezdy on assays oftrypsin and its inhibition. We thank J. Westley, F. J. Kezdy, L. S.Seiden, and H. Nikaido for suggestions concerning the manuscript. Theinspiration of Diane C. Markovitz is acknowledged. This research wassupported by U.S. Public Health Service Grant AI-06966 from theNational Institute of Allergy and Infectious Diseases (to A.M.) andAmerican Cancer Society Grants VC116 (to A.M.) and PF1296 (toR.C.G.).

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