multiple intracellular peptidases in neurospora crassa - journal of

JouRNAL OF BACTERIOLOGY, Mar. 1979, p. 1324-13320021-9193/79/03-1324/09$02.00/0

Vol. 137, No. 3

Multiple Intracellular Peptidases in Neurospora crassaSAI-TEE TANt AND GEORGE A. MARZLUF*

Department ofBiochemistry, Ohio State University, Columbus, Ohio 43210

Received for publication 28 November 1978

Neurospora crassa possesses multiple intracellular peptidases which displayoverlapping substrate specificities. They were readily detected by an in situstaining procedure for peptidases separated in polyacrylamide gels, within whichthe auxilliary enzyme, L-amino acid oxidase, was immobilized. Eleven differentintracellular peptidases were identified by electrophoretic separation and verifiedby their individual patterns of substrate specificities. Most peptide substratestested were hydrolyzed by several different peptidases. The multiple intracellularpeptidases may play overlapping roles in several basic cell processes which involvepeptidase activity. The amount of peptidase activity for leucylglycine present incrude extracts of cells grown under widely different conditions was relativelyconstant, suggesting that this enzyme may be constitutive, although alterationsin the amounts of individual peptidase isozymes may occur. A single enzyme,designated peptidase II, was partially purified and obtained free from the otherpeptidase species. Peptidase II was found to be an aminopeptidase with activitytoward many peptides of vWied composition and size. It was more active withtripeptides than homologous dipeptides and showed strong activity toward me-thionine-containing peptides. This enzyme, with a molecular weight of about37,000, was thermolabile at 65°C and was strongly inhibited by p-hydroxymer-curibenzoate, Zn2+, Co2+, and Mn2+, but was insensitive to the serine proteaseinhibitor phenylmethylsulfonyl fluoride. Peptidase II apparently possesses anessential sulihydryl group and may be a metalloenzyme.

Neurospora crassa can utilize exogenous pro-teins as its sole source of nitrogen, sulfur, orcarbon (5, 6). An extracellular protease is syn-thesized and secreted in response to an exoge-nous protein and a simultaneous limitation fornitrogen, sulfur, or carbon (3, 5-7). The extra-cellular protease apparently hydrolyzes externalproteins to a mixture of peptides and aminoacids, which are transported and used forgrowth. Various tripeptides are also known toserve as a source of required amino acids formutant strains (21, 22). An oligopeptide trans-port system has been demonstrated to exist ingerminated conidia (23) and is necessary for theutilization of extracellular peptides such asglycly-L-leucyl-L-tyrosine. A mutant strainwhich lacks this permease for peptides has alsobeen characterized (23).Synthesis of the extracellular protease is reg-

ulated in a complex manner and requires bothinduction and derepression (3, 5-7). It was ofinterest to determine whether the oligopeptidetransport system and one or more intracellularpeptidases might be similarly regulated in acoordinate fashion to permit the efficient utili-

t Present address: School of Biological Sciences, UniversitiSains Malaysia, Penang, Malaysia.

zation of exogenous proteins and peptides. Intra-cellular peptidase activity plays a significant rolein protein maturation. Cytoplasmic protein syn-thesis in eucaryotes is initiated by methionine,and maturation of many nascent proteins re-quires limited aminopeptidase action to removethe terminal methionine and perhaps a few ad-jacent residues (18). This maturation processrequires the activity of a consitutive peptidase,perhaps associated with ribosomes. Such a pep-tidase may be expected to show high activitywith methionine-containing peptides. It is notclear whether or not a peptidase which functionsin protein maturation might also have a role inthe metabolism of extracellular peptides oncethey have been transported into the cell.

In this paper we report investigations concem-ing the peptidase species present as intracellularenzymes in Neurospora. It is difficult to detectthe presence of multiple enzymes with overlap-ping specificities when they occur together in acrude extract. This problem can be largely over-come by use of gel electrophoresis, with theenzymes being selectively stained within the gelafter their separation. We devised and reporthere an efficient staining method to detect pep-tidase activity in situ in which the auxilliary

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PEPTIDASES IN NEUROSPORA 1325

enzyme required for staining, L-amino acid oxi-dase, is immobilized within the gel. After elec-trophoresis the gels are incubated with the de-sired peptide substrate and other components ofthe staining reaction to yield intense, sharp bluebands of precipitated diformazan at the sites ofpeptidase activity.With this staining technique, eleven different

intracellular peptidases were detected in Neu-rospora. Partial purification and characteriza-tion of one of the major peptidases is also re-ported.

MATERIALS AND METHODS

Chemicals. Gly-Leu-Gly-Leu was obtained fromCyclo; Leu-His and His-Leu were from Bachem; andLys-Trp, Glu-Trp, Met-Glu and Leu-Trp-Met-Arg-Phe-Ala were from Schwarz/Mann. Other peptidesand carbobenzoxy-peptides were purchased fromSigma Chemical Co. All amino acid residues of allpeptides were in the L configuration except wherespecifically stated otherwise. Aquacide was from Cal-biochem, and L-amino acid oxidase was from Sigma.Other chemicals were obtained from common com-mercial sources.

Strains and growth conditions. The Emerson awild-type strain of N. crassa and the cys-3, met-2, andnit-2 mutants were obtained from Fungal GeneticsStock Center, Arcata, Calif. Neurospora cultures wereobtained by inoculation of 100 ml of liquid mediumcontained in 250-ml Erlenmeyer flasks with conidia.After 3 days of growth at 25°C on a reciprocatingshaker, the cells were harvested. When larger amountsof mycelia were needed, 400-ml cultures contained in1-liter flasks were grown as described above. Vogelmedium (4) was routinely used, and in some studiesmodified Vogel medium lacking nitrogen, sulfur, orcarbon was utilized and was supplemented with theappropriate nutrients. Extracellular growth mediumwas concentrated at 4°C with an Amicon ultrafiltra-tion unit using a 43-mm PM10 membrane.Enzymes assays. Mycelia were harvested by fil-

tration and were washed several times with distilledwater before they were pressed dry. The mycelial padswere used immediately or were frozen until needed.The pads were ground in acid-washed sand in an ice-cold mortar. One milliliter of 20 mM Tris-hydrochlo-ride buffer, pH 7.5, was added per gram (wet weight)of mycelia, and the extract was centrifuged for 20 minat 20,000 x g for 4°C. The supernatant fluid wasretained.

Peptidase activity was assayed as described byBinkley et al. (1), with some minor modifications. Inthis method, free amino acids liberated by peptidaseactivity react with trinitrobenezenesulfonic acid in thepresence of Cu2", which greatly retards the rate ofreaction of peptides with trinitrobenzenesulfonic acid.The assay mixture contained 36mM borate buffer, pH8.0, 2 mM peptide, and 0.1 mi of enzyme preparationcontaining 2.0 to 2.5 mg ofprotein/ml in a total volumeof 1.25 ml. The substrate and buffer were preincubatedfor 20 min at 37°C, and then 0.1 ml of enzyme wasadded; portions of 0.1 ml were then withdrawn at

regular time intervals for the colorimetric assay (1). Azero-time sample was always taken for use as a blank.It should be noted that this assay will measure totalpeptidase which is active toward the particular peptideused as substrate, whether this represents a singleenzyme or a mixture of several within a crude extract.One unit of enzyme activity is defined as that amountwhich catalyzes formation of 1 umol of amino acid permin per mg of protein. Protein was determined by themethod of Lowry et al. (10), with bovine serum albu-min as the standard.

Hydrolysis of L-leucine-p-nitroanilide was deter-mined by following the absorption of p-nitroanilide at405 nm in a reaction mixture containing 0.05 M boratebuffer, pH 8.0. L-Leucyl-jl-naphthylamide hydrolysisat pH 8.0 was similarly monitored at 340 nM.

Thin-layer chromatography. Amino acids, pep-tides, and certain peptidase reaction mixtures werespotted onto cellulose thin-layer sheets (Eastman Or-ganic Chemicals). The plates were developed in asolvent consisting of ethanol-acetic acid-water (65:1:34, vol/vol). After approximately 3 h, the sheets wereremoved, dried, and sprayed with ninhydrin (21).Polyacrylamide gel electrophoresis. A method

was devised for in situ staining of peptidase activitywithin gels. The auxilliary enzyme, L-amino acid oxi-dase, was mixed with the acrylamide (0.67 mg ofenzyme per ml of solution) before addition of ammo-nium persulfate. To obtain rapid setting of the gel,12.5 mg of ammonium persulfate and 50 ,l ofN,N,N',N'-tetramethylethylenediamine were used per25 ml of gel solution. Higher concentrations of am-monium persulfate were avoided because they wereinhibitory for enzyme activity.

Polyacrylamide gels (5%) in 0.37 M Tris-hydrochlo-ride buffer, pH 8.9, were cast in glass tubes; the upperand lower chambers of the Buchler electrophoresisunit contained 0.04 M Tris-0.005 M glycine, pH 8.3.The enzyme extracts (10 to 50 ul) containing sucroseand bromophenol blue were layered onto the gel sur-face. After an initial current of 1 mA per gel duringwhich the samples entered the gel, electrophoresis wasconducted at 3 mA per gel. The gels were stained byincubating at 37°C in a mixture of 170 yg of peptide,170,g of Nitro Blue Tetrazolium, and 17 jg of phen-azine methosulfate per ml of 0.1 M Tris-hydrochloridebuffer, pH 7.0. Bands of peptidase activity appearedwithin 30 to 120 min of staining.Aminopeptidase activity was revealed in gels by

incubating them at room temperature in 20 mM Tris-hydrochloride buffer, pH 8.5, containing 0.33 mg ofleucyl-,B-naphthylamide/ml and 0.83 mg of Fast garnetGBC/ml; aminopeptidase activity was also detectedby incubating gels in the same buffer containing 0.33mg of L-leucine-p-nitroanilide/ml.Partial purification of peptidase II. Step 1. Am-

monium sulfate was added to the crude cell extract(prepared as described above) at 4°C to bring it to 60%saturation. After standing for 1 h at 4°C, the samplewas centrifuged and the supernatant fluid was ad-justed to 90% saturation with additional ammoniumsulfate. After 1 h at 4°C, the precipitate recovered bycentrifugation was retained and dissolved in a smallvolume of buffer.

Step 2. The solution was applied to a column of

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1326 TAN AND MARZLUF

Sephadex G-150 (34 by 2.0 cm, ID), and eluted with0.02 M Tris-hydrochloride, pH 7.5, at a flow rate of 38ml/h. Fractions (150 drops each) were collected andspot-tested for peptidase activity. Fractions with ac-tivity toward Leu-Leu-Leu were pooled.

Step 3. The pooled fractions from step 2 wereplaced in a dialysis bag, concentrated against Aqua-cide, and then applied to a column (26 by 1.5 cm, ID)of DEAE-cellulose. Elution was achieved with a lineargradient from 0.05 to 0.5 M NaCl in 125 ml of 0.05 MTris-hydrochloride buffer, pH 7.5, at a flow rate of 75ml/h. Fractions of 70 drops were collected and spot-tested, and those containing activity for Leu-Leu-Leuwere pooled and concentrated with Aquacide.

Step 4. The material from step 3 was applied to a

column (27.5 by 1.5 cm, ID) of Sephadex G-200 andeluted with 0.02 M Tris-hydrochloride buffer, pH 7.5.Fractions (70 drops each) were collected at a flow rateof 18 ml/h and spot-tested for peptidase activity. Ac-tive fractions were pooled and concentrated.

RESULTS

Levels of total peptidase activity. Synthe-sis of an extracellular protease in Neurospora is

regulated by induction and simultaneous de-repression. It was of interest to determinewhether or not peptidase synthesis would besimilarly controlled since peptidase activity ap-pears to be involved in the final stages of thehydrolysis of proteins to the constituent aminoacids. It also seemed possible that various pep-tides added to the growth medium might di-rectly induce peptidase synthesis.Wild-type Neurospora was grown under var-

ious conditions which might be expected to alterthe rate of peptidase synthesis. Total peptidaseactivity in extracts was assayed with trinitroben-zenesulfonic acids as described in Materials andMethods, with either leucylglycine or trileucineas substrates. There was no indication of in-creased total peptidase activity for these pep-

tides due to growth of the cells on mediumlacking nitrogen and supplemented with an ex-ogenous protein (Table 1). Similarly, when wild-type cells were limited for sulfur and providedwith bovine serum albumin or Met-Met as asulfur source, peptidase activity was not higherthan in control cells. Substantial peptidase ac-

tivity was found in all cases in crude cells ex-tracts. No peptidase activity was observed in theconcentrated extracellular medium of cells

grown on minimal medium although smallamounts were detected in the medium of cellsgrown under "inducing" conditions. However, inthis case there was also evidence of leakage ofenzymes, presumably by cell lysis, as monitoredby the concomitant appearance of constitutivealkaline phosphatase (an intracellular enzyme).When bovine serum albumin served as both a

nitrogen and methionine source for the met-2

TABLE 1. Peptidase activity in Neurospora cellsgrown under various conditions

Sp act

Strain Growth conditions' Pepti- Pro-

daseb tease"

Wild type Minimal medium 0.30 0.0Wild type Minimal - N + BSA 0.20 5.1Wild type Minimal + 1 mM Leu-Gly 0.21 0.0Wild type Minimal - S + 0.3 mM 0.21 NDd

Met-Met-MetWild type Minimal - S + BSA + 0.2 0.24 4.7

mM MetWild type Minimal - S + BSA + 5 0.22 0.1

mM Metmet-2 Minimal - N + BSA 0.45 NDmet-2 Minimal - N + BSA + 0.38 ND

0.1 mM Metmet-2 Minimal - N + BSA + 0.34 ND

0.5 mM Met

aCultures were grown as described in Materials and Meth-ods. Bovine serum albumin (BSA) was filter sterilized andused at 1 mg/ml.

h Peptidase activity was measured in cell extracts by thetrinitrobenzenesulfonic acid assay with L-leucylglycine as sub-strate. Specific activity is defined as release of 1 umol of aminoacid per min per mg of protein.

' Protease was assayed as described by Hanson and Marzluf(6).

d Not done.

mutant, no obvious difference in peptidase activ-ity was observed, whether or not additional me-thionine was also provided (Table 1). Peptidaseactivity of control cells grown on minimal me-dium was not increased by including the dipep-tide Leu-Gly as a possible inducer (Table 1).Finally, when wild-type cells were first grown onminimal medium for 12 h and then transferredto fresh medium, the specific activity of pepti-dase did not noticeably vary when the cells werelimited for N, S, or C and provided with eitherbovine serum albumin or a peptide (Table 2).These experiments and others not detailed hereindicate that total peptidase activity specific forLeu-Gly (or Leu-Leu-Leu) as measured in crudeextracts does not change significantly under con-trasting growth conditions, including those spe-cific conditions known to cause derepression ofnitrogen- and sulfur-controlled enzymes (3, 5-7,12). Nor is Leu-Gly (or Leu-Leu-Leu) peptidaseactivity increased when peptides or exogenousproteins are present in the growth medium, evenwhen they are being used to fulfill a growthrequirement.Three aminopeptidases could be detected in

crude cell extracts by separation on polyacryl-amide gels and staining with leucyl-/?-naph-thylamide (Fig. 1). No new aminopeptidase spe-cies were found when cells were subjected topossible induction or derepression conditions asdescribed above.

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VOL. 137, 1979

TABLE 2. Peptidase activity in Neurospora cellstransferred to new growth conditions

Growth conditions" PeptidaseSp acth

Minimal medium .................... 0.30Minimal-N + BSA ................ 0.38Minimal - N + Leu-Gly ............. 0.26Minimal-C + BSA ................. 0.33Minimal - C + Leu-Gly 0.20Minimal - S + BSA ................. 0.18Minimal - S + Met-Met ........ 0.15

'Wild-type cultures were grown on minimal me-dium for 12 h and then transferred to the indicatednew medium for 2 days of additional growth. Bovinesetum albumin (BSA) was present at 1 mg/ml; peptideconcentration was 1 mM.

Assays and specific activity as described in Table1.

+

A B C

FIG. 1. Diagram of peptidase activity visualizedinpolyacrylamide gels ofwild-type cell extracts. Afterelectrophoresis in 7.5% acrylamide gels, leucine ami-nopeptidase activity was stained with L-leucyl-/3-naphthylamide as described in Materials and Meth-ods. Growth conditions: (A) stationary phase, mini-mal medium; (B) logphase, minimal medium; (C) logphase, minimal medium lacking nitrogen plus I mgof bovine serum albumin/ml. No activity bands couldbe detected even after 1.5 h of staining in identicalexperiments but with the concentrated, dialyzed ex-

tracellular growth medium obtained in the samethree growth conditions.

The cys-3 mutant of Neurospora is missing anumber of sulfur-related enzymes, because thislocus encodes a positive-acting regulatory ele-ment (12). The nit-2 gene is believed to be an

analogous regulatory gene for nitrogen metabo-lism, and nit-2 mutants are deficient in a numberof nitrogen-related enzymes (11). Unlike wildtype, the cys-3 and nit-2 mutants fail to synthe-size extracellular protease under conditions ofsulfur and nitrogen limitation, respectively (7).However, when grown under various conditions,these two mutants both displayed normal levelsof peptidase activity for Leu-Gly and for Leu-


Leu-Leu (data not given). Since a relatively uni-form level of peptidase activity for these partic-ular peptides was found in crude extracts of wild-type cells, as well as the two regulatory mutants,after growth under contrasting conditions, wesuspect that the peptidases active for Leu-Glyand Leu-Leu-Leu are constitutive enzymes.However, this conclusion must be regarded astentative since changes might occur in the rela-tive amounts of the multiple peptidase isozymeswhich possess activity toward Leu-Gly and Leu-Leu-Leu (see below). We have no informationabout possible regulation of peptidases whichpossess other substrate specificities.Multiple intracellular peptidases. To de-

termine whether Neurospora possesses multiplepeptidase species, we used polyacrylamide gelsto separate peptidases in crude extracts. Thegels were stained for peptidase activity, with aspectrum of different peptides used as sub-strates, by a technique which reveals activity assharp blue bands. Immobilizing the couplingenzyme, L-amino acid oxidase, within the gelsignificantly improved resolution, in terms ofboth the intensity and the sharpness of bands,over previously described methods (9, 15).The results demonstrate the presence of at

least 11 electrophoretically distinct peptidasesin crude extracts derived from wild-type mycelia(Table 3, Fig. 2). These peptidases were arbi-trarily numbered in order of their decreasingmobility. Peptidases II and IIIA showed partic-ularly strong activity toward a large number ofdi-, tri-, and tetrapeptides. Peptidase II dis-played highest activity toward tripeptides,whereas IIIA showed stronger activity with di-peptides. Peptidase IIIB was also a very activeform, but it only hydrolyzed dipeptides. All ofthe peptides tested, except for Glu-Trp, weresubstrates for more than one peptidase; e.g.,Leu-Gly was hydrolyzed by seven different pep-tidases (Table 3). Peptides containing a prolineresidue were cleaved by peptidases IV, V, andVI. Glu-Trp, which has an acidic amino-terminalresidue, was hydrolyzed only by peptidase X.

Extracts derived from conidia were analyzedin an identical fashion. Conidia possess the samepattern of peptidases as found in mycelia, exceptthat conidia lack peptidases I, IIIA, IV, VI, andVIII. Conidia do not have any unique peptidasespecies which are not present in mycelia.Effect of inhibitors and cations. Potential

inhibitors and cations were added to the stainingmixture (1 mM final concentration) used to re-veal the peptidases in gels. All of the peptidases(II, IIIA, IIIB, V, VI, VIII, and IX) which hydro-lyze Leu-Gly or Met-Ala were completely in-hibited by p-hydroxymercuribenzoate; EDTAalso inhibited all ofthese same peptidases except

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1328 TAN AND MARZLUF J. BACTERIOL.

TABLE 3. Multiple Neurospora peptidase species revealed by polyacrylamide gel electrophoresisaPeptidase species

SubstrateI II IIIA IIIB IV V VI VII VIII IX X

Met-Phe, Leu-Gly, Met-Leu,Met-Mla + ++++ +++ + + + +

Gly-Leu-Tyr.++ + + +Met-Ala-Ser.+++ + + + +Met-Pro + + +Lys-Try, Try-Glu, Leu-Leu-Leu.+++ +Glu-Try. +Gly-Leu-Gly-Leu.++Gly-Met.+ + + + +Gly-Ala-Leu.++ + +Met-His.+ ++Leucyl-p-nitroanilide ++Leucyl-13-naphthylamide ++ + +

Rf ...................... 0.79 0.69 0.65 0.63 0.59 0.57 0.56 0.53 0.52 0.44 0.27

a See Materials and Methods for the staining procedure for peptidase activity. The relative intensity of theindividual peptidase bands is indicated by the number of plus signs.

b Peptidase species are arbitrarily numbered in order of decreasing electrophoretic mobility, as indicated bytheir Rf values, which were calculated relative to that of the bromophenol blue marker.

t- i; for II and IX, which showed only moderate.....

iniWbition. Azide (0.1%) did not inhibit any ofthe 11 intracellular peptidases. Zn" inhibited all

i1twrr 0 X h« of the peptidases, except that IIIA and IIIB wereonly slightly affected.

Purification of peptidase HI. Peptidase II(as designated hi Table 3) was particularly active

1 ~~~~~~~~~~towardtripeptides. Sfince Wolfinbarger and Mar-Izluf (21-23) demonstrated that Neurospora can

utilize a variety of tripeptides for growth andpossesses an active transport system capable of

.......1- 1F taking up tripeptides, this peptidase was selectedfor additional study. Peptidase II was purifiedsufficiently to isolate it free from all of the other10 intracellular peptidases by use of a number ofconventional enzyme purification steps summa-rized in Table 4. The purification was followedwith Leu-Leu-Leu as the substrate for in vitroassays; since Leu-Leu-Leu is also a substrate forpeptidase hIA, the amount of purification wasactually somewhat greater than is obvious fromTable 4. When the partiaUly purified enzyme was

- subjected to electrophoresis and the gels were-~~~~ -~~~ then stained with a variety of peptides as sub-t:S:g20f t strate, peptidase II was the onl detectable ac-

tivity.Properties ofpeptidase II. The enzyme was

strates. After electrophoresis of wild-type extracts inA B C D) E 7.5% acrylamide gels, individual gels were stained

with the coupled system as described in Materialsand Methods. Peptide substrates: (A) Leu-Gly, (B)

FIG. 2. Gel pattern of peptidase activity revealed Gly-Leu-Tyr, (C) Met-Ala-Ser, (D) Leu-Leu-Leu, (E)in polyacrylamide gels with different peptide sub- Met-His.---9 - --.- ------v- --

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TABLE 4. Purification ofpeptidase II

Activity' (U! Total activ- Protein Sp act (U!Step Vol (ml) nml) ity (U) Recovery (mg/nl) mg of pro-Recovery(~) (mg/mi) tein)

Crude extract ................. 100 0.335 33.5 100 2.34 0.14360-90% ammonium sulfate precip-

itation ..................... 8.0 2.77 22.2 66 8.40 0.33Sephadex G-150.50 0.42 20.9 62 0.96 0.44DEAE-cellulose 35 0.30 10.5 31 0.36 0.84Sephadex G-200 29.3 0.14 4.13 12.3 0.24 0.59

a Activity was assayed in vitro with Leu-Leu-Leu as substrate by the trinitrobenzenesulfonic acid proceduredescribed in Materials and Methods. Analysis of step 5 preparation by gel electrophoresis showed that onlypeptidase II was present. Bands of activity at the position corresponding to peptidase II were observed in gelsstained with Leu-Leu-Leu and Met-Ala-Ser, and weaker bands were observed at this same site with Gly-Leu-Tyr, Gly-Leu, and Leu-Gly. Bands of activity corresponding to the positions of the other peptidase species werenot detected.

determined to have a pH optimum of about 8.0and showed maximum activity at 370C (data notshown). Peptidase II activity was quite heatlabile at 650C, with a half-life at this tempera-ture estimated to be 6 min (Fig. 3). Peptidase IIactivity was completely inhibited by Zn2+,whether or not the enzyme was dialyzed,whereas several other metal ions were inhibitorsonly with dialyzed preparations. Co2+ and Mn2"inhibited peptidase II by 96% and 89%, respec-tively, whereas Ca2" and Mg2" showed only mod-erate inhibition (Table 5). The serine-proteaseinhibitor phenylmethylsulfonyl fluoride did notinhibit peptidase II. The enzyme was completelyinhibited by the sulfhydryl agent (p-hydroxy-mercuribenzoate, and this inhibition was largely

100 > 2.0

>840 _ 1.2

1e10.6

> a:

O

60 x

<O, 0.060 ~~~~0*. 0 .0 20 30 40 5

z ~~~~~~~~~~~~~TIME(mein)

z40-

o-e20

0_0 o - 20 30 40 50

TIME (min)FIG. 3. Thermal lability ofpeptidase II. Partially

purified peptidase II was incubated for various timeintervals at 65°C; then samples were removed andassayed for remaining activity by the trinitroben-zenesulfonic acid method, utilizing Leu-Leu-Leu as

substrate. The insert shows a semilogplot from whichthe half-life of 6 min was determined.

TABLE 5. Effect of divalent cations and potentialinhibitors upon peptidase II activity

Addition'

None .......................

Mg2+ .......................

Ca2. ........................

Mn2 .......................

Co2. ........................

Zn ........................

Phenylmethylsulfonyl fluorideEDTA ......................o-Phenanthroline .............2-Mercaptoethanol (2-ME) ....

p-Hydroxymercuribenzoate(pHMB) ..................

pHMB (10-4 M) ..............

pHMB (10-4 M) plus 2-ME (10-3M) ........................

Percentage of controlactivity

Nondi- Dialyzedalyzed sample"sample100 10095 6199 3893 1180 40 0

90603462

04

56

'Potential inhibitors were preincubated with theG-200 enzyme preparation for 30 min at 250C prior toassay with trileucine as substrate. All compounds wereadded to a final concentration of 1 mM except wherenoted differently.

b The enzyme was dialyzed against 0.05 M boratebuffer pH 8.0 for 18 h.

reversed by 2-mercaptoethanol, which by itselfwas slightly inhibitory as well (Table 5). Themolecular weight of peptidase II was estimatedto be 37,000 by gel filtration with standard pro-teins as references (data not given).The following results indicate that peptidase

II is an aminopeptidase and does not have eitherendopeptidase or carboxypeptidase activity. Noindication of endopeptidase activity was de-tected when peptidase II was tested with thebeta chain of insulin. To determine whether thisenzyme acted as an aminopeptidase or carbox-ypeptidase, peptidase II was incubated with the

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insulin beta chain or the hexapeptide Leu-Trp-Met-Arg-Phe-Ala as substrates, and the reactionmixtures were analyzed by thin-layer chroma-tography. The results showed that only aminoacids at the amino terminus (Phe and perhapsVal) were released from the insulin beta chainand that the carboxyl-terminal amino acid wasnot released from the insulin chain nor the hex-apeptide even after prolonged incubation pe-riods (Fig. 4). Peptidase II could not hydrolyzecarbobenzoxy-Leu-Gly, carbobenzoxy-Leu-Phe,or carbobenzoxy-Gly-Leu, all substrates for car-boxypeptidase activity (Table 6); yet, these samedipeptides without the blocked amino terminuswere substrates for the enzyme. Finally, thesequence of appearance of hydrolysis productswith Met-Ala-Ser and Gly-Leu-Tyr as substrateswas analyzed with thin-layer chromatographssimilar to that shown in Fig. 4; the results indi-

MINUTES OF INCUBATIONrI Il

0 !5 30 60 80 120 180240300360i.0-

0.8 0 0 O 0

0.6

0.

LU MINUTES OF INCUBATION-J lTlFFTl TI> 0 10 60. 90 12C 150 180

1.0

_ 0 00000.8

STANDARDS

PHE VAL ASN PRO LYS ALA

00

0

STANDARDSLEU TRP MET ARG PHE ALA

000 0

I®0.2

FIG. 4. Diagram of thin-layer chromanalysis of the reaction products derived f

dase II activity upon the insulin beta ch4defined hexapeptide. Reaction mixtures (2itained 50mM borate buffer, pH 8.0, and eiiLeu-Trp-Met-Arg-Phe-Ala or 250 pg of tbeta chain/ml plus 50 IlI of peptidase II (G200 fraction) and were incubated at 37°Cwere removed at various time intervals anately frozen until they were analyzed by s

,tl samples onto cellulose thin-layer sheetscm) and chromatographed as described inand Methods. (A) Insulin beta chain; the thacids at the amino terminus, phenylalanvaline (Val), and asparagine (Asn), and Xboxy terminus, proline (Pro), lysine (Lys), ai

(Ala), were included as standards. (B) LeiArg-Phe-Ala hydrolysis by peptidase II.

J. BACTERIOL.

TABLE 6. Substrate specificity ofpeptidase IIRelativeSp act rate of

Substrate (,ttmo/nun hydroly-per mg) sis

Leu-Leu-Leu ................ 0.664 100Leu-Leu .................... 0.650 98Lys-Trp .................... .644 97Gly-Ala-Leu .................

0.480 72Leu-Gly-Gly ................. 0.302 45Gly-Leu-Gly-Leu ............ 0.282 42Met-Glu .................... 0.258 39Gly-Leu-Tyr ................ 0.258 39Tyr-Glu .................... 0.258 39Leu-Trp-Met-Arg-Phe-Ala 0.146 22Gly-Met .................... 0.141 21Met-Met-Met ............... 2.110 317Met-Met ....................

1.490 224Met-Leu .................... 1.060 160Met-Gly .................... 0.780 117Met-Ala .................... 0.752 113Met-Ala-Ser ................. 0.848 128Ala-Ala-Ala-Ala-Ala ......... 2.32 350Ala-Ala-Ala-Ala ............. 1.41 212Ala-Ala-Ala ................. 1.36 205Ala-Ala .....................

0.71 107Gly-Gly-Gly-Gly ............. 0.31 47Gly-Gly-Gly ................. 0 0N-carbobenzoxy-Gly-Leu ..... 0 0Gly-Leu .................... 0.127 19Gly-D-Leu .................. 0.018 3N-carbobenzoxy-Leu-Phe 0 0Leu-Phe .................... 0.420 63N-carbobenzoxy-Leu-Gly ..... 0 0Leu-Gly .................... 0.150 23D-Leu-Gly ..........0........OL-Leucine-p-nitroanilhde ...... 0.172 26L-Leucine-,B-naphthylamideHCl ...................... 2.623 395

a The rate of hydrolysis relative to that of Leu-Leu-Leu is reported as determined with the assays de-scribed in Materials and Methods.

cate that cleavage occurs from the amino-ter-minal end of the peptide. In each case, theamino-terminal amino acid was released prior to

atograPhy the appearance of the other two constituents ofrom pepti- the tripeptide. This peptidase was inactive to-ain and a ward peptides which have a D-amino acid as the00/4l) COfl amino-terminal residue (Table 6).ther 5mM amhe insulin Peptidase II was found to act upon a broad(Sephadex range of peptides as substrates and to hydrolyze5. Portions dipeptides and larger oligopeptides (Table 6).d immedi- Methionine-containing peptides were observedEpotting 5- to be particularly good substrates. In general,s (20 by 20 larger peptides tended to be better substratesMaterials than dipeptides; this is obvious in the hydrolysisiree amino rate ofthe alanine peptide series, ofwhich penta-itntehe car' alanine is the best peptidase II substrate, fol-nd alanine lowed in order by tetra-alanine, tri-alanine, andn-Trp-Met- di-alanine (Table 6). Similarly, Met-Met-Met

was cleaved more rapidly than Met-Met.

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DISCUSSION

Under certain conditions of derepression andinduction, N. crassa secretes an alkaline pro-tease which hydrolyzes extracellular proteinsand permits their use as a source of nitrogen,sulfur, or carbon (3, 5-7). The work reportedhere shows that Neurospora also possesses aspectrum of intracellular peptidases with over-lapping specificities. We do not yet have anydetailed information about possihe regulationof these multiple peptidase species. Crude ex-tracts of cells grown under various contrastingconditions which it was anticipated might causeinduction or derepression of peptidase failed toshow any increased activity toward Leu-Gly orLeu-Leu-Leu. However, we do not knowwhether alterations occur in the amounts ofindividual peptidase isozymes which are activetoward these peptides. No changes were ob-served in the qualitative isozyme pattern of pep-tidases which hydrolyze leucyl-fl-naphthylam-ide.Although the extracellular protease is regu-

lated in a complex fashion, the peptidases mayin fact be constitutive enzymes because they arerequired for a variety of cellular functions, onlyone of which is the use of external proteins andpeptides. The oligopeptide transport system waspreviously found to be constitutive (23). Thecontinuous presence of the peptide uptake sys-tem and the multiple intracellular peptidaseswould permit immediate capture and hydrolysisof peptides from the environment as a preformedand readily usable source of amino acids. Metab-olism of extracellular proteins likewise involvesthese same activities, but also requires the "turn-ing on" of extracellular protease.The results presented here show that Neuro-

spora possesses multiple intracellular pepti-dases. That these various activities representseparate and distinct enzymes seems clear be-cause of the patterns of substrate specificity andinhibition profiles. Other microorganisms havesimilarly been found to possess a spectrum ofpeptidases (11, 13, 15). The mutational loss inSalmonella typhimurium of specific peptidasesis usually tolerated quite well without detectablechanges in the growth rate (13, 14). When sev-eral peptidase mutants are combined, however,deleterious effects, such as decreased growthrate, become apparent (14). These observationssuggest that the multiple peptidase species withoverlapping specificities present in Salmonella,Neurospora, and other microorganisms may col-lectively participate in a number of functions inwhich peptidase activity is involved, rather thaneach enzyme having only a single role. Thecellular functions of the peptidases include (i)

maturation of nascent proteins, (ii) use of extra-cellular proteins and peptides for growth, (iii)normal turnover of endogenous proteins, (iv)hydrolysis of defective cell proteins and frag-ments (2), and (v) protection against the toxiceffects caused by certain peptides (16, 20). Be-cause of the diverse requirements for peptidaseactivity and the importance of several of theseprocesses, it should not be considered surprisingthat the cell possesses a variety of peptidases.A relatively abundant peptidase, designated

peptidase II, was shown in this study to be anaminopeptidase with activity toward a variety ofpeptides of different composition and sizes. Theenzyme cannot hydrolyze substrates whoseamino-terminal amino acid is substituted or is aD isomer. Since peptidase II activity results inthe initial release of the N-terminal amino acidfrom peptides, its identity as an aminopeptidaseis obvious. Peptidase II is particularly activetoward methionine-containing peptides andcould play a role in the limited removal of aminoacids during the maturation of nascent proteins,although its almost equally good activity withother peptides argues against this possibility.The strong inhibition of this enzyme by p-hy-droxymercuribenzoate indicates the presence ofan essential sulfhydryl group for activity. Pep-tidase II may also be a metalloprotein, in viewof its strong inhibition by Zn2+, Co2+, and Mn2+and its partial inhibition by EDTA and o-phen-anthroline. Peptidase II was shown to have amolecular weight of about 37,000 by gel filtra-tion. Analysis of more highly purified enzymewith sodium dodecyl sulfate-polyacrylamide gelsindicated the presence of a single protein with amolecular weight of 40,000 (S.-T. Tan, unpub-lished data), suggesting that peptidase II mayconsist of a single polypeptide chain of this size.Johnson and Brown (8) described two ami-

nodipeptidases from N. crassa. One of these, animidodipeptidase, hydrolyzes only dipeptidescontaining proline carboxy-terminal residuesand might be the same as peptidase V detectedhere, although the properties of the two enzymesare not identical. The other enzyme, identifiedby Johnson and Brown as a methionyl dipepti-dase, hydrolyzes various dipeptides, with a pref-erence for methionine in the N-terminal posi-tion, but it has no activity toward tripeptides orlarger substrates. This enzyme, with a molecularweight of 110,000, very clearly differs from pep-tidase II in a number of basic properties; itappears to correspond to peptidase IIIB.

Seipen et al. (17) reported partial characteri-zations of three peptidases from N. crassa, onecarboxypeptidase and two aminopeptidases.Their amino peptidase Al, with a molecularweight of about 96,000, displayed activity toward

VOL. 137, 1979

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dipeptides with lysine at the amino terminus,but not sufficient sbstrates were tested for usto determine which, if any, ofthe multiple activ-ities of Table 3 it might represent. Aminopepti-dase A2, with a molecular weight of about100,000, displayed a broader substrate specificitybut clearly is different from peptidase II studiedin this paper. The presence of four protein bandsupon electrophoresas of aminopeptidase A2 (22)could indicate the presence of several species ofsimilar size, particularly in view of our observa-tion that Neuro8pora possesses multiple pepti-dases.The technique described in this paper for

staining peptidase activity in situ within poly-acrylamide gels yields sharp banding patternsand is useful since it permits visuization of anentire sectrum of peptidases and yet distin-guishes between them on the basis of differentspeficities for peptide substrates. One limita-tion is that the peptides employed as substratesmust contain amino acids which are substratesfor i-amino acid oxidase. This is not a seriousdrawback since many L-amino acids are sub-strates for the oxidase (19). We observed 11different bands of activity coreponding to pep-tidase isozymes. It seems probable that evenmore peptid species could be identified byusing additional peptides as substrates. It is ob-vious that Neurospora possse a wealth ofintracellular peptidases, and they appear to beinvolved in a number of important cellular func-tions.

ACKNOWLDGMENTSThis research was supported by Public Health Service

grant 5 ROI GM-23367 from the National Institute of GeneralMedical Sciences. S.-T.T. was the recipient of a Fulbright-Hays Predtoral Fellowship during the tenure of these stud-is. GA.M. is supported by Public Health Service CareerDevelopment Award K4-GM00052.We acknowledge many useful discussions with Lloyd Wol-

finbarger.

LITERATURE CITED1. Binkley, F., F. Leibach, and N. King. 1968. A new

method of peptidse asay and the separation of threeleucylglycinases of renal tissues. Arch. Biochem. Bio-phys. 138:397405.

2. Bukhrl, A. I., and D. Zipsor. 1973. Mutants of Eswhe-hia coli with a defect in the degradation of nonsense

fragments. Nature (London) New Biol. 243:238-241.3. Cohen, B. L., J. E. Morris, and HL Dnrue. 1975.

Rgulation oftwo exzacellula proteam ofNeuro8poracrassa by induction and by carbon-nitrogen and sulfur

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metabolite repression. Arch. Biochem. Biophys. 169:324-330.

4. Davi, R. E, and F. J. DeSerres. 1970. Genetic andmicrobiological research techniques for Neurosporacrana. Methods Enzymol. 17A:79-143.

5. Drucker, H. 1972. Regulation of exocellular proteass inNeurospora crassa: induction and repression ofenzymesynthesis. J. Bacteriol. 110:1041-1049.

6. Hanson, AL A., and G. A. Marzluf. 1973. Regulation ofa sulfur-controlled protease in Neurospora crassa. J.Bacteriol. 116:786-789.

7. Hanson, AL A., and G. A. Marzluf. 1975. Control of thesynthesis of a sine enzyme by multiple regulatorycircuits in Neurospora crassa. Proc. Natl. Acad. Sci.U.S.A. 72:1240-1244.

8. Johnon, G. L, and J. L Brown. 1974. Partial purifi-cation and characterization oftwo peptidase from Neu-rospora crassa. Biochim. Biophys. Acta 370:630-0.

9. Lewis, W. H. P., and H. Harris. 1967. Human red cellpeptidase. Nature (London) 215:351-355.

10. Lowry, 0. H., N. J. Rosebrough, A. L Farr, and R. J.RnL 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

11. McHugh, G. L, and C. G. Miller. 1974. Isolation andcharacterization of prolinie peptidase mutants of Sal-monela typhinuriunL J. Bacteriol. 120:364-371.

12. Marzlu, G. A. 1977. Regulation of gene expression infiugi, p. 196-242. In J. C. Copeland and G. A. Marzluf(ed.), Regulatory biology. Ohio State Univ. Press, Co-lumbus.

13. Miller,C.G. 1975. ProteasesandpeptidaseofEscherdchiacoli and Sabnonella tphimurium. Annu. Rev. Micro-bioL 29:4865-04.

14. Mier, C. G., C. Heiman, and C. Yen. 1976. Mutants ofSalmoneUa tphimurium deficient in an endoprotease.J. Bacteriol. 127:490-497.

15. MIr, C. G., and L MacKinnon. 1974. Peptidase mu-tents of Salmonella typhimuriun. J. Bacteriol. 120:355-363.

16. Payne, J. W., and C. Gilvarg. 1971. Peptide transport.Adv. Enzymol. 85:187-244.

17. Seipen, D., P. Yu, and M. Kula. 1975. Proteolytic en-zymes of Neurospora crassa. Eur. J. Biochem. 66:271-281.

18. Smith, A. E., and K. A. Marcker. 1970. Cytoplasmicmethionine transfer RNAs from eukaryotes. Nature(London) 226:607-610.

19. Thayer, P. S., and N. H. Horowitz. 1951. The L-aminoacid oxidae of Neurospora. J. Biol. Chem. 192:755-767.

20. Von der Harr, R. A., and H. E. Umbarger. 1972.Isoleucine and valine metabolism in Eschenchia coli.XIX. Inhibition of isoleucine biosynthesis by glycyl-leucine. J. Bacteriol. 112:142-147.

21. Wolfinhbarger, L, Jr, and G. A. Marzuf. 1974. Peptideutilization by amino acid auzotrophs of Neurosporacrassa. J. Bacteriol. 119:371-378.

22. Wo}lfnbarger, L., Jr., and G. A. Marzluf. 1975. Sizerestriction on utilization of peptides by amino acidauxotrophs of Neurospora crassa. J. Bacteriol. 122:949-956.

23. Wolfnbarger, L, Jr., and G. A. Marzluf. 1976. Speci-ficity and regulation ofpeptide tansport in Newroeporacrassa. Arch. Biochem. Biophys. 171:637-644.

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