Characterization of a novel phenylalanine-specific aminopeptidase from Schizophyllum commune

Mycol. Res. 100 (4): 462-466 (1996)

462

Printed in Great Britain

Characterization of a novel phenylalanine-specific aminopeptidase from Schizophyllurn commune

ROBERT E. BILBREY, ALAN R. PENHEITER, ALLEN C. G A T H M A N A N D WALT W. LILLY Department of Biology, Southeast Missouri State University, Cape Girardeau, MO 63701, U.S.A.

An arninopeptidase (APFI) with high specificity for phenylalanine and tyrosine in the active site P, position was purified from mycelial extracts of the wood-decaying basidiomycete Schizophylluni commune. A two-dimensional preparative electrophoretic scheme resulted in a 60-fold purification of the enzyme, which was found to have an M , of approximately 60 kDa by size exclusion chromatography. SDS-PAGE resolved two closely migrating subunits of approximately 30 and 31 kDa. Its pI was found to be 5.5 and its activity optimum was pH 6.5. Of 11 aminoacyl-p-nitroanilide (-pNA) substrates tested, activity was found only against PhepNA. APFl also showed activity against Phe-p-naphthylamide (-PNA) and Tyr-PNA; however, the activity against Tyr-PNA was less than one third of that against Phe-PNA. No other AA-PNA substrates were hydrolysed. APFl hydrolysed a variety of dipeptides with Phe in the P, position; the amino acid in the Pi position aflected the rate. No activity was found against dipeptides where Phe was not the N-terminus nor against substrates that were N-blocked. APFl was partially inhibited by 1,10-phenanthroline, and this inhibition was reversible by the addition of Zn2+.

Schizophyllum commune Fr. is a common basidiomycete which decays wood. Because wood is a poor nitrogen source (Park, 1976; Watkinson, 1984) we have hypothesized that the extended growth of the mycelial margin is a result of autolysis of older mycelia and subsequent translocation of amino acids. This has been demonstrated for colonies grown in culture under nitrogen-limited conditions (Lilly,Wallweber & Higgins, 1991). The autolytic degradation of proteins requires a complex system of proteolytic activities. In 5. commune this system comprises a ubiquitin-mediated system in the cytoplasm (Higgins & Lilly, 1993) and a variety of exo-and endopeptidase activities (Lilly et al., 1994). At least some of these latter activities may be associated with the vacuolar compartment (Venable, 1993). Many of the putative vacuolar enzymes have their specific activities elevated under conditions of nitrogen deprivation (Lilly et al., 1994) and the upregulation of one of them, ScPrB, has been specifically localized to regions of autolysis (Gordon & Lilly, 1995). Crude extracts of S. commune show aminopeptidase activity against a variety of aminoacyl-p-nitroanilide substrates, including Phe-pNA, Lys-pNA, Arg-pNA, Met-pNA, LeupNA, Gly-pNA and Val-pNA. In general the activity against these substrates was maximal at pH 6.0. Phe-pNA was the most rapidly hydrolysed substrate of the group and the specific activity of the enzyme was increased two-fold during nitrogen starvation (Lilly et al., 1994). This enzyme activity has been named APF and it exists in two molecular forms which migrate closely together in native polyacrylamide gels. The role of APF in nitrogen-limited growth has yet to be determined; however, the enzyme is not secreted, suggesting it participates solely in intracellular protein degradation or

modification. Here we report the biochemical characteristics of the most active isoform of the enzyme, APFI.

MATERIALS A N D M E T H O D S Fungus and culturing methods. Schizophyllum commune homokaryotic strain 4-39 (A41/B41) was used throughout this study. Stock cultures were maintained on cellophanecovered minimal medium as previously described (Lilly, Higgins & Wallweber, 1990). Squares of mycelia, 3 x 3 mm, were taken from the advancing mycelial margin and transferred to minimal medium without membranes in which normal nitrogen source, 1 g 1-' L-asparagine, had been replaced by 1 g 1-' gelatin. After 96 h, mycelia and agar from two colonies were mixed with 50 ml of liquid gelatin-containing medium and macerated with two 30 s bursts in a Waring blendor. Nine ml of this macerate were used to inoculate 50 ml of liquid gelatin-containing medium, and the mycelia were grown for 2-3 d at 21 OC with rotary shaking. These mycelia were then transferred to 1 1 liquid gelatin-containing medium in a Fembach flask and shaken at 50 rpm for 1-2 d. Mycelia were harvested from this flask by filtration through - cheesecloth and exhaustively washed with glass distilled water to remove the extracellular polysaccharide slime and frozen at - 85' until used.

Purification of aminopeptidase activity. All of the following procedures were performed at 4'. Freshly thawed mycelia were suspended in 0.05 M Tris buffer, pH 7.0 containing 0.1 mM phenylmethylsulphonyl fluoride (PMSF); ( I ml buffer 1 g-' F.W. fungus) and homogenized at high speed for 10 min

R. E. Bilbrey and others

463

with a Kunkel Ultra-Turrox tissue homogenizer. After this homogenate had been clarified by centrifugation at 20000 g for 45 min, the supernatant was concentrated approximately two-fold by dialysis against dry polyethylene glycol 8000 (PEG-8000).One ml of 0.05 % (w/v) bromophenol blue 40% glycerol (v/v) was added to 15 ml of this crude extract and the mixture was subjected to preparative native gel was electrophoresis in a BioRad Model 491 Prep Cell. A 9 cm-long separating gel (7.5 % T, made from 37.5: 1 acrylamide stock) and a 2 cm-long standard stacking gel were used. The gel was run at 55 rnA constant current and bands were continuously eluted with Tris-glycine native running buffer at a flow rate of 0.75 ml min-l. 100-drop factions were collected and assayed for APF activity. The 10 fractions containing the highest APF activity were subjected to analytical native-PAGE, and the three fractions showing only APFl activity by this analysis were pooled. The APFl pool was concentrated approximately two-fold against PEG-8000 and then desalted in 3 ml aliquots on BioRad 10-DG columns, with elution in 10 mM Tris buffer, pH 7.0. Preparative isoelectric focusing was performed in a BioRad Rotofor cell. The focusing solution consisted of 2% (v/v) pH 3-10 arnpholytes, 11% (v/v) glycerol in deionized water. After 1 h of pre-focusing the cell (12 W constant power), the APFl pool was added and focusing was performed at 12 W constant power for 4-5 h. Following fractionation of the gradient, the two fractions containing the highest APFl activity were pooled and the ampholytes were removed by desalting on a BioRad 10-DG column with elution in 10 mM tris buffer, pH 7.0.

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Analytical PAGE and molecular weight determination. Analytical native and SDS polyacrylamide gel electrophoresis was routinely performed in 10% T, mini-format gels according to standard protocols (Laemmli, 1970 for SDS gels; LaemmIi buffer system without SDS for native gels). Following electrophoresis, gels were stained for protein with Coomassie Blue or with silver using a BioRad kit. Molecular weight estimation for subunits was obtained from a standard curve based on the relative migration of 10 kDa ladder proteins (Amersham) during SDS-PAGE. APF activity in native gels was detected by staining the gel with 250 pl of 20 mM PhePNA 150 PI 30 mg ml-I fast garnet GBC in 25 ml of 0.05 M citrate buffer, pH 6.5, following a 15 rnin wash in water and two successive 15 rnin equilibrations in 0.05 M citrate, pH 6.0. The method of Gordon & Lilly (1995) was used to reactivate APF activity in SDS gels. The gels were incubated 30 rnin in 0.05 M citrate buffer, pH 6.5, 2.5 % (v/v) triton X-100. This was followed by two successive 15 rnin washes in 0.05 M citrate buffer, pH 6.5 and staining with phe-PNA as above.

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Determination o f native molecular weight by size exclusion chromatography. Purified APFl activity was chromatographed on a 1.5 crn x 70 cm Sephacryl S-200 column equilibrated in 0.1 M Tris buffer, pH 7.0. Relative molecular weight was determined by comparing the V,/V, of APFl to that of the following standards: P-amylase, Mr = 200 000; alcohol dehydrogenase, M, = 150000; carbonic anhydrase, Mr = 29000; and cytochrome c, Mr = 14500.

Protein and APF enzyme assays. Protein in samples was determined using the method of Bradford (1976) with bovine serum albumin as the standard. APF activity was measured by determining the rate of hydrolysis of phen~lalanine-pnitroanilide in 0.1 M citrate, pH 6.5. Routinely, assay solutions contained 930 p1 buffer 50 pl 20 mM phe-PNA 20 p1 sample. The reactions were allowed to run from 30 rnin to 3 h at 30' and A,,, was determined. The reaction kinetics were linear over this time span. One unit of APFl activity is defined as the amount of enzyme needed to release 1 pmol pnitroaniline per rnin at 30'. Activity against other pNA substrates was measured in a similar manner. Activity against PNA substrates was determined by incubating 20 p1 sample in a solution containing 260 PI 0.1 M citrate buffer, pH 6.0 20 pl of the appropriate 20 mM PNA substrate for 2 h at 30°. Colour development was initiated by the addition of 600 p1 of 0-75 mM fast garnet GBC in 4 % (v/v) Tween 20. After 10 rnin the absorbance at 500 nm was determined. Activity of APFl against dipeptide substrates was determined using an enzymecoupled method similar to that of Logan (1987). Twenty pl (ca 15 mUnits) of sample was incubated in a reaction mixture of 50 p1 20 mM dipeptide substrate 200 pl 0.1 M citrate buffer, pH 6.5. Following incubation for 4-10 h, the reaction mix was heated to 80° for 10 min, allowed to cool, and 730 p1 of solution consisting of 36 pg ml-' L-amino acid oxidase, 6 ~g ml-l horseradish peroxidase, and 30 pg ml-lo-dianisidine was added. This solution was incubated 30 rnin at room temperature and A,,, was determined. Blanks for each substrate were used to zero the spectrophotometer; however, they showed no colour development. Because L-aminoacid oxidase oxidizes different amino acids at different rates, standards for each single amino acid were run. By determining the relative contribution to colour development for each amino acid it was possible to represent the rate of hydrolysis of the dipeptides in terms of the release of the N- or the C-terminal phenylalanine o n l h All data which follow represent the mean of two or, more typically, three replicate assays. The total range around these means was normally less than 10% of the mean.

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RESULTS APF activity exists in two forms in S. commune (Fig. I). APFl activity predominates in culture extracts of young mycelia that are not nitrogen limited; APF2, while present in all colonies, has its activity increased somewhat during nitrogen starvation (Lilly et al., 1994). Colonies grown on gelatincontaining medium have three- to four-fold higher specific activity than those grown on minimal medium. Most of this specific activity increase is due to decreased protein production overall by the colonies, meaning that gelatin-grown mycelia are already enriched in APF. The results of the two-step purification of APFl are shown in Table I. Various conditions for both Prep Cell and Rotofor runs were tested, those indicated above are representative of the typical conditions that give the best yield and purification. The apparent low yield of APF activity is a result of our selecting only the fractions with the highest activity of APFl and no contaminating APF2 following the Prep Cell run. Typically, APF activity was spread over 20 fractions from which we

Schizophyllum commune phenylalanine aminopeptidase

464 Table 2. Hydrolysis of P-naphthylamide substrates by S. commune APFl Substrate

Relative rate1

Phe-PNA Tyr-PNA Lys-PNA Gly-PNA Leu-PNA Met-PNA Val-PNA GlyPhe-PNA GlyPro-PNA LysAla-PNA

I00 29 5

2

0.1 0 0 0 0 0

Rate of hydrolysis of Phe-PNA = 69.9 A (min)-I (mg protein)-'. Assays performed with 20 munits of APFI. Table 3. Hydrolysis of di- and tripeptides by S. commune aminopeptidase APF 1 Hydrolysis rate m o l (min)-' (mg protein)-'

Fig. 1. Gel A: Native polyacrylamide gel stained for Pheaminopeptidase activity using Phe-P-naphthylamide and Fast Garnet GBC. lane I, crude extract; lane 2, purified APFl activity. Gel B: SDSpolyacrylamide gel silver stained for protein. Lane 1, crude extract; Lane 2, pooled Prep Cell fractions; Lane 3, Blank; Lane 4, Purified APFl activity following Rotofor.

selected three for the Rotofor separation. It was necessary to include the irreversible serine protease inhibitor PMSF in the extraction buffer due to the presence of a low amount of some contaminating protease in the purified APFl sample. This protease was detected by increased activity of purified APFl in the presence of PMSF during initial inhibitor studies. That the contaminating protease activity was destroyed by the initial PMSF treatment is supported by subsequent inhibitor studies (Table 4). The purified APFI activity was substantially free of other contaminating proteins as demonstrated by SDS-PAGE (Fig. I). In replicate Rotofor runs, the pI of APFI activity was consistently found to be between 5.5 and 5.65. The native M,. of APFI, estimated by size exclusion chromatography, was 60000. Silver stained SDS-PAGE gels revealed two closely migrating bands with M, of 30 kDa and 31 kDa (Fig. I). At least one of these putative subunits could be renatured to give APF activity. The activity was much lower than in native gels, however, and the long staining time resulted in a large enough loss of resolution that it was impossible to determine if the restored activity occurred with both subunits. These results imply that native APFI is a dimer with two similar but not identical subunits. Our earlier studies of the activity of APF in crude extracts indicated that the enzyme had a pH optimum of 6.0. Careful measurement of the pH optimum of purified APFI showed

PhePhe PheAla PheTyr PheMet PheLeu PheVal PheCly PhePro TyrPhe LeuPhe AlaPhe PhePhePhe N-cbz-PheLeu

that it is maximally active at pH 6.5, although the range of activity is broad between pH 6.0 and pH 8.0. All of the kinetic data we obtained were determined at pH 6-5. APFl was highly active against Phe-pNA. No activity was found against any other -pNA substrates tested including Leu-, Val-, Met-, Gly-, Pro-, Agr-, Lys-, GlyPhe-, GlyPro-, N-succinyl-Phe-, or N-succinyl-AlaAlaProPhe-pNA. The extent of this specificity was also tested with -PNA substrates (Table 2). Again, PhePNA was hydrolysed most rapidly. Activity against Tyr-PNA was approximately 70% lower. In addition, a very small amount of activity was detected against Lys-PNA and GlyPNA. No activity was detected against Leu-, Val-, Met-, GlyPhe-, GlyPro-, or LysAla-PNA. The hydrolysis of dipeptides b y purified APFl showed an effect of the amino acid present in the S,' position of the substrate (Table 3). Activity was highest when the S,' amino acid was Phe, Ala or Tyr. Other aliphatic amino acids in the S,' position were hydrolysed at a slower rate (Met > Leu > Val > Gly). Very little hydrolysis of PhePro occurred.

Table I. Purification of aminopeptidase APFI from Schiwphyllum commune

Concentrated crude extract Prep cell fractions 6 5 4 7 Rotofor

Volume Total protein (mg) (ml)

Total activity Specific activity units1 units mg protein-'

Purification- Yield (70) fold

15 10.2

3200 163 74.4

1 12 66

4.8

28.6 0.12 0.01

One unit of activity = 1 pmol Phe-pNA hydrolysed min-I at pH 6.5, 30'.

112 1336 7440

100 5 2.5

R. E. Bilbrey and others Table 4. Effect of mechanistic inhibitors on APFl activity

None 1,10 phenanthroline

Table 7. Kinetic properties of S. commune aminopeptidase, APFI

Concentration (m)

Activity control (%)I

na

100 94 78 42 101 102 90 99 100 100

0.5 5.0 20.0 PMSF 0.2 10.0 trans-epoxysuccinyl-L-leucylamido- 0.03 (4-guanidino)butane (E-64) 0.15 Pepstatin A 0.007 0.035

Assays performed with 20 munits enzyme activity. Table 5. Effect of divalent cations on S. commune APFr activity Concentration ( m ~ )Activity control (%)I Zn2+

0.01 0.05 1.00 3.00 0.10 1.00 2.00 0.10 1.00 2.00 0.05 1.00

100 91

Phe-pNA Phe-Phe Phe-Ala

li, ( m ~ )

VmIXm o l (min)F1 (mg protein)-'

0.24 0036 0041

7456 1521 1739

reactivate the enzyme. A combination of the two ions did not substantially increase the reactivation over Zn2+ alone. Kinetic constants for APFl were determined from Lineweaver-Burk plots and plots of s/v v. s (Henderson, 1992) for hydrolysis of Phe-pNA, PhePhe and PheAla. The data from the latter analysis are shown in Table 7; however, both analyses gave similar results. The K, for both dipeptides was considerably lower than that for artificial substrate Phe-pNA. . ' , of the enzyme was much higher with the In contrast, the I pNA substrate.

DISCUSSION

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Purification of active enzymes from Schizophyllum commune is hampered by the presence of a viscous polysaccharide slime which is released from the cell walls upon extraction. The slime interferes with column chromatography and with Co2+ standard precipitation techniques such as salting out. To overcome this problem it is possible to precipitate the slime Ca2+ using ethanol or acetone prior to protein separation. This was successfully applied to the purification of acid phosphatase Assays performed with 15 mUnits enzyme. isozymes of S. commune (Wallweber & Lilly, 1992). In the case of APF, activity is destroyed by treatment with acetone and Table 6. Reactivation of 1,10 phenanthroline inhibited APFl activity the subsequent dialysis necessary to remove it. For this reason we have resorted to the use of preparative electrophoretic Activity methods for initial purification steps. We found that the slime control (%) can be effectively removed from extracts using either the Control BioRad 491 Prep Cell or the Rotofor as a first step. While I 0 InM 1,10 phenanthroline using the Rotofor first has the advantage of a larger sample 10 mM 1,IO phenanthroline+ I mM ZnC1, load, it suffers from having the core plugged by slime during 10 mM 1,10 phenanthroline + 5 mM ZnC1, run if the extract concentration is high. In addition, we the 10 mM I,IO phenanthroline+ I mM MgCI, 10 mM 1,10 phenanthroline + 5 mM MgC1, found that a Rotofor-first protocol resulted in a less stable 10 m~ 1,IO phenanthroline + I mM ZnCI, + 1 mM MgCl, enzyme preparation, although yield and purification were 10 mM 1,10 phenanthroline + 5 mM ZnCI, + 1mM MgC1, about the same (data not shown). Assays performed with 10 munits of enzyme. Native APFl is a dimeric protein with two similar subunits of approximately 30 kDa each. One, if not both, of these Hydrolysis of the dipeptide TyrPhe occurred at approximately subunits is active when dissociated from the other based on 36% of the rate of PheTyr, corroborating the results for the ability to reactivate them following SDS-PAGE. Arninospecificity of the P, active site position found using PNA peptidases are structurally diverse among species (cf. Taylor, substrates. AlaPhe was not hydrolysed nor was the N-blocked 1993) and even within species. In yeast, which arguably has substrate N-CBZ-PheLeu. The tripeptide PhePhePhe was the best characterized proteolytic system, aminopeptidases hydrolysed at a slow rate although this may partially be due ranging in sizes from 43 kDa (Met aminopeptidase; Chang, Teichert & Smith, 1992) to 640 kDa (yscl; Frey & Rohm, to its insolubility compared to other substrates. The mechanism of APFl was investigated using class- 1978) have been reported. This diversity extends to both specific inhibitors (Table 4). AFPl was found to be inhibited substrate specificity and biochemical mechanism, with the only by I,IO-phenanthroline, suggesting that it is a metallo- former representing one of the principal means of classifying protease. Assays with added divalent cations provided mostly aminopeptidases (Taylor, 1993). With respect to substrate inconclusive evidence regardmg the nature of the involved specificity, APFI appears to be unique. While aminopeptidases cation species (Table 5), although high concentrations of Zn2+ have been described which preferentially hydrolyse N-terminal completely inhibited the enzyme. We found it was possible to Leu and other hydrophobic amino acids, including phenylpartially reactivate 1,lo-phenanthroline-inhibitedAPFl by alanine, none has been described which has such a high addition of Zn2+ (Table 6). Addition of Mg2+ did not specificity for Phe. Aromaticity evidently is the determining Mg2+

0 105 108 100 97 99 100 103 104

Schizophyllum commune phenylalanine aminopeptidase factor in specificity of APFI, given that the only amino acid other than Phe that can appear in the S, position of the substrate and still give significant activity is Tyr. Differences in hydrolysis rates based on the second amino acid residue do not follow an apparent pattern, thus its significance is unclear. Most of the described aminopeptidases are metallo-enzymes, many of which utilize Zn2+as a cofactor (Taylor, 1993).Based on inhibition and reactivation studies, APFl falls into this class. Our data also suggest that Zn2+ is the only cofactor, because addition of Ma2+ in combination with Zn2+ had no effect. The role of APFl in the physiology of S. commune is unknown. Its high degree of specificity suggests that it may be used principally as a modifying enzyme or as a constituent of a multienzyme protein degradation system in the vacuole. It has been proposed that aminopeptidases might have a role in regulating in vivo degradation rate of proteins (Bachmair, Finley & Varshavsky, 1986). In what is known as the N-End rule, the rate of degradation of specific proteins is determined by the N-terminal amino acid. The amino acids Met, Ser, Ala, Thr, Val and Gly have relatively long half-lives (ca 20 h). In contrast, Arg, Lys, Phe, Leu and Asp tend to destabilize proteins and reduce half lives significantly (less than 3 min). What is the role of an aminopeptidase which would apparently lengthen the half-life of proteins upon which it acts? This is certainly enigmatic; however, since most N-End rule protein degradation proceeds via ubiquitin mediated pathways (Richter-Rouff, Heinmeyer & Wolf, 1992), the APFl would have to be cytoplasmic to have this effect. The subcellular localization of APFl activity has not been rigorously examined. In a study by Venable (1993) it was found to copurify in sucrose density gradients with putative vacuolar marker enzymes. This is consistent with the localization of the major aminopeptidases of yeast (Rendueles & Wolf, 1988; Yasuhara, Nakai & Ohashi, 1994). If the vacuole is the location of APFI, then its effects on N-End rule protein degradation might be minimal. On the other hand, one recent study using yeast deficient in the major vacuolar proteases showed that ubiquitin-conjugated proteins accumulate in the vacuole (Simeon et a]., 1992), suggesting that such conjugates may be degraded in the vacuole as well as by the normal multicatalytic proteinase (Richter-Ruoff et al., 1992). This work was supported by grants from the National Science Foundation (DMB-9303879)and from the Grants and Research Funding Committee of Southeast Missouri State University to W. W.L. The authors thank Ms Bobbie Arnold for excellent technical assistance.

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(Accepted 5 September 1995)

466 Bradford, M. M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248-254. Teichert, U. &Smith, J. A. (1992).Molecular cloning, sequencing, Chang, Y.-H., deletion, and overexpression of a methionine aminopeptidase gene from Saccharomyces cerevisiae. ]ournu1 of Biological Chemistry 267, 8007-8011. Frey, J. & Rohm, K.-H. (1978). Subcellular localization and levels of aminopeptidases and dipeptidases in Saccharomyces cerevisiae. Biochimica et Biophysics Acta 527, 31-41. Gordon, L. J. & Lilly, W. W. (1995). Quantitative analysis of Schizophyllum commune metalloprotease ScPrB activity in SDS-gelatin PAGE reveals differential mycelial localization of nitrogen-limitation autolysis. Current Microbiology 30, 337-343. Henderson, P. J. F. (1992). Statistical analysis of enzyme kinetic data. In Enzyme Assays: A Practical Approach (ed. R. Eisenthal & M. J. Danson), pp. 277-316. Oxford University Press: Oxford, U.K.. Higgins, S. M. & Lilly, W. W. (1993). Multiple responses to heat stress by the basidiomycete Schiwphyllum commune. Current Microbiology 26, 123-127. Laemmli, U. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 6 8 H 8 5 . Lilly, W. W., Bilbrey, R. E., Williams, 8. L., Loos, L. S., Venable, D. F. & Higgins, S. M. (1994). Partial characterization of the cellular proteolytic system of Schizophyllum commune. Mycologia 86, 564-570. Lilly, W. W., Higgins, S. M. & Wallweber, G. J. (1990). Uptake and translocation of 2-aminoisobutyric acid by Schizophyllum commune. Experimental Mycology 14, 169-177. Lilly, W. W., Wallweber, G. J. & Higgins, S. M. (1991). Proteolysis and amino acid recycling during nitrogen deprivation in Schiwphyllum commune. Current Microbiology 23, 27-32. Logan, D., Naider, F. & Becker, J. (1987). Partial purification and characterization of intracellular carboxypeptidase of Candida albicans. Eiperimental Mycology 11, 115-121. Park, D. (1976). Carbon and nitrogen levels as factors influencing fungal decomposers. In The Role of Terrestrial and Aquatic Organisms in Decomposition Processes (ed. J. M. Anderson & A. MacFayden), p p 41-59. Blackwell: Oxford, U.K. Rendueles, P. S. &Wolf, D. H. (1988).Proteinase function in yeast: biochemical approaches to a central mechanism of post-translational control in the eukaryotic cell. FEMS Microbiological Reviews 54, 17-46. Richter-Rouff, B., Heinrneyer, W. & Wolf, D. H. (1992). The proteosome/ multifunctional proteinase. In uivo function in the ubiquitin-dependent Nend rule pathway of protein degradation in eukaryotes. FEBS Letters 302, 192-196. Simeon, A., Van der Klei, I. J., Veenhuis, M. & Wolf, D. H. (1992). Ubiquitin, a central component of selective cytoplasmic proteolysis, is linked to proteins residing at the locus of non selective proteolysis, the vacuole. FEBS Letters 301, 231-235. Taylor, A. (1993). Aminopeptidases: structure and function. FASEB journal 7, 29G298. Venable, D. F. (1993). Subcellular localization of protease activities of Schiwphyllum commune. M.S. thesis, Southeast Missouri State University, Cape Girardeau, MO. Wallweber, J. G. & Lilly, W. W. (1992). Purification and characterization of the two constitutively produced acid phosphatase isoymes from Schiwphyllum commune. Mycological Research 96, 792-797. Watkinson, S. C. (1984). Morphogenesis of the Serpula lacrimans colony in relation to its function in nature. In The Ecology and Physiology of the Fungal Mycelium (ed. D. H . Jemings & A. D. M. Rayner), pp. 165-184. Cambridge University Press: Cambridge, U.K. Yasuhara. T., Nakai, T. & Ohashi, A. (1994). Aminopeptidase Y, a new aminopeptidase from Saccharomyces cerevisiae. journal of Biological Chemistry 269, 13644-13650.