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Vol. 137, No. 3
Multiple Intracellular Peptidases in Neurospora crassa SAI-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 display overlapping substrate specificities. They were readily detected by an in situ staining procedure for peptidases separated in polyacrylamide gels, within which the auxilliary enzyme, L-amino acid oxidase, was immobilized. Eleven different intracellular peptidases were identified by electrophoretic separation and verified by their individual patterns of substrate specificities. Most peptide substrates tested were hydrolyzed by several different peptidases. The multiple intracellular peptidases may play overlapping roles in several basic cell processes which involve peptidase activity. The amount of peptidase activity for leucylglycine present in crude extracts of cells grown under widely different conditions was relatively constant, suggesting that this enzyme may be constitutive, although alterations in the amounts of individual peptidase isozymes may occur. A single enzyme, designated peptidase II, was partially purified and obtained free from the other peptidase species. Peptidase II was found to be an aminopeptidase with activity toward many peptides of vWied composition and size. It was more active with tripeptides than homologous dipeptides and showed strong activity toward me- thionine-containing peptides. This enzyme, with a molecular weight of about 37,000, was thermolabile at 65°C and was strongly inhibited by p-hydroxymer- curibenzoate, Zn2+, Co2+, and Mn2+, but was insensitive to the serine protease inhibitor phenylmethylsulfonyl fluoride. Peptidase II apparently possesses an essential sulihydryl group and may be a metalloenzyme.
Neurospora crassa can utilize exogenous pro- teins as its sole source of nitrogen, sulfur, or carbon (5, 6). An extracellular protease is syn- thesized and secreted in response to an exoge- nous protein and a simultaneous limitation for nitrogen, sulfur, or carbon (3, 5-7). The extra- cellular protease apparently hydrolyzes external proteins to a mixture of peptides and amino acids, which are transported and used for growth. Various tripeptides are also known to serve as a source of required amino acids for mutant strains (21, 22). An oligopeptide trans- port system has been demonstrated to exist in germinated conidia (23) and is necessary for the utilization of extracellular peptides such as glycly-L-leucyl-L-tyrosine. A mutant strain which lacks this permease for peptides has also been characterized (23). Synthesis of the extracellular protease is reg-
ulated in a complex manner and requires both induction and derepression (3, 5-7). It was of interest to determine whether the oligopeptide transport system and one or more intracellular peptidases might be similarly regulated in a coordinate fashion to permit the efficient utili-
t Present address: School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia.
zation of exogenous proteins and peptides. Intra- cellular peptidase activity plays a significant role in protein maturation. Cytoplasmic protein syn- thesis in eucaryotes is initiated by methionine, and maturation of many nascent proteins re- quires limited aminopeptidase action to remove the terminal methionine and perhaps a few ad- jacent residues (18). This maturation process requires the activity of a consitutive peptidase, perhaps associated with ribosomes. Such a pep- tidase may be expected to show high activity with methionine-containing peptides. It is not clear whether or not a peptidase which functions in protein maturation might also have a role in the metabolism of extracellular peptides once they have been transported into the cell.
In this paper we report investigations concem- ing the peptidase species present as intracellular enzymes in Neurospora. It is difficult to detect the presence of multiple enzymes with overlap- ping specificities when they occur together in a crude extract. This problem can be largely over- come by use of gel electrophoresis, with the enzymes being selectively stained within the gel after their separation. We devised and report here an efficient staining method to detect pep- tidase activity in situ in which the auxilliary
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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 of the staining reaction to yield intense, sharp blue bands of precipitated diformazan at the sites of peptidase 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.
Chemicals. Gly-Leu-Gly-Leu was obtained from Cyclo; Leu-His and His-Leu were from Bachem; and Lys-Trp, Glu-Trp, Met-Glu and Leu-Trp-Met-Arg- Phe-Ala were from Schwarz/Mann. Other peptides and carbobenzoxy-peptides were purchased from Sigma Chemical Co. All amino acid residues of all peptides were in the L configuration except where specifically 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 a wild-type strain of N. crassa and the cys-3, met-2, and nit-2 mutants were obtained from Fungal Genetics Stock Center, Arcata, Calif. Neurospora cultures were obtained by inoculation of 100 ml of liquid medium contained in 250-ml Erlenmeyer flasks with conidia. After 3 days of growth at 25°C on a reciprocating shaker, the cells were harvested. When larger amounts of mycelia were needed, 400-ml cultures contained in 1-liter flasks were grown as described above. Vogel medium (4) was routinely used, and in some studies modified Vogel medium lacking nitrogen, sulfur, or carbon was utilized and was supplemented with the appropriate nutrients. Extracellular growth medium was 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 distilled water before they were pressed dry. The mycelial pads were 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 min at 20,000 x g for 4°C. The supernatant fluid was retained.
Peptidase activity was assayed as described by Binkley et al. (1), with some minor modifications. In this method, free amino acids liberated by peptidase activity react with trinitrobenezenesulfonic acid in the presence of Cu2", which greatly retards the rate of reaction of peptides with trinitrobenzenesulfonic acid. The assay mixture contained 36mM borate buffer, pH 8.0, 2 mM peptide, and 0.1 mi of enzyme preparation containing 2.0 to 2.5 mg ofprotein/ml in a total volume of 1.25 ml. The substrate and buffer were preincubated for 20 min at 37°C, and then 0.1 ml of enzyme was added; portions of 0.1 ml were then withdrawn at
regular time intervals for the colorimetric assay (1). A zero-time sample was always taken for use as a blank. It should be noted that this assay will measure total peptidase which is active toward the particular peptide used as substrate, whether this represents a single enzyme or a mixture of several within a crude extract. One unit of enzyme activity is defined as that amount which catalyzes formation of 1 umol of amino acid per min per mg of protein. Protein was determined by the method 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 at 405 nm in a reaction mixture containing 0.05 M borate buffer, pH 8.0. L-Leucyl-jl-naphthylamide hydrolysis at pH 8.0 was similarly monitored at 340 nM.
Thin-layer chromatography. Amino acids, pep- tides, and certain peptidase reaction mixtures were spotted onto cellulose thin-layer sheets (Eastman Or- ganic Chemicals). The plates were developed in a solvent consisting of ethanol-acetic acid-water (65:1: 34, vol/vol). After approximately 3 h, the sheets were removed, dried, and sprayed with ninhydrin (21). Polyacrylamide gel electrophoresis. A method
was devised for in situ staining of peptidase activity within gels. The auxilliary enzyme, L-amino acid oxi- dase, was mixed with the acrylamide (0.67 mg of enzyme 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 of N,N,N',N'-tetramethylethylenediamine were used per 25 ml of gel solution. Higher concentrations of am- monium persulfate were avoided because they were inhibitory for enzyme activity.
Polyacrylamide gels (5%) in 0.37 M Tris-hydrochlo- ride buffer, pH 8.9, were cast in glass tubes; the upper and lower chambers of the Buchler electrophoresis unit contained 0.04 M Tris-0.005 M glycine, pH 8.3. The enzyme extracts (10 to 50 ul) containing sucrose and bromophenol blue were layered onto the gel sur- face. After an initial current of 1 mA per gel during which the samples entered the gel, electrophoresis was conducted at 3 mA per gel. The gels were stained by incubating 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-hydrochloride buffer, pH 7.0. Bands of peptidase activity appeared within 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 of leucyl-,B-naphthylamide/ml and 0.83 mg of Fast garnet GBC/ml; aminopeptidase activity was also detected by incubating gels in the same buffer containing 0.33 mg 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 sample was centrifuged and the supernatant fluid was ad- justed to 90% saturation with additional ammonium sulfate. After 1 h at 4°C, the precipitate recovered by centrifugation was retained and dissolved in a small volume of buffer.
Step 2. The solution was applied to a column of
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Sephadex G-150 (34 by 2.0 cm, ID), and eluted with 0.02 M Tris-hydrochloride, pH 7.5, at a flow rate of 38 ml/h. Fractions (150 drops each) were collected and spot-tested for peptidase activity. Fractions with ac- tivity toward Leu-Leu-Leu were pooled.
Step 3. The pooled fractions from step 2 were placed 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 linear gradient from 0.05 to 0.5 M NaCl in 125 ml of 0.05 M Tris-hydrochloride buffer, pH 7.5, at a flow rate of 75 ml/h. Fractions of 70 drops were collected and spot- tested, and those containing activity for Leu-Leu-Leu were 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 and eluted with 0.02 M Tris-hydrochloride buffer, pH 7.5. Fractions (70 drops each) were collected at a flow rate of 18 ml/h and spot-tested for peptidase activity. Ac- tive fractions were pooled and concentrated.
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 determine whether or not peptidase synthesis would be similarly controlled since peptidase activity ap- pears to be involved in the final stages of the hydrolysis of proteins to the constituent amino acids. 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 alter the rate of peptidase synthesis. Total peptidase activity in extracts was assayed with trinitroben- zenesulfonic acids as described in Materials and Methods, with either leucylglycine or trileucine as substrates. There was no indication of in- creased total peptidase activity for these pep-
tides due to growth of the cells on medium lacking nitrogen and supplemented with an ex- ogenous protein (Table 1). Similarly, when wild- type cells were limited for sulfur and provided with bovine serum albumin or Met-Met as a sulfur source, peptidase activity was not higher than in control cells. Substantial peptidase ac-
tivity was found in all cases in crude cells ex- tracts. No peptidase activity was observed in the concentrated extracellular medium of cells
grown on minimal medium although small amounts were detected in the medium of cells grown under "inducing" conditions. However, in this case there was also evidence of leakage of enzymes, presumably by cell lysis, as monitored by the concomitant appearance of constitutive alkaline 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 cells grown under various conditions
Sp act
daseb tease"
Wild type Minimal medium 0.30 0.0 Wild type Minimal - N + BSA 0.20 5.1 Wild type Minimal + 1 mM Leu-Gly 0.21 0.0 Wild type Minimal - S + 0.3 mM 0.21 NDd
Met-Met-Met Wild type Minimal - S + BSA + 0.2 0.24 4.7
mM Met Wild type Minimal - S + BSA + 5 0.22 0.1
mM Met met-2 Minimal - N + BSA 0.45 ND met-2 Minimal - N + BSA + 0.38 ND
0.1 mM Met met-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 and used at 1 mg/ml.
h Peptidase activity was measured in cell extracts by the trinitrobenzenesulfonic acid assay with L-leucylglycine as sub- strate. Specific activity is defined as release of 1 umol of amino acid 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). Peptidase activity 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 on minimal medium for 12 h and then transferred to fresh medium, the specific activity of pepti- dase did not noticeably vary when the cells were limited for N, S, or C and provided with either bovine serum albumin or a peptide (Table 2). These experiments and others not detailed here indicate that total peptidase activity specific for Leu-Gly (or Leu-Leu-Leu) as measured in crude extracts does not change significantly under con- trasting growth conditions, including those spe- cific conditions known to cause derepression of nitrogen- and sulfur-controlled enzymes (3, 5-7, 12). Nor is Leu-Gly (or Leu-Leu-Leu) peptidase activity increased when peptides or exogenous proteins are present in the growth medium, even when they are being used to fulfill a growth requirement. 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 to possible induction or derepression conditions as described above.
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TABLE 2. Peptidase activity in Neurospora cells transferred to new growth conditions
Growth conditions" Peptidase Sp acth
Minimal medium .................... 0.30 Minimal-N + BSA ................ 0.38 Minimal - N + Leu-Gly ............. 0.26 Minimal-C + BSA ................. 0.33 Minimal - C + Leu-Gly 0.20 Minimal - S + BSA ................. 0.18 Minimal - S + Met-Met ........ 0.15
'Wild-type cultures were grown on minimal me- dium for 12 h and then transferred to the indicated new medium for 2 days of additional growth. Bovine setum albumin (BSA) was present at 1 mg/ml; peptide concentration was 1 mM.
Assays and specific activity as described in Table 1.
FIG. 1. Diagram of peptidase activity visualized inpolyacrylamide gels ofwild-type cell extracts. After electrophoresis 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) log phase, minimal medium lacking nitrogen plus I mg of bovine serum albumin/ml. No activity bands could be detected even after 1.5 h of staining in identical experiments but with the concentrated, dialyzed ex-
tracellular growth medium obtained in the same three growth conditions.
The cys-3 mutant of Neurospora is missing a number of sulfur-related enzymes, because this locus 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 number of nitrogen-related enzymes (11). Unlike wild type, the cys-3 and nit-2 mutants fail to synthe- size extracellular protease under conditions of sulfur and nitrogen limitation, respectively (7). However, when grown under various conditions, these two mutants both displayed normal levels of 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, we suspect that the peptidases active for Leu-Gly and Leu-Leu-Leu are constitutive enzymes. However, this conclusion must be regarded as tentative since changes might occur in the rela- tive amounts of the multiple peptidase isozymes which possess activity toward Leu-Gly and Leu- Leu-Leu (see below). We have no information about possible regulation of peptidases which possess other substrate specificities. Multiple intracellular peptidases. To de-
termine whether Neurospora possesses multiple peptidase species, we used polyacrylamide gels to separate peptidases in crude extracts. The gels were stained for peptidase activity, with a spectrum of different peptides used as sub- strates, by a technique which reveals activity as sharp blue bands. Immobilizing the coupling enzyme, L-amino acid oxidase, within the gel significantly improved resolution, in terms of both the intensity and the sharpness of bands, over previously described methods (9, 15). The results demonstrate the presence of at
least 11 electrophoretically distinct peptidases in crude extracts derived from wild-type mycelia (Table 3, Fig. 2). These peptidases were arbi- trarily numbered in order of their decreasing mobility. Peptidases II and IIIA showed partic- ularly strong activity toward a large number of di-, 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 active form, but it only hydrolyzed dipeptides. All of the peptides tested, except for Glu-Trp, were substrates for more than one peptidase; e.g., Leu-Gly was hydrolyzed by seven different pep- tidases (Table 3). Peptides containing a proline residue were cleaved by peptidases IV, V, and VI. Glu-Trp, which has an acidic amino-terminal residue, was hydrolyzed only by peptidase X.
Extracts derived from conidia were analyzed in an identical fashion. Conidia possess the same pattern of peptidases as found in mycelia, except that conidia lack peptidases I, IIIA, IV, VI, and VIII. Conidia do not have any unique peptidase species which are not present in mycelia. Effect of inhibitors and cations. Potential
inhibitors and cations were added to the staining mixture (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; EDTA also inhibited all ofthese same peptidases except
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TABLE 3. Multiple Neurospora peptidase species revealed by polyacrylamide gel electrophoresisa Peptidase species
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…