Specificity of the collagenolytic enzyme from the fungus Entomophthora coronata: Comparison with the bacterial collagenase from Achromobacter iophagus

ARCHIVES OF RIOCHEMISTRY AND BIOPHYSICS Vol. 192, No. 2, February, pp. 438-445, 1979

Specificity

of the Collagenolytic

Entomophthora Bacterial NICOLE

Collagenase

HURION,’

coronata:

Enzyme from the Fungus Comparison with the

from Achromobacter

HUGUETTE

FROMENTIN,

AND

iophagusl BORIVOJ

KEIL

Received August 3, 1978 The specificity nf the rollagenolytic enzyme from the fungus E)~towophfhorcrco~o~fn toward Some inhibitors and the B chain of oxidized insulin was investigated and compared to that of the bacterial collagenase from Achromohacfer iophagrts. The fungal enzyme was completely inhibited by diisopropylfluorophosphatc, tosyl-L-lysine chloromethyl ketone, and tosyl-amino-2-phenylethyl chloromethyl ketone but not at all by ethylenediaminetetraacetate. This indicates that it is not a metalloenzyme like the bacterial Acl~w~~~obacter collagenase. The B chain of insulin was not hydrolysed at all by the bacterial enzyme under conditions Mhere exlensive digestion was observed with the Eutowophthom enzyme. The fungal enzyme cleaves preferentially the bonds Leu-Tvr and Leu-Val as determined hy automatic Ii. I”6 II 12 sequencing; the secondary cleavages were identified by a systematic analysis of the digestion mixture: thus, the fungal collagenolytic enzyme from E~forrto/~i~f/~orn comuatn differs both structurally and functionally from the bacterial Acl~ro~trut~crcfer collagenase.

Data presented in a previous paper (1) have shown that the pathogenic fungus EHtowoplrthom coro~lata produces an extracellular protease which possesses collagenolytic activity. This enzyme was found to be capable of degrading native calf skin collagen in its helical parts, and of hydrolyzing the Leu-Gly bond in the synthetic pentapeptide Pz-Pro-Leu-Gly-Pro-D-Arg.:’ In that first approach, the specificity appeared to be similar to that of the bacterial collagenase from Achrowobactev iophagus which has been extensively studied in our laboratory (Z-8). Like vertebrate collagenases the Achromobacter collagenase is a Zn metalloenzyme with a very narrow specificity toward protein substrates. This paper is an ’ This investigation was supported in part hy the grant No. 76-7-0079 of D.G.R.S.T. and hy la Fondation pour la Recherche Medicale Frangaise. y To whom correspondence should be addressed. :I Abbreviations used: DFP, diisopropylfluorophosphatc; TLCK, 1-tosyl~L~lysine chloromethyl ketone; TPCK, 1-tosyl-amino-2-phenylethyl chloromethyl ketone; Pz-Pro-Leu-Cly-Pro-D-Arg, 4-phenylazo-henzylox~carbon~l-~-prolyl-~-leucyl-glycyl-~-proly~-~~~ine. 438

0003.98611791020438.08$02.00/O Copyright All rights

D 1979 by Academic Press. of reproduction in any form

Inc. reserverl.

attempt to compare the fungal and the bacterial enzymes, with regard to the nature of their active sites and their specificity toward the B chain of oxidized insulin. The fact that the collagenolytic activity of the fungal enzyme was not inhibited by EDTA, which inhibits bacterial collagenases (2, 9) and most of the vertebrate collagenases (lo), suggested that this enzyme could be a serine protease, probably related to other serine proteases from molds (11). The present results confirm this suggestion. Although the overall action of the fungal and bacterial enzymes on collagen and the synthetic substrate seemed to be comparable, the analysis of the action on the B chain of insulin has shown that their specificity is entirely different: No splitting of any bond of the B chain occurred with the bacterial collagenase, whereas the fungal collagenolytic enzyme degraded the same substrate readily. To determine the sites of the early action of the enzyme we found it advantageous to apply automatic sequencing to the whole digestion mixture, independently or simultaneously with the time-consuming separation and characterization of the fragments.

COLLAGENOLYTIC MATERIALS

ENZYME

AND METHODS

FROM Enloitiophthoru

439

&teiiiiinatio), ftt’ l/le points of clcnc~age ijl tile R oforidized inscclirl. In preliminary experiments, 3 mg of the B chain in 0.1 M ammonium carbonate, pH 7.8, were incubated at the following enzyme-substrate ratios: 1:20, 1:50, l:lOO, 1:200, and 1:600 for 1 or 4 h at room temperature or at 37°C. The reaction was stopped by freezing; after lyophilization the digest was analyzed by the fingerprinting technique on Whatman No. 1 paper, first by high-voltage electrophoresis (Gilson apparatus, Model D) in acetic acidlpyridinei water (30:3:867, v/v/v), pH 3.7, at 55 V/cm for 30 min followed by chromatography in tt-butanol/pyridinei acetic acid/water (15:10:3:12, v/v/v/v) and staining with 0.2? collidine/ninhydrin solution in acetone. For the systematic analysis of fragments resulting from the digestion of the B chain by the fungal enzyme, the following procedure was adopted: 25 mg of the B chain of oxidized insulin dissolved in 4 ml of 0.1 M ammonium carbonate at pH 7.8 were mixed with 80 ~1 of the enzyme solution (enzyme-substrate ratio, 1:200) and incubated at room temperature for 1 h. After freezing and lyophilization, the digest was dissolved in 2 ml of 0.5 M pyridine-acetate buffer, pH 3.0, and applied to a column (20 x 1.2 cm) of Dowes 5OW-X2 equilibrated with the first buffer, at room temperature. A system of volatile elution buffers of increasing molarity and pH was adopted (16); a peristaltic pump maintained a constant flow rate of 10 ml/h and fractions of 2 ml were collected at 12.min intervals. One-fifth of the eluate from every second tube was evaporated and analyzed by paper chromatography. Fractions were pooled and evaporated to dryness in a rotary evaporator 1~ I’UCUO at 35°C. Pooled fractions containing mixtures were further fractionated by preparative paper chromatography. The isolated pure peptides were subjected to acid hydrolysis with 6 M hydrochloric acid at 110°C for 24 h in sealed, evacuated tubes and analyzed on a Beckman Multichrom B analyzer, using a long column system (17). When necessary, the amino-terminal residues were identified by dansylation according to Hartle! (18) (7.5 x 7.5.cm polyamide sheets were used). To determine the very early stages of proteolytic degradation, two assays were carried out under conditions of limited digestion: 300 nmol of the B chain in 300 ~1 of 0.1 M ammonium bicarbonate, pH 7.8, were incubated with 6..5 ~1 of the enzyme solution (enzymesubstrate ratio, 1:200), at 0°C for 15 and 60 min, respectively. The digestion was stopped by lowering the pH with 20% formic acid, and the lgophilized samples were submitted to automatic degradation (Beckman apparatus, Model X90 C) using parvalbumin as carrier and the dimethylbenzylamine program recommended by Ericsson (personal communication). The phenylthiohydantoins of the amino acids were analyzed either by thin-layer chromatography on silica gel 60 F 254 plates (10 x 10) eluted with chloroform/methanol (9911. v/v, and 98112, v/v) or by high-pressure liquid chromatography (Siemens) using columns 300 x 2.8 mm, filled with Spherisorb and Lichrosorb Si 60 as described by chui~

Elrzyrnes. The collagenolytic enzyme of E. coror~ato was isolated from the culture medium concentrate by the procedures described previously (1). The purified fraction (peak III) obtained after the second ion-exchange chromatography was used for the assays, the concentration of the sample being determined by the method of Lowry (12). The crude collagenase from A. io~J~ng/t.s was obtained from Institut Pasteur Production and purified to homogeneity according to Lecroisey et trl. (2). f)ti/r,, ,t/crtc~rirrl.s. Chemicals were purchased from the following sources: Pz-Pro-Leu-Gly-Pro-D-Arg, diisopropylfluorophosphate, sodium ethylenediamine tetraacetate, n’-cr.benzoyl-I,-arginine ethyl ester, and Dowex 5OW-X2 (200-400 mesh) from Fluka; l-tosglI>-lysine chloromethyl ketone, 1-tosyl-amino-Z-phenylethyl chloromethyl ketone, casein, Lichrosorb Si 60, and silica gel 60 F 254 HPTLC plates from Merck: (‘ongo red elastin. from Sigma; insulin chain B oxidized form (bovine insulin) and Spherisorb from Boehringer. Hake parvalbumin was prepared in our laboratory according to Pechere (13). All chemicals used Lvere of analytical grade. Etf-,~/~nic c~s.sn!,.s.The activity toward Pz-Pro-LeuGly-Pro-D-Arg, and the caseinolytic and the elastolytic activities were determined as described elsewhere (1). As the cleavage of the synthetic pentapeptitle and the degradation of native collagen by the enzyme from g. cof.o,/rrftr was found to be due to the same catalytic action, the expression “collagenolytic” is used throughout the text for the pentapeptide assay for the sake of brevity. /~hihitio~/. All the inhibition assays were done at room temperature. For the inactivation assay by EDTA. the sample was first dialyzed against 50 rnx Tris, l)H 7.0, to remove the Ca” ions. The substrate xvas prepared in the usual Verona1 buffer, pH 8.5, without CaCl>, and the solutions used made 10 L M in EDTA. Appropriate blanks were prepared for each assay, containing 50 mM Tris, pH 7.0, made lo-’ M in EDTA, instead of the enzyme. The dialyzed enzyme was incubated with EDTA at a final concentration of 10 ” M, and the reaction was stopped by freezing and immediately assayed toward the synthetic peptide. For the inactivation by DFP, a 10 ’ M stock solution in isopropanol was mixed with the enzyme solution at pH 6.0 to give final concentrations of DFP of 10 :< and 10 -I M. At time intervals, samples were withdrawn, the reaction was stopped by freezing, and the inhibition was evaluated by the appropriate enzymic assay. The inhibitions by TLCK and TPCK were determined according to (14) and (15): 10 ’ M stock solutions of either reagent in appropriate solvent (50 mM Tris 10-l M CaCl,, pH 6.0, for TLCK, and methanol for TPCK) were used. For the measurement of the residual activity, substrate solutions were adjusted to the same inhibitor molarity as the reaction mixture.

corolratn

440

HURION,

FROMENTIN.

Franck and Struber (19). For the calculation of the relative yields of cleavage, a blank was run under identical conditions, except that the formic acid was added to the B chain solution before the enzyme.

RESULTS

Effects of various inhibitors on proteolytic and collagenolytic activities. The abil-

ity of various inhibitors to block the activities of the fungal enzyme from E. coronata and of the bacterial collagenase from A. iophagus are compared in Table I. EDTA, which completely inhibits the bacterial collagenase, was ineffective against the fungal proteinase, while DFP inhibits the fungal proteinase partially at 10e4M within 60 min and completely at lo-” M within 30 min. It does not affect the collagenase from A. iophagus. The caseinolytic and elastolytic activities are more susceptible to DFP, as both activities are blocked at 1O-4M within 30 min and at 10m3M within 5 min. Both TLCK and TPCK, potent inhibitors of mammalian trypsin and chymotrypsin, respectively, inhibit the collagenolytic activity of E. coronata enzyme at 5 x lop2 M within 60 min. Determination of the bonds cleaved in the B chain of oxidized insulin. The collagenase from A. iophagus was without action on the

B chain up to the enzyme-substrate ratio 1:600 at 37°C for 4 h of incubation. On the TABLE

AND KEIL

contrary, under the same conditions, the enzyme degraded the B chain readily. When the B chain was incubated at 0°C for 15 min and the digestion mixture was submitted to automatic Edman degradation, two N-terminal residues appeared at each step, one from the N-terminal sequence of the B chain, the other from a new N-terminal, Tyr-Leu-Val- , formed by the preferential proteolytic cleavage. It indicated that the bond Leu-Tyr was hydrolyzed much faster than Eny b6ther one in the chain; the splitting of this bond occurred with a 97.5% yield, calculated from the amount of substrate used. When the time of digestion was increased to 60 min at the same temperature and the same enzyme-substrate ratio, a second new N-terminal appeared resulting from the cleavage of the bond Leu-Val with 11 12 92% yield. Digestion of the B chain for 1 h at room temperature led to a mixture of small peptides, reflecting extensive breakdown of the chain. The positions of the peptides eluted from the Dowex 5OW-X2 column and their Rf values after subsequent paper chromatography are shown in Fig. 1; their recoveries and sequential assignments after systematic chromatographic separations are shown in Table II. From these data, the major, intermediate, and minor cleavages E. coronata

I

EFFECT OF VARIOUS INHIBITORS ON THE DIFFERENT ACTIVITIES OFTHE COLLAGENOLYTICENZYME FROME.coronata Percentage

Inhibitors

Final concentration mf)

Time (min)

Synthetic peptide

EDTA

10-Z

30 60

DFP

10-a 10-S

30 60 5 15 30

25 55 (0) 59 98.7 100 (0)”

TLCK

5 x 10-Z

60

100

TPCK

5 x 10-Z

60

100

” The results of the inhibition

inhibition

Casein

against

Elastin

0 0 (loo)” 100

100

100

100

assays on the collagenase from A. iophagus according to Lecroisey

et al. (2).

COLLAGENOLYTIC

ENZYME

FROM E~tfowophfhorn

441

cororcafrr

‘H

8

6

4

.2

0

Ibo

I 400

3’00

2bo FRACTION

500

6

-0 0

NUMBER

FIG. 1. Peptide map of the digest of B chain of insulin by E’~~towophtho~rc collagenase. Peptides are designated by Roman numerals in the order of their elution from the Dowex column; the peptides obtained by subsequent paper chromatography are designated by an additional subscript. Abscissa: fraction number. Ordinate: pH values (A __ A) of the eluate and R, of individual peptides.

occurring along the B chain are represented in Fig. 2. The major cleavage points are the bonds Leu-Tyr and Leu-Val as shown by 13 limited dige$ion of ihe chain. Two additional cleavages giving significant yields of peptides occurred at Tyr-Thr and Lys-Ala; 26 27 29 30 another cleavage took place at the AZig-Gly “3 bond giving the peptide Tvr-Arg in 5%) l”6 22 yield. Minor cleavages were observed at the bonds His-Leu and Ser-His near to the Nterminal par; of the B ch:in. Other minor peptides (I to III) were obtained only in traces, and thus they cannot influence the overall picture of the degradation scheme. Peptides IV and IX, appeared in two different positions of the chromatogram, with different yields; this is probably due to partial deamidation of the glutamine residue occurring during either digestion or the elution procedures, as has already been observed (20, 21).

DISCL3SION

The collagenolytic enzyme from E. corerruta may be regarded as a collagenase according to their definition as enzymes capable of degrading native collagen in its helical parts (22). Nevertheless, during this study it appeared that the specificity of the fungal enzyme toward some inhibitors was quite different from that of the bacterial ones: collagenases from Clostridirc )i/ 11isfol~yticrcrn and A. iophagm are inhibited by EDTA and not by DFP (9, 2) like other metalloproteinases, whereas the collagenase from E. coro7lcrfcr is very sensitive to DFP and not to EDTA. In addition, it is inhibited by TLCK and TPCK which suggests that it is a serine proteinase and that a histidine residue may be implicated in its catalytic site. The assumption that the collagenase from E. comuata might rather be a serine proteinase of broad specificity which is capable

442

HURION,

FROMENTIN, TABLE

AND KEIL II

AMINO ACID COMPOSITIONOF PEPTIDES ISOLATEDFROMTHE DIGE~TOF OXIDIZED IN~TJLINB CHAIN BYTHE COLLAGENOLYTICENZYME FROME. coronafa

Peptides

Composition

Sequence

IV

Val”

V

&TM” (1.0) Cys (0.6) Gly (1.5) Ser (0.6) His (0.2) Arg (0.2) Ala (0.2)

VI VII

(1.6) Glu” (1.0) Ala (0.9) Leu (1.1)

Yield (%I

Val-Leu

3

LZu-SZr 8 9

1

Phe” (1.0) Vai (1.1) Asp (1.2) Gln (1.4) His (1.8) Leu (2.3) Cys (1.4) Gly (1.7) Ser (2.1)

Phe-Leu

6

Tyr (0.6)Leu (0.8)Val (0.4)

Tir- Alig

Cys (0.4) Gly” (1.0) Gln (1.1) Arg (1.0)

VIII,

Thr (0.9) Pro (1.5) Lys (1.1) Ala” (1.0)

Tir-A,p

VIII,

Le!c (1.1) Val” (1.0) Cys (0.7) Gly (1.8) Glu (0.9) Arg (0.8) Phe (0.9)

Leu-Phe

1x1

Glyh (1.0) Gln (1.0) Arg (0.7)

d;y - A;;g

1x2

Ala

A?a

5 10 0.8 0.8 14.6

1x3

Phe (0.9) Val (1.1) Asp (0.9) Gin” (1.0) His (0.9)

PFe-His

0.7

1x4

Lecc (1.8) Cys (0.9) Gly (1.6) Ser (1.5) His* (1.0)

Lku-LEu

2.5

1x5

Val (1.0) Gln (1.0) Ala” (1.0) Leu (0.9) His (0.2)

&LZu

8

X

His (1.0) Leu” (1.0) Gly (0.3) Phe (0.2)

I&-Lil

4.25

XIII

Lecc (1.1) Val” (1.0) Cys (0.8) Gly (1.9) Gln (0.9) Arg (0.9) Phe (2.0)

L:u-Pie

1.5

XIV

Gly (0.9) Phe (2.1) Tyr”


0.8

(1.0) Arg (0.3) Ser (0.7)

a The italicized amino acids were identified as N-terminal obtained in quantities too small to be detected. b Arbitrarily taken as one residue.

of action on native collagen was reinforced by the comparative study of the bond cleaved by the bacterial and the fungal collagenase in a polypeptidic substrate. Although both proteinases from A. ioph,agus and E. coronata cleave the same pentapeptide substrate and native collagen (l), they differ profoundly in their specificity toward the B chain of insulin. This polypeptide is not attacked by the collagenase from A. iopagus, in accordance with the narrow specificity of the enzyme which is known to be preferentially directed toward the bonds Yi Gly-Ala and YiGly-Pro in collagen and in some other proteins (3,6,23). However, the fungal enzyme cleaves the same polypeptidic substrate readily, and the first bond to be hydrolyzed, as automatic sequencing clearly showed, is the bond Leu-Tyr, which is also cleaved preferentially’by most of the serine proteinases from microorganisms (11, 32,33). The other bond readily cleaved, LeuVal, occurs twice in the B chain, in positions

by dansylation;

Peptides I-III,

XI and XII were

Leu-Val and Leu-Val, but only the former 17 18 12 is’iusceptible to splitting. This is in agreement with a similar observation on the degradation of the B chain by pepsin made previously by one of the authors (24) and which can be explained by the differences in the subsites surrounding the susceptible bond (25, 26). As has been shown by Morihara et al. (27) fungal proteinases are in fact considered to have large binding sites capable of interacting with large substrates in a selective way. Obviously, the bond specificity of the collagenase from E. coronata is not highly selective: Some relationships can be established with other proteinases from molds and microorganisms, such as the preferential splitting of the bonds at the large hydrophobic residues. Although the enzyme can be considered as homogeneous, we have shown in our earlier work that the enzyme also has elastolytic and carboxypeptidasic activities, and that these activities can be

COLLAGENOLYTIC

ENZYME

FROM Entornophthora

FIG. 2. Sites of cleavage of B chain of oxidized insulin by by other enzymes. Upper part: the thickness of the rectangles from the cleavages by the proteinase from E. coromta (peptide enzyme, solid arrows denote major cleavages; broken arrows

cororzata

443

the collagenolytic enzyme from E. coronnfa and is proportional to the yield of peptides resulting V = 1%). Lower part: in the case ofE. corounta represent minor cleavages.

attributed to the same enzyme (1). In the ficity from that of the proteinase from E. case of the B chain of insulin, the hydrolysis co?‘onata, and this result cannot be influof the Ser-His bond is analogous to that ob- enced by the fact that the conditions of the digestion were different. However, another served ‘wit: the pancreatic elastase (28). proteinase from A. oryxae, aspergillopeptiThe release of free alanine by the cleavage dase B (32), devoid of collagenolytic activity, ofthe bond Lys-Ala as we11as the cleavage showed a narrow specificity toward the 29 30 bond, when the incubation took of the Arg-Gly is reminiscent of the action Leu-Tyr L3 Ifi 22 2.3 of trypsin, although our enzyme does not place at 0°C for 30 min. Our findings are degrade benzoyl arginine ethyl ester, the similar to the earlier results of Morihara typical synthetic substrate for trypsin (1). and Tsuzuki (11) who reported the broad This release of alanine could also be inter- specificity of aspergillopeptidase B on the preted as carboxypeptidasic activity of the same substrate when digestions were carried out for extended periods of time. The fungal enzyme (1, 29). specificity of another enzyme, with slight Among the extracellular enzymes pro- collagenase-like activity, thermomycolase, duced by fungi, few have been found to have from the fungusMalbra)?chea f.‘r~lclrella var. collagenase-like activity. In Fig. 2, the spec- s!Ll@ea (33) is, in a way, rather similar to ificities of some selected mammalian and that of the collagenase from E. coronata: mold proteinases toward the B chain of in- it is inhibited by DFP, but not by EDTA sulin are compared; only an approximative (34); the first attack occurs at the Leu-Tyr 1J l-6 comparison can be made, as the conditions of digestion were different. The serine pro- bond, under restricted conditions, and some additional cleavages occur after proteinase, aspergillopeptidase C from Asperlonged digestion, namely, at the Phe-Phe and gillus oryxae (30, 31), degrades native col24 2.5 lagen, but it has an entirely different speci- Tyr-Thr bonds. By its action on collagen and 26 27

444

HURION,

FROMENTIN,

its sensitivity to DFP, TLCK, and TPCK, the collagenase from E. coronata may be compared with an extracellular collagenolytic proteinase from the hepatopancreas of fiddler crab, Uca pugilator (35): This enzyme of broad bond specificity is capable of cleaving the helical parts of native collagen; it is insensitive to EDTA, but is inhibited by DFP. It can therefore be concluded that the action of the enzyme from E. coron,ata, as well as from many other eucaryote species, on native collagen, can be considered only as a specific feature of their broad proteolytic activity. The information on the specificity of the enzyme from E. coronata is consistent with the suggestion that collagenolytic enzymes play a specific role in the pathogenicity of fungal strains. In some ocular infections caused by Pseudomonas aerlhginosa the collagen structure is severely damaged by an enzyme with collagenase-like activity (36). Rippon and Peck demonstrated the relation between collagenase production and the pathogenicity of different mutants of an actinomyceteActi)lomadlcra madwae (37). In our recent work (38) we have found remarkable differences in the qualitative and quantitative production of the extracellular enzymes by different strains of E. coronata which seem to indicate the synergistic role of the proteolytic and collagenolytic activities of the enzyme from the pathogenic strain toward connective tissue. ACKNOWLEDGMENTS We wish to thank Mr. A. De Wolf for his skilled assistance in automatic sequencing, Mr. N. T. Tong for performing the numerous amino acid analyses, and Mr. I. Emod for preparing hake parvalbumin. REFERENCES 1. HURION, N., FROMENTIN, H., AND KEIL, B. (1977) Camp. Biochem. P/~ysiol. 56B, 259-264. 2. LECROISEY, A., KEIL-DLOCHA, V., WOODS, D. R., PERRIN, D., AND KEIL, B. (1975) FEBS Lett. 59, 2, 167-172. 3. KEIL, B., GILLES, A. M., LECROISEY, A., HURION, N., AND TONG, N. T. (1975) FE&S’ Lett. 56, 2, 292-296. 4. SVENSSON, B., SIFFERT, O., AND KEIL, B. (1975) E/w. J. Biochem. 60, 423-425. 5. KEIL-DLOUHA, V. (1976)Bioclrim. Biopl?ys. Acta 429, 239-251.

9. 10. 11. 12.

13.

14. 15.

AND KEIL GILLES, A. M., AND KEIL, B. (1976) FEBS Lett. 65, 3, 369-372. KEIL-DLOUHA, V., MISRAHI, R., AND KEIL, B. (1976) J. Mol. B&l. 107, 293-305. KEIL-DLOIJHA, V., AND KEIL, B. (1978) Bioc//irn. Biophys. Acta 522, 218-228. GALLOP, P. M., SEIFTER, S., AND MEILMAX, E. (1957) J. Biol. Chew. 227, 891-906. EISEN, A. %., BAUER, E. A., AND JEFFREY, J. J. (1970) J. Ir~vest. De?.inatol. 55, 359-373. MORIHARA, K., AND TSUZUKI, H. (1969) Arch. Biochem. Biophys. 129, 620-634. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951)J. Biol. Cherry. 193, 265-275. P&H&RE, J. F., DEMAILLE, J., AND CAPONY, J. P. (1971) Biochim Biopllys. Acta 236, 391408. SHAW, E., AND GLOVER, G. (1970)Arch. Biochem. Biophys. 139, 298-305. SCHOELLMANN, G., AND SHAW, E. (1963) Biochemistry

2, 252-255.

16. KEILOVA, H., AND KEIL, B. (1964) Collecf. Czecli. Chem. Cotnm~cn. 29, 2272-2275. 17. DI?VI?NYI, T. (1969) Acta Biochem. Bioplqs. Acad. Sci. H/cng. 4, 297-299. 18. HARTLEY, B. S. (1970) Bioclre)rl. J. 119, 805-822. 19. FRANCK, G., AND STRUBER, W. (1973) Chrontntographia

6, 522-524.

20. EBATA, M., AND TAKAHASHI, K. (1966) Biochi~~/. Biophys. Acta 118, 201-203. 21. JOHANSEN, J. T., AND OTTESEN, M. (1968) C. R. TraL!. Lab. Carlsberg 36, 15, 265-283. 22. SEIFTER, S., AND HARPER, E. (1971) i?l The Enzymes (Boyer, P. D., ed.), Vol. 3, 3rd ed., pp. 649-697, Academic Press, New York. 23. KEIL, B. (1977) in Solid Phase Methods in Protein Sequence Analysis (Previero, A., and ColettiPreviero, M. A. eds.), pp. 287-292, Elsevierl North-Holland Biochemical Press, Amsterdam. 24. KEIL, B., AND KEILOVA, H. (1964) Collect. Czech. Chem. Commun. 29, 2206-2215. 25. SCHECHTER, I., AND BERGER, A. (1967) Biochem. 27, 157-162. Biophys. Res. Cowmun. 26. INOUYE, K., AND FRUTON, J. S. (1967) Biochemistry 6, 1765-1777. 27. MORIHARA, K., OKA, T., AND TSUZUKI, H. (1971) Arch. Biochem. Biophys. 146, 297-305. 28. NAUGHTON, M. A., AND SANGER, F. (1961) Biothem. J. 78, 156-163. 29. PRESCOTT, J. M., AND BOSTON, J. D. (1967)Arch. Biochem. Biophys. 121, 555-562. 30. NORDWIG, A., ANDJAHN, W. F. (1966)Z. Physiol. Chem. 345, 284-287. 31. NORDWIG, A., AND JAHN, W. F. (1968) Eur. J. Bioch,em. 3, 519-529. 32. SPADARI, S., SUBRAMANIAN, A. R., AND KALNITSKY, G. (1974) Biochim. Biophys. Acta 359, 267-272.

COLLAGENOLYTIC

ENZYME

33. STEVENSON, K. J., AND GAUCHER, M. (1975) Biothem. J. 151, 527-542. 34. ONG, P. S., AND GAUCHER, M. (1975) Canad. J. Microbial. 22, 165-176. 35. EISEN, A. Z., HENDERSON, K. O., JEFFREY, J. J., AND BRADSHAW, R. A. (19’73) Biochemistq 12, 1814- 1822.

FROM Entomophthora

coronata

445

36. SCHOELLMANN, G., AND FISHER, E., JR. (1966) Biochirn. Biophys. Acta 122, 557-559. 37. RIPPON, J. W., AND PECK, G. L. (1967) J. ITTvest. Dematol. 49, 371-378. 38. FROMENTIN, H., HURION, N., AND MARIAT, F. (1978)Ann. Microbial. Inst. Pasteur 129A, 425431.