some properties of neutral proteinases from

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trypsin- and irypsin-like activities were not delected: azocasein-degrading aclivily ... Elastase activity was also stimulated by high ionic strength buffers and KCI. but not as ... Previous reports had indicated that rabbit leucocytes contain esterase activity ... ester (Starkey, 1977) but Dewald et al (1975) found no correlation.
AJEBAK 59 (Pt. 1) 63-75 (1981)

©SOME PROPERTIES OF NEUTRAL PROTEINASES FROM LYSOSOMES OF RABBIT POLYMORPHONUCLEAR LEUCOCYTES by M. L. BRITZ* AND D . A. LOWTHER (From the Department of Biochcmislry, Monash University, Clayton, Vic, 3168, Australia.) (Accepted for puhlicaiion November 3, 1980.) Summary. Neutriil prolsinases capLibIc of degrading proteoglyciin were found in lysosomes of rabbit polymorpbonuclear leucocytes exlriictecl wilb 0 0 1 M citric acid. Eslerase uclivily againsi an elast;ise substrate was also present bui chymotrypsin- and irypsin-like activities were not delected: azocasein-degrading aclivily was poor. Proteoglycanase activity was stimulated by high concentrations of salts (0.2 M KCI) and divalent cations (Ca, Mg. Mn, Zn) but was inhibited by Cu + +. Elastase activity was also stimulated by high ionic strength buffers and KCI. but not as much by divalent cations, and was inhibited by Cu+-i-. Proleoglycanase in crude extracts w.is inhibited by EDTA. pbenylmetbanesiilpbonyillnoride (Pms-F). cell cytosol. "i-antitrypsin. gold tbiomalate and N-acetyl-di-l.-alanyl-Lprolyl-L'Valine chloromcthyl ketone (AAAPVCK). Partial inhibition by N-a-p-tosylL-Iysine cblorometbyl ketone (TLCK) and L-l-tosylamide-2-phenylethyl chloromethyl ketone (TPCK) occurred. Elastase adsorbed to CM-cellulose and was eluted by 06-0-7 .M NaCi: a metallo-proteinase failed to adsorb completely but was retarded by tbe CM-cellulose. (soelectric focusing showed that the major proteinases had pi's of 5-5, 8-5 and y I; the activity witb pi 8 5 was a melalloprotemase. and the pi 9-1 activity was an elastase. The apparent molecular weight of the elastase, determined on Sephadex CM00. was 8.000 to I ].0(K) daltons.

INTRODUCTION There have been relatively few reports describing the properties of neutral proteinases of rabbit polymorphonuclear leucocytes (PMNL) (review by Starkey, 1977). One of the first was that of Davies, Krakauer and Weissmann (1970), who showed that neutral proteinase activity was localized exclusively in the azurophil granules. A neutral histonase occurring as the major proteinase here was partially purified by Davies et al. (1971) by Sephadex G-75 chromatography and isoelectric focusing fpl 4 2 - 5 - 2 ) . Histonase activity in crude extracts was partially inhibited by the chymotrypsin inhibitor L-l-tosylamide-2-phenylethyl chloromethyl ketone and by soy bean trypsin inhibitor. Dewald et al. (1975) confirmed that casein- and histone-degrading proteina.ses occurred in the azurophil granules, and further • Present address: School of Microbiology, University of Melbourne, Parkville, Vic. 3052, Australia.

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M. L. BRITZ AND D . A. LOWTHER

demonstrated esterase activity against the elastase substrate, t-butyloxycarbonyl alanine 4-nitrophenyl ester. The elastase activity followed the same distribution as the casein- and histone-degrading proteinases within the cell. Previous reports had indicated that rabbit leucocytes contain esterase activity against the chymotrypsin substrate N-acetyl-DL-phenylalanine 2-naphthyl ester (Starkey, 1977) but Dewald et al (1975) found no correlation between the distribution of this esterase activity and the proteolytic activity, thus failing to demonstrate proteinase activity corresponding to eathepsin G of human leucocytes. Since these reports, there have been no further publications that have led to an understanding of the types of proteinases in rabbit PMNL, so that available data reveal no clear relationship of the proteinases described above to the elastase and eathepsin G of human leucocytes. Defining such a relationship is of some interest, considering the broad use of rabbits in experimental models of inflammation and pathological processes involving ti.ssue destruction, where PMNL proteinases potentially have an important role. This communication attempts, in part, to clarify some of the properties of rabbit PMNL elastase, describing the partial purification of this enzyme and a lysosomal neutral metallo-proteinase. The properties of proteinases from lysosomes of rabbit PMNL are compared with those of the human, in relation to previously published data on the rabbit enzymes.

MATERIALS AND METHODS Preparation of lysosomal proleinases from rabbit polymorphonuciear leucocytes Peritoneal-extidate PMNL were induced according to the method of Cohn and Hirsch (1960). Adult rabbits were injected intraperitoneally wilh 150 ml of 0 1 % ( w / v ) glycogen (Type IL oyster. Sigma) in 0 1 5 M NaCl. Afler 18 h. the peritoneal cavity was washed out wilh 100 ml of 0 1 5 M NaCl and the exudiile collected into flasks on ice. The suspension was filtered ihrough gauze and (he cells ( > 9 0 % of which were PMNL) ccnlrifiiged (500 X^', 10 min., 4 ° ) . washed once in 0 3 4 M sucrose, then rcsuspendeil in 0-34 M sucrose, concentrating the cells 20-fold in comparison with the original exudate. Cells were lysed by mixing on a vortex (Baggiolini, Hirsch and deDuve. 1969) and cell debris removed by low speed centrifugation (500 X,;'. 10 min.. 4 ° ) . Lysosomes were sedimented by high speed centrifugalion (8.0(10 X,;'. 20 min.. 4 ° ) , the post-granule supernatant fluid removed and the lysosomes washed once with 0 34 M sucrose. Lysosomes were resuspended finally in 0 01 M citric acid (typically, lysosomes from 4(H) m! of exudate were resuspended in 5 to 10 ml citric acid) and gently stirred at 4° for 18 to 24 h (Higuchi, Honda and Hayashi. 1975). Lysosomal debris was removed by centrifugation (11.000 Xi'. 20 min.. 4 " ) ; the supernatant fluid was collected and stored at 4° as such, or dialysed (cellulose tubing, 6000-8000 molecular weight cul-ofT) 18 h at 4" against 100 volumes of 0 05 M sodium phosphate buffer. pH 7-2. before storage at 4°. Detection of proteo^lycan-dei-rtidina activity {"*''S]-labelled rabbit articular cartilage was the substrate, which WLIS prepared by injecting 6-week-old rabbits intravenously with 1 to 2 mCi of {-'^'SJ-sodium sulphate (Amershamt 20 h before death. Shavings of articular cartilage were removed from ail joints and washed extensively with 0-15 M NaCI, followed by heating at 60° for 30 min. Heating lessened autolysis during storage at 20° in 0 15 M NaCl and decreased the 'bleed" of -'"'S from the substrate during long-Ierm incubations (Hauser and Vaes, 1978). Assays contained 5 to 10 mg of L'artilage, 0 1 M potassium phosphate buffer, pH 7 4, 0 25 M KCl and enzyme in a

RABBIT LYSOSOMAL PROTETNASES

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tot:il volume of I ml. When incubation periods exceeded 6 h penicillin and streptomycin (Glaxo) were iiuJcd ai lOO fg/ml to minimize microbial attack of the substrate; this level of antihiotics did not alter PMNL proteinase acliviiy. Incubation mixtures were placed at 37° for 10 min and reactions started hy ihe addition of enzyme; 0 1 ml samples were removed at I, 2, 4, 6 and 18 to 24 h for radioactivity measurement. At the end of the incuhation period Ihe cartilage was washed extensively with 0 1 5 M NaCl. then 1 4 ml of O-S M ncetiile buffer. pH 5 7. containing 25 mM EDTA added together with 10 mg cystcine-hydrochloride and 0 1 ml pap;iin (5 mg/ml in 0 2 M NaCI). The cartilage was digested at 60" for 16 h. and a sample removed for radioactivity measurement. Activity was calculated as the rate of release of !''"'S;-fragments from the cartilage, and expressed as a percentage of the total amount of ='-^S initially present in the cartilage: one unit released \% of the total radioactivity per hour. All activities were corrected for the release of -'''S occurring in the absence of enzyme. Other buffers used in assaying protcoglycanase activity are detailed in 'Results'. Hydroh.si\ of .yvrUlwlic suhstratv.s Llastiise-like activity (subsequently termed clastase) was determined by the rate of liberation of p-nitrophenol from t-hutyIoxycarbonyl-L-al;inine 4-nitrophenyl ester (BOC-AIaONp) (Sigma). Assay mixtures contained 0 1 5 M sodium or potassium phosphate buffer. pH 7 4. 0-5 M KCl and enzyme in a finul volume of I ml. Reaction was started by the addition of 20 f-l of substrate (10 mg/ml in dimethyl sulphoxide). and increase in iibsorbance at 347.5 nm followed for 3 to 5 min at IT. The amount nf /j-nitrophenol released was determined from a standard curve of p-nilropheno! (Sigma) in the assay buffer, and activity expressed as wmoles p-nitrophenol releasecl/min/m! of enzyme (units/ml). All rates of reaction were corrected for the spontaneous release of /i-nitrophenol in assay huffer lacking enzyme; other buffers used in this assay are detailed in 'Results'. Assays of other estenise activities h:ive heen described in detail previously. Assay using N-acetyl-tri-L-alanine methyl ester was performed according to Janoff and Basch (1971). Hydrolysis of N-benzoyl-L-tyrosine ethyl ester and N-benzoy!-L-arginine ethyl ester were assayed as described hy Gerber. Carson sind Hadorn (1974). Hydrolysis of N-benzoyl-DLarginine 4-nitroanilide and N-acetyl-L-phenylalanine 2-naphthyl ester were assayed as described by Starkey and Barrett (1976). f'ffeci.y of inliUniors af-tiinsl proteoi-lycanase and elasta.se Appropriate volumes of test agents were added to incubation mixtures of the proteoglycanase and elastase :iss;iys and these incubated at 37° for I h before Ihe addition of substrate. EDTA (disotlium salt) was used al 5 or 10 mM and 1,10-phenanthroline at 1 mM. Pms-F was prepared as a stock solution of 0-1 M in propanol and used at I mM. TLCK (Sigma) was used at 0-2 mM and L-I-tosylamide-2-phenylethyl chloromethyl ketone (TPCK) (Sigma) was prepared as a stock solution of 20 mM in dimethyl sulphoxide and also used at 0-2 mM. AAAPVCK (obtained from A. Sriratana. Dcp:irtment of Biochemistry. Monash University. Melbourne) was dissolved at 0 Olf^f (w/v) in methanol and diluted with 0 1 M sodium phosphate buffer. pH 7 4. to 1 mM then used at (M mM. CiolJ thiomalate (May and Baker) was used at K)-- to KH' M. Cell cylosoi. from the post-granule supernatant fluid of rabbii PMNL. was used :it 100 /'g protein and 7 5 ng of purified at-antitrypsin protein (obt;iincd from M. Nagiishima. Dep:irtment of Biochemistry. Melbourne University) was used. lon-i.wihani'c ciiroiuato^raphy on CM-ci'lluIose The CM-celliilose column (]-5 x 30 cm) was equilibrated in 0 05 M potassium phosphate. 0 2 M NaCi huffer. pH 7 4. Crude citric acid extracts were either dialyscd against this buffer or used direclly. m;tking the volume up to 25 ml with column buffer before application of sample. The column was washed extensively following application of sample and then a linear gradient of 0 2-1.0 M NaCI in 0 0 5 M potassium phosphate buffer. pH 7.4. applied; the flow rate throughout was 60 ml/b and the procedure was performed at room temperature. The concentration of NaCI in fructions was determined from conductivity measurements.

66

M. L. BRITZ AND D. A. LOWTHER

Isoelectric focu.sin^ This was performed using an LKB 21 17 Multiphor system and was carried out according to the instructions supplied by the mantifacturer. Ampholines (pH 3 5-10, LKB) were used as a S% solution in 100 ml water containing 5% ( w / v ) Sephadex G-75 (Pharmacia). An area of the dry gel was removed from the middle of the bed and mixed with the sample (3 ml containing 5% (v/v) ampholines) and the mixture reapplied to the gel. Electrofocusing was performed at 400 mV, 20 va.\ initially and cooled by a water flow at 4°. The gel was fractionated after 16 !o 20 h. then eluted with 3ml of 2 M NaCI. 0 - 1 % ( w / v ) Brij 35 (Sigma) or 0 1 M potassium phosphate bufTer, pH 7-4. Gel filtration on Sephadex G-lOO A column of Sephadex G-lOO (Pharmacia) ( 2 . 2 x 90 cm) was equilibrated at 4=' in 0.05 M sodium phosphate bufTer, 0 1 5 M KCl. 0 - 1 % Brij 35, pH 7 2 , and flowed at 14 ml/h. The column was calibrated with markers of known molecular weight, blue dextran (Pharmacia), bovine serum albumin (67.000), ovalbumin (43,000), soy bean trypsin inhibitor (21,000) (all from Sigma) and Naiv'^'SO.,. Other analy.ses Radioactivity was measured using a Phillips liquid scintillation counter with external standardization for quench correction. Aqueous samples were made up to 2 ml with water and 8 ml of scintillation fluid ( 0 . 8 % , w/v, 2.5-diphenyloxazole dissolved in 5 0 % . v/v, Triton X-114 in toluene) added. Protein was determined by the method of Lowry et al. (1951), wish bovine serum albumin as the standard.

All chemicals were analytical grade reagents. Trypsin (type III from bovine pancreas) and elasiase were supplied by Sigma. Papain was prepared by J. Sandy (Department of Biochemistry, Monash University).

RESULTS Stability of proteolytic enzymes Citric acid extracts of lysosomes fpH 2 5) could be stored at 4° for up to 28 days without significant lo.ss in total proteoglycanase activity. However, holding these extracts at —20° caused a 50% loss of proteoglycanase activity in 15 days and only 20% of the original activity remained after 28 days at —20\ When the extracts were dialysed against phosphate buffer (0 1 M, pH 7.4), proteoglycanase activity was stable for up to 28 days at 4" or —20°. Protein levels in the extracts tested were 0 3 to 3 mg/ml. Enzymes eluted from CM-cellulose and G-lOO were unstable to storage at 4°, even when 0 1% Brij 35 was included; protein levels in these preparations were less than 50 Hydrolysis of synthetic substrates In an attempt to define the substrate specificity of the proteinases in the crude lysosomal extract, artificial substrates of known specificity were examined for their possible hydrolysis. Neither N-benzoyl-DL-arginine 4-nitroanilide or N-bcnzoyl-L-arginine ethyl ester were hydrolysed, although satisfactory hydrolysis of these substrates by trypsin was observed. Tbis is consistent with the report by Davies et al. (1971), wbo also failed to deteet trypsin-like activity in rabbit PMNL lysosomes. N-Benzoyl-L-tyrosine ethyl

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ester was not hydrolysed and N-acetyl-L-phenylalaninc 2-naphthyI ester was hydrolysed slowly, even when large volumes of extract were used (up to 1 mg total protein), implying that only low levels of chymotrypsin-like esterase aetivity were present in these extraets. Although the elastase substrate N-acetyl-tri-L-alanine methyl ester was readily hydrolysed by commereially available elastase, the lysosomal extracts showed poor aetivity against this substrate. In contrast, elastase-like activity against BOC-Aia-ONp wa.s readily detected, having an average specific activity of 0 0 7 9 U/mg protein. Dewald ei al (1975) reported that casein-degrading activity was present in rabbit PMNL lysosomes. but the specific activity was low in comparison with aetivity in human PMNL. Poor azocaseinase activity was found here in citric acid extracted lysosomes; azocasein was not used routinely as the assay was considered wasteful of enzyme. Effects of salts and ionic strent^lh of buffers on neutral proteinase and elastase activities Proteoglycan degrading activity in crude lysosomal extracts was stimulated by 0-2 M NaCI or KCl (Table I ) : increasing the concentration of KCl to 0 5 or 1 0 M further stimulated the aetivity (200 to 250% of the control in 0 05 M phosphate) but also caused increased spontaneous release of radioactivity from the I''''SI-cartilage substrate. Divalent cations, with the exception of copper, stimulated proteoglycanase activity. Magnesium chloride was the most effective salt tested: even when the concentration of MgCl:TABLE i Effects of salts on proteo!"!ycana.se activity from rabbit PMNL ly.w.iomes. BufTers and concentrations 0.05 M phosphate 0 05 M Tris

0 05 M Tris, 0 2M KCl

Salti. KCl (0-2 Ml NaCI {"0-2 M) KCl (0 2 M) MgCl.. MgSO4 MnCIo ZnSO4 CiiCU CuCl-. CuSO4 MgCI-. MnCI'. ZnSO, CuCI..

% conlrol activity'^ 100 179 181 287 316 260 196 154 290

9 30 126 110 146

25 5

* Activity relative lo that ft)und when the listed buffer was used alone. t All sails were used at 50 mM final concentration, except CaCli.. which was at 10 mM. i-''''S)-labelled cartilage was incubated at 37° in bufTers at pH 7-2, with 30 lo 60 ^'g of protein from cilric acid extracts of lysosomes. Aliquots were removed after 2. 4 and 24 h. The rate of |''"'SJ-peptide release was determined relative to the total counts originally present and figures e.xpressed as ii percentage of the appropriate control activity. Each figure is the average obtained for four dilTercnt enzyme preparations, each tested in duplicate.

68

M. L. BRTTZ AND D . A. LOWTHER TABLE 2 Effects of salts and ionic strenf^th of phosphate btiffers on ela.stase from rabhit PMNL Ivso.somes. Buffers ;ind concentrations 0-05 M phosphate 0-1 M phosphate 0' 15 M phosphate 0-5 M phosphate 0.05 M phosphate 0 • 1 M phosphate 0 05 M Tris

0 05 MTris. 1 5 M KCl

Salt

— — — KCl 11 5 M) KCl ( 1 5 M)

% control activity 100* 189 279 417

150

loot

KCl ( 1 . 5 M )

198

MgSOit MnCl.. ZnS04 CaCI.. CUSO4

122 142 85

CuSO-i

75

110 15

* Activity relative to that found in 0-05 M phosphate buffer. t Activity relative to that found in 0.1 M phosphate bufFer. t 50 mM final concentration was used for all salts. All assays were performed at pH 7-2 and 22°. and contained 30 lo 50 extracts of lysosomes.

of citric acid

was decreased from 50 mM to 0 5 mM, the proteoglycanase was stimulated to 2509c of the control activity. Copper ions dramatically inhibited proteoglycanase activity: the minimum concentration required for 50% inhibition was 1 to 5mM CuCl-. The stimulatory effects of the divalent cations and inhibition by CuCl- were abolished, in part, by 0-2 M KCl. Elastase activity was also stimulated by high concentrations of KCl {Table 2), but this effect was only significant when tested against a background of low ionic strength phosphate buffers or in Tris buffer: changing the concentration of KCl from 0 1 to 1 5 M did not alter the elastase activity in 0 1 M phosphate buffer. Increasing the concentration of Tris buffer from 0 05 to 0 I M stimulated elastase activity by 3O9r ; further increases in concentration (up to 0 3 M) failed to enhance this stimulation. In contrast, increasing the concentration of phosphate buffer from 0 05 to 0 5 M markedly stimulated elastase activity (Table 2). High concentrations of phosphate buffer were not used routinely for assaying elastase, as the rate of spontaneous hydrolysis of BOC-Ala-ONp was also elevated in these buffers. Divalent cations did not stimulate elastase to the same extent as seen for proteoglycanase. but copper ions did significantly inhibit elastase. Again, the inhibition by copper was reversed in the presence of KCl. Effects of selected inhibitors on neutral proteltmse activity Proteoglycanase activity in crude citric acid extracts of lysosomes was inhibited 40 to 60% by 10 mM EDTA or I mM Pms-F, indicating the presence of metal-dependent and serine proteinase activities. Proteolytic activity was almost totally inhibited by 0 1 niM AAAPVCK, inhibited by

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30-40% by 0 2 mM TPCK, and by 20% by 0 2 mM TLCK. The last two compounds did not inhibit elastase activity when this was assayed using BOC-AIa-ONp, whereas AAAPVCK completely abolished elastase activity. Both elastase and proteoglycanase activities were completely inhibited by the cell cytosol (100 /*g of post-granule supernatant protein used) and «iantitrypsin (7 5 yg u.sed). Inhibition of rabbit PMNL histonase by the cell cytosol was previously reported by Davies et al (1971). Gold thiomalate (1 mM) inhibited elastase by 80%, and proteoglycanase by 30 to 50%; assays here lacked KCl. lon-e.xehange chromatography on CM-eellulose Fig. I shows a typical elution profile for a lysosomal extract applied to CM-celluIose. This particular extract contained 2-83 mg protein, 0 22 units of elastase and 220 units of proteoglycanase; 60% of the original elastase 4a

4b o

10

2 Q

n

/ -

0 a

CO

0.6

m

u 2

C

-^

o m 2 ^

3

ca

O

0 4

o O

o

2

0,2

2

CQ

10

20

30

40

50

FRACTION NUMBER Fig. 1. Ion-exchange chromatography of a citric acid extract of rabbit PMNL lysosomes. The column was equilibrated in 0-05 M potassium phosphate, 0 2 M NaCl buffer. pH 7-4. A 6 ml extract of PMNL lyso^iomes was diluted lo 25 ml in the same buffer and applied lo the column and 6 ml fractions collected first as the sample was applied. The column was washed until the absorbance at 280 nm was less than 0 02. Elution was performed with a 150 ml linear gradient of 0-2-i-O M NaCl in 0.05 M potassium phosphate buffer. Fractions were tested for absorbance at 2K0 nm ( ). proteoglycanase (PGase) activity (• •), elastase activity ( • • ) and conductivity ( ). Designated fractions were pooled.

M. L. BRITZ .^ND D. A. LOWTHER

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and greater than 100% of the proteoglycanase activities applied were eluted. The apparent increase in total proteoglycanase can be accounted for by the elevated levels of NaCI in assays of fractions eluted by the NaCI gradient, where 0 5 ml of the fractions was required to detect activity. The specific activity of elastase was increased 50-fold by CM-cellulose chromatography. Four peaks of proteoglycanase were usually recovered from CM-cellulose: peak I. comprising here 36% of the elastase recovered, eluted immediately after the extract was applied; peak 2 eluted during the initial stages of washing, whereas peak 3 eluted much later in the wash, after most of the protein had already eluted. The late elution of peak 3 suggested that this activity was partially retarded by the CM-cellulose. However, when the gel was equilibrated and washed in 0 05 M phosphate buffer lacking 0 2 M NaCl. activity corresponding to peak 3 still failed to bind and continued to be eiuted late in the wash. Elastase bound to the CMcellulose and was eluted by 0 6 to 0 7 M NaCl (peak 4). The proteoglycan-degrading activity associated with this activity normally appeared as a broad peak centred about the elastase activity: such apparent attenuation of activity reflects some of the peculiarities of the assay method. Inhibitor profiles were performed in an attempt to classify the proteinases separated on CM-cellulose (Table 3). A worrying point was that the elastase inhibitor (AAAPVCK) appeared to significantly inhibit all of these activities. However. AAAPVCK failed to inhibit neutral metalloproteinascs from cultured rabbit cartilage (Britz and Lowther, 1980), implying that this agent was acting selectively and that the dual inhibition of peak 3 activity by EDTA and AAAPVCK was a characteristic of this enzyme. Elastase/proteoglycanase activity (peak 4, a and b) was only sensitive to inhibition by AAAPVCK. CM-cellulose chromatography could therefore separate the two major proteinases in the lysosomal extracts, viz. the metallo-proteinase (peak 3) and the serine-dependent elastase (peak 4). Activity eluted during application of the extract and initial washing (peaks 1 and 2) is made up of any proteinases that failed to adsorb to the gel. Althoush the inhibitor profiles show that these activities are largely metalloand serine-proteinases. it is impossible to say how many enzymes and what TABLE 3 Sensitivity of protvo\:lycann!:e from CM-cellulose to proteinase inhibitors. % inhibition

Peak number TPCK

TLCK

AAPVCK

EDTA

4b

10

60 0 0 0 0

98 34 100 100 95

69

4a

49 26 28 0

1

2 3

96 100 7 0

TPCK and TLCK were used ill a final concenlration of 0 2 mM, AAAPVCK at 0 1 mM and EDTA ;il 5 mM. Figures are expressed as percentiige inhibition relative to activity in the absence of inhibitor and in the presence of the solvent used for the inhibitor.

RABBIT LYSOSOMAL PROTEINASES

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types are present here. There is no evidence at this stage to indicate if elastase activities in peaks 1 and 4 are different, or if peak 1 corresponds to unbound peak 4 elastase activity. Peak I was usually 25-30% of the total elastase eluted. Isoelectric focusing (lEF) of lysosomal proteinases After several attempts at isoelectric focusing of lysosomal extracts, it became apparent that the same enzymes were not present in the same proportions when difl'erent preparations were used. It also became obvious that, if low ionic strength buffers were used to elute the proteinases following focusing, then the elastase activity was not detected. Recovery of proteinase activity was poor, being 20 to 50% of that applied, and the highest yield of elastase activity was 10%. It is therefore dillicult to say if the profile shown in Fig. 2 represents all of the proteinases originally present, and if the relative proportions reflect the original preparation. This profile, however, shows the enzymes typically recovered, in the proportions normally seen. The crude extract applied here contained 0 22 units of elastase, 190.H units of proteoglycanase and 2 25 mg of protein; 10%' of the elastase and 50% of the proteoglycanase were recovered. Three major peaks of protcoglycan-dcgrad-

o o o

TO

o —1

X

"i

m

O O

1-5

I—

O

1.0

0-5

pH F i g . 2 . Isoelectric f o c u s i n g of r a h b i t P M N L lysosomal proteinsises in a 3 - 5 to 10 p H g r a d i e n t . T h e crude^ e n z y m e p r e p a r a t i o n in I ) ( H M citric acid w;is d i a l y s e d a g a i n s t lOO v o l u m e s of 0 - 0 5 M s o d i u m p h o s p h a t e . O l ' ^ r Brij 35 bufTer. p H 7 2 ;ind 2 X 5 ml upplicd t o ihe S e p h a d e x G - 7 5 - a m p h o l i n e gel. A v o l t a g e of 4()t) m V w a s a p p l i e d , t h e c u r r e n t c h a n g i n g f r o m 2 0 to 2 m A o v e r 18 h. T h u gel w a s divided into 30 f r a c t i o n s w h i c h w e r e e l u t e d with 2 x 1 5 ml of 2 M N a C l . 0 ' \'','r Brij 3 5 . Aficr r e c o r d i n g the p H of e a c h f r a c t i o n , these w e r e diiilysed a g a i n s t 2 I of 0 - 0 5 s o d i u m p h o s p h a t e . 0 I ' c Brij 35 buffer. p H 7 - 2 . f o r 8 h at 4 " a n d tested for p r o t e o g l y c a n a s e ( • • ) and e l a s l a s e ( • • ) activity.

72

M. L. BRITZ AND D. A. LOWTHER

30

40

50

FRACTION

60

70

80

NUMBER

Fig. 3. Gel chromatography on Sephadex G-100. Five ml of a crude enzyme preparation was dialyzed against 100 volumes of 0-05 M sodium phosphate, 0-15 M KCl. 0 - 1 % Brij 35 buffer, pH 7-2. then applied to a column of Sephade.x G-IOO equilibrated at 4° in the same buffer. Fractions (3-6 rnl) were collected and assayed for proteoglycanase (• • ) and elastase (• • ) activities, and absorbance at 280 nm recorded ( ). The column was calibrated using blue dextran (V,,). bovine serum albumin (BSA), ovalbumin (Ov), soy bean trypsin inhibitor (SBTI) and Naj-'^'SO., ( V T ) .

ing activity were seen (pH 5 5. 8 5 and 9 1 ) plus several minor peaks (pH 4 2. 6 2. 7-2 and 7-7). The proteinases focusing at pH 5 5 and 8 5 could be readily eluted from the gel by low ionic strength buffers and were frequently recovered as the major proteinases. The proteinase focusing at pH 5-5 was partially inhibited by TPCK, AAAPVCK and TLCK. but not by EDTA: it is apparently a serine-proteinase but is sensitive to a range of differential inhibitors. One would not expect this proteinase to bind to CMcellulose. The proteinase focusing at pH 8 5 was completely inhibited by 10 mM EDTA. I mM 1. 10-phenanthroline and AAAPVCK. and partially inhibited by TLCK. This activity probably corresponds to peak 3 from CMcellulosc. Elastase focused in a single peak coincident with proteoglycanase activity at pH 9.1. No other metallo-proteinases were recovered from the gel and the minor peaks were all partially inhibited by the serine-proteinase inhibitors. All of the proteinases above also degraded histone (Sriratana, personal communication). Gel chromatography on Sephadex G-100 Gel chromatography was performed on part of the same preparation used above for electrofocusing: 0-27 units of elastase. 231 units of proteoglycanase and 2-74 mg of protein were applied. Fig. 3 shows the elution

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profile, where 20% of the elastase and approximately 30% of the proteoglycanase were recovered. Total activity recovered from this column was normally low, with elastase as the major activity eluted; recovery was enhanced by 0-1% Brij 35. Although the protein associated with the elastase was low ( < 2 0 iig/m\), the loss in total activity meant that the specific activity did not increase. The two minor peak.s of proteoglycan-dcgrading activity had molecular weights of approximately 80.000 and 35.000; activity was low and did not survive storage at 4° for 48 h, so that inhibitor profiles were not determined. The molecular weight of the elastase was 8.000 to 10,000. The chromatography was repeated in 1 5 M NaCl in 0 05 M sodium phosphate, 01%i Brij 35 buffer, pH 7 2. in an attempt to eliminate possible non-specific adsorption to the gel: molecular weight of the elastase here was about 11,000. DISCUSSION The major neutral proteinases of rabbit PMNL lyso.somes are two serine proteinases and one metallo-proteinase. all of which can degrade rabbit cartilage proteoglycan and histone. These activities could be separated by isoelectric focusing (IEF) and CM-cellulose chromatography and differentiated on the basis of sensitivity to inhibitors. The proteinase of pi 8 5 was a metallo-proteinase which failed to adsorb completely to CM-cellulose. but was retarded by this to elute after the majority of protein had washed through. Elastase was also a cationic protein of pi 9 1, and could be partially purified by CM-cellulose and Sephadex G-100 chromatography. The molecular weight determined by gel chromatography was 8.000 to 11,000 daltons. However, it must be pointed out that this figure is only an indication of the true molecular weight of the enzyme, as elastases have been shown to be anomalously retarded on Sephadex gels even in the presence of high salt concentrations (Starkey and Barrett, 1976). The rabbit elastase indeed proved to be 'sticky', requiring high salt concentrations to elute it following IEF. Even so, the molecular weight of the rabbit PMNL elastase is substantially lower than previously reported figures for human neutrophil elastases, which range from 22,000 to 37,000 daltons (Starkey, 1977). The protein levels associated with the elastase following partial purification were too low to allow alternative determinations of size by methods such as amino acid analysis. The third major proteinase was a serine-proteinase with a pi about 5 5. Several minor activities were also present, none of which hydrolysed synthetic substrates for trypsin or chymotrypsin and were not characterized beyond IEF because of low activity. It is interesting to compare the spectrum of proteinases reported here with those .seen by Davies et al (1971) and Dewald et al (1975). The latter reported that rabbit PMNL azurophil granules contained, on the basis of electrophoretic patterns of Triton X-lOO extracts, one major elastase plus 3 or 4 very minor activities, but no chymotrypsin-like proteinase activity. We also found only one major elastase and only low chymotrypsin-like

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M. L. BRITZ AND D . A. LOWTHHR

esterase activity in citric acid extracts of lysosomes. The major neutral histonase seen by Davies et al (1971) had a pi of 4 2 - 5 2 and, if one assumes that the major enzyme eluting from Sephadex G-75 was the same activity, a molecular weight of 30.000 to 50,000 (calculated from the data presented). Starkey (1977) suggested that the pi estimated by Davies et al (1971) was aberrantly low because it was determined in the presence of heparin. However, any ionic complex between the histonase and heparin would probably dissociate during IEF, so that the pi 4 2-5 2 histonase of Davies et al. (1971) may correspond to the pT 5 5 activity reported in the present communication. This is contrary to the further prediction of Starkey (1977) that the pi 4 2-5 2 histonase activity was the rabbit PMNL elastase. Davies et al. (1971) did not look at the inhibitor profiles of the focused enzymes or activities eluted from the Sephadex G-75. and only assayed for histonase activitity after showing that histonase was located exclusively in the azurophil granules. If one re-examines the data presented here and compares it with data in the present communication, it becomes, apparent that Davies et al (1971) were detecting the same range of enzymes but in different proportions. Besides the major histonase activity, they showed several minor proteolytic activities focusing at pH 6 3. 7.0. 8 5 and 9 0, but. because elastase was not assayed and inhibitor profiles were not performed, it is diOicult to correlate these activities to our elastase (pi 9 1 ) and metallo-proteinase (pi 8 5). Furthermore, the Sephadex G-75 profile of Davies et al. (1971) shows a minor peak of histonase eluting after cytochrome C and lysozyme which probably is the elastase/proteoglycanase/ histonase (MW 8.000-fl.OOO) seen on our Sephadex G-100 profiles. The differences in the proportions of proteinases reported here and those seen by Davies et al (1971) may reflect the different methods of preparing crude extracts of lysosomes. The major neutral proteinases of human neutrophil leucocytes are the serine proteinases. elastase and eathepsin G, which are described in some detail by Starkey (1977). The rabbit PMNL neutral proteinases differ in several ways from the human enzymes, tbe most obvious disparity being the lack of a eathepsin G analogue in the rabbit PMNL, plus the presence of a cationic neutral metallo-proteinase. Furthermore, elastase in the human PMNL is a composite of three closely related activities together with several minor elastases. whereas the rabbit cells have one major elastase activity (data presented here and by Dewald et al, 1975). The rabbit and human elastases share certain properties, such as similar isoelectric points, stimulation by high salt concentrations, and a tendency to adsorb non-specifically to insoluble surfaces at low ionic strength. The inhibition of rabbit PMNL elastase by copper ions has not been reported for human elastase. A major difference in the elastases from rabbit and human PMNL may be in the size of the molecules. This is of some interest regarding the potential physiological roles of these enzymes in the two species, as the molecular weight may influence the ability of the molecules to penetrate macromolecular matrices. To clarify this point, further purification of the rabbit elastase would be

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required. This is made difficult by the relatively small starting levels of enzyme (10- to 30-fold lower than in human leucocytes; Dewald et al, 1975) plus significant losses in activity at several stages during purification which was compounded by the instability of the partially purified enzymes (also seen by Davies (?rfl/.,1971). REFERENCES and enzyme from human neutrophil leucodeDuvn, C. (1969): 'Resolution of cytes". Biochim. Biophys. Acta. 364. 103granules from rabbit heterophil leuco112. cytes inio distinct populations by zonal H/vusrR, P.. and VAES. G . (1978): 'Desedimenlalion.' J. Cell. Biol., 40, 529-541. gradation of cartilage proteoglycans by a BR[TZ, M. L., and LOWTHEK. D . A. (1980): neutral proteinase secreted by rabbit 'Effects of medium constituents on secrebone-marrow macrophages in culture.' tion of latent collagenase and proteoglyBiochem. /., 173. 275-284. canase by cultured rabbit articular carHioucHi, Y.. HONDA, M . . and HAYASHI, tilage.' Proc. Au.st. Biochem. Soc. 13. 19. H. (1975): 'Produclion of chemotactic COHN, Z. A., and HIRSCH. J- G. (1960): faclor for lymphocytes by neutral SH'1 he isolation and properties of the dependent protease of rabbit PMN leuspecific cylopliismic granules of rabbit cocytes from immunoglobulins, especially polymorphonuclear leucocytes.' /. Exp. ]gM.' Cellular Immunol., 15, 102-108. Med.. 112. 983-1004. JANOEF. A . , and BASCH, R . S. (1971): DAVIHS. P,, KRAKAUER. K.. and WEISSMANN, 'Further studies on elastase-like esterases G. (1970): 'Subcellular distribution of in human leucocyte granules.' Proc. Soc. neutral protease and peptidases in rabbit Exp. Biol. Med., 136. 1045-1049. polymorphonuclear leucocytes.' Nature. l-owRv, O. H.. RoSENBROuriH, N. J., FARR, Lond., 228, 761-762. A. L.. and RANDALL, R. J. (1951): 'ProDAVIES, P., RITA, G . A.. KRAKAULR, K., and tein measurement with the Folin phenol WEISSMANN, G . (1971): 'Characterization reagent". /. Biol. Chem., 193. 265-275. of a neutral protease from lysosomes of STARKEY. P. M. (1977): 'Elastase and rabbit polymorphonuclear leucocytes.' eathepsin G; the serine proleinases of Biochem. /.. 123, 559-569. human neutrophil leucocytes and spleen.' D F . W A L D , B . , RlNULER-LUDWKl, R.. B R E T Z . In "'Proteinases in Mammalian Cells and U.. and BAGGIOLINI, M. (1975): 'SubcelTissues", A. J. Barret (ed.). North lular localization and heterogeneity of Holland Publishing Company, Amsterneutral proteases in neutrophilic polymordam, pp. 57-89. phonuclear leucocytes.' J. E.xp. Med., STARKrv. P. M., and BARRETT, A. J. (1976): 141, 709-723. •Neutral proteinases of human spleen. GERBER, A . C , CARSON. J. H., and HADORN, Purificalion and crileria for homogeneity B. (1974): 'Parlial purification and of elastase and eathepsin G." Biochem. characterization of a chymolrypsin-like J.. 155, 255-263. BAGGIOLINI.

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