A collagenase from Pseudomonas aeruginosa

PRELIMINARY NOTES

557

611o9 A collagenase from Pseudomonasaeruginosa BBA

In a relatively recent review MANDL1 states that in spite of several claims appearing in the literature "all evidence points to the conclusion that collagenolytic activity (in bacteria) is uniquely associated with Clostridium histolyticum, Clostridium perfringens and possibly Clostridium capitovale." Ocular infections with Pseudomonas aeruginosa frequently have a destructive effect upon corneal tissue. Because of the severe damage to the collagen structure of cornea it was reasonable to assume the presence of an enzyme capable of breaking down undenatured native collagen 2. In extensive work with the clostridial collagenase it has been established that only amino acid sequences of the general structure X - P - R - G l y - P - Y (X and Y, amino or carboxyl protecting groups; P, proline or hydroxyproline; Gly, glycine; R, any amino acid residue) are susceptible to enzyme hydrolysis by cleavage of the R-Gly bond. Data reported below indicate the occurrence of an enzyme in P. aeruginosa capable of specifically hydrolyzing synthetic peptides with structures susceptible to collagenases. Organism and growth condition. P. aeruginosa, strain 7, was used throughout these experiments. Cells were grown at 37 ° in peptone rich (5%) medium prepared according to M A C L E N N A N , MANDL AND HOWESa. After growth for 18 h the cells were removed by centrifugation at 6000 × g for 30 min, and protein was harvested from the supernatant by fractionation with solid ammonium sulfate at concentrations of 3o%, 5o%, and 750/0. Enzyme assays. Non-specific proteolytic activity was determined with diazocasein and azocoll as substrates by standard procedures. The synthetic substrate Cbz-Gly-Pro-Gly-Gly-Pro-Ala (Mann Res. Laboratories, New York, N.Y.) was used following the method of GRASSMANN AND NORDWIG4. A preparation of clostridial collagenase (EC 3.4.4.I9) was obtained form Nutritional Biochemical Corp. Cleveland, Ohio. i O.Sp

06

--~ Protein 2 8 0 m l J . . . . . . Diczocasein 4 2 0 mlJ ~........~: Azocoll 5 4 0 m p

F

g ~ 0.4 < ©.2 v i

i

i

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q

vlr i

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i

~

. . . .

;

o Fraction number

Fig. I. E l u t i o n p a t t e r n f r o m a D E A E - S e p h a d e x A-5 o c o l u m n of a c r u d e p r e p a r a t i o n f r o m P . aeruginosa. 200 m g l y o p h i l i z e d m a t e r i a l e l u t e d w i t h 0.05 M Tri s -H C 1 (pH 7.4), NaC1 g r a d i e n t s t a r t e d a f t e r i o o o m l were collected. C o l u m n size: 1.9 c m × 60 c m ; flow r a t e : I m l / m i n ; f r a c t i o n size: IO ml. A b b r e v i a t i o n : Cbz-, c a r b o b e n z o x y group.

Biochim. Biophys. Acta, 122 (1966) 557-559

55 ~

PRELIMINARY

NOTES

T A 131~E I RELATIVE

COLLAGENOLYTIC

A C T I V I T I t g S OF F R A C T I O N S

ELUTED

FROM

DEt~E-SEPHADEX

COLUMN

AS DETI*;RMINI{DWITH C b z - G l y - P r o G l y - G l y - P r o -Ala

ltmoles/h per mg protein Fractions l and 11 F r a c t i o n 111 Fraction IV F r a c t i o n VI Collagenase f r o m C. hislolylicum

o

I. 5 2. 7 o. 7

7.6

Thin-layer chromatography. Peptide separations were carried out on plates prepared with silica gel G (F. Merck AG, Darmstadt, Germany). Peptides were most easily detected with the chlorine/tolidine test. Column chromatograph,. For further purification chromatography of crude enzyme preparations was performed on columns prepared with DEAE-Sephadex A-5o. Elution was carried out with 0.05 M Tris-HC1 (pH 7.4) as buffer and a concentration gradient established with NaC1 ranging from o to 2.0 M. Effluents from the columns were tested ff~r ultraviolet absorption at 280 m# and for enzymic activities against diazocasein and azocoll. After exhaustive dialysis and lyophilization of appropriately pooled fractions enzymic activity against synthetic substrates could be measured. Fig. I sho\vs a typical separation on a DEAE-Sephadex column using as starting material a preparation which was obtained from cell-free cultures by precipitation with solid ammonium sulfate (3o% saturation). A brown pigment which all preparations contain is retained on top of the column. All caseinolytic and azocoll activity appeared in the first peak. For the determination of collagenase-like activity the elution curve was divided into seven Fractions I V I I as indicated in Fig. I. Fractions I I I , IV, and VI showed definite increase in ninhydrin color after incubating flw (~ h with hexapeptide Cbz-Gly-Pro G l y - G l y - t ' r o Ala (Table I). Preliminary results indicate that Ca "t do not promote this activity. A collagenase

O

f, :

O <

Q

[)

Q Z

e

O

o

•

Q I

K

Fig. 2. Thin-layer c h r o m a t o g r a p h y of peptides f r o m hydrolysis with collagenases. Solvent nb u t a n o l - a c e t i c acid- 5 % a m m o n i a (55:3 o : I 5 , v/v/v). A, s t a n d a r d solution C b z - G l y - P r o - G l y G l y - P r o - A l a (I); B, (I) plus collagenase f r o m C , histolyticum; C, (I) plus F r a c t i o n IV from P. aeruginosa; I), s t a n d a r d solutions Cbz-Gly-Pro Gly (II) and G l y - P r o - A l a (III).

Biochim. Biophvs. Acta, 122 (1966) 557 559

559

PRELIMINARY NOTES

should split this hexapeptide at the Gly-Gly bond forming Cbz-Gly-Pro-Gly and the tripeptide Gly-Pro-Ala. Aliquots from incubation mixtures were applied to thinlayer plates prepared with silica gel G together with the proper controls. The formation of the expected peptides could be shown without any doubt, thus providing strong evidence for the presence of a collagenase-like enzyme in these preparations (Fig. 2). As was shown by parallel experiments the collagenase form C. histolyticum also cleaves the hexapeptide at the identical bond. Fzactions containing collagenolyric activity did not show any activity when measured with diazocasein or azocoll; moreover these fractions exhibited no esterolytic activity with trypsin- or chymotrypsin-specific substrates. It is, however, clear from the distribution of the collagenolytic activity and the azocoll activity that the collagenase under investigation is heterogeneous in nature. A heterogeneity of clostridial collagenase has been noted by GRANTAND ALBURN5 and confirmed in several recent papers 6-9. It is believed that the situation in P. aeruginosa is similar and that the multiplicity we find, either reflects a dissociation into subunits with some limited changes in specificity and/or activity or constitutes different collagenases. Further work is needed to clarify this point. This investigation was supported by a research grant from the U.S. Public Health Service (NB 03215-05). We thank Mr. R. BOZEMAN for his able technical assistance.

Department of Ophthalmology, Tulane University, School of Medicine, New Orleans, La. (U.S.A.) I z 3 4 5 6

GUENTHER SCHOELLMANN EARL F I S H E R , JR.

I. MANDL, Advan. Enzymol., 23 (1961) 163. E. FISHER AND J. I-I. ALLEN, Am. J. Ophthal., 46 (1958) 249. J. D. MACLENNAN, I. MANDL, AND E. L. HOWLS, J. Clin. Invest., 32 (1953) 1317. W. GRASSMANN AND A. NORDWlG, Z. Physiol. Chemie, 322 (196o) 267. 1N. H. GRANT AND H. E. ALBURN, Arch. Biochem. Biophys., 82 (1959) 245E. HARPER, G. SEIFTER AND V. D. HOSPELHORN, Bioehem. Biophys. Res. Commun., 18 (19(75)

627 . 7 I. MANDL, S. KELLER AND J. MANAHAN, Biochemistry, 3 (1964) 17378 M. C. SCHAUB AND L. STRAUCH, Biochem. Biophys. Res. Commun., 2I (1965) 34. 9 I-I. NODA, E. YOSHIDA AND S. YAGISAWA, Collagen Currents, 4 (1963) 365.

Received May 2oth, 1966 Biochim. Biophys. Acta, 122 (1966) 557-559

BBA 6IIO 7

The substitution of placental 'mitochondrial' extracts for adrenal 'mitochondrial' factors in steroid l|[B-hydroxylation BovineZ, 2 and rat 3 adrenal mitochondria contain steroid ilfl-hydroxylase (EC 1.99.1.7) which can be extracted from dried powders of the mitochondria z,4-6. Enzyme purification and carbon monoxide inhibition studies of the reaction indicate Biochim. Biophys. Acta, 122 (1966) 559-563