BIOCHIMIE, 1979, 61, 385-391.

Characterization of an inhibitor of the intracellular protease from Bacillus subtilis. Jacqueline M I L L E T ~ and J osiane GREGOIRE. (31-10-1978).

Unitd de Physiologie Cellulaire, D~partement de Biochimie el G~n~tique Microbienne, Institut Pasteur, 28, rue da Docleur Roux, 75724 Paris Cedex 15.

R~sum~.

Summary.

Un inhibiteur sp6cifique de la s 6 r y l p r o t 6 a s e intracellulaire de Bacillus subtilis a &t6 isol6 darts les cellules en c r o i s s a n c e et en vole de spornlation. C o m m e d ' a u t r e s inhibiteurs isol6s & partir de cellules e u c a r y o t e s , rinhibiteur d6crit d a n s cet article est u n e prot6ine thermostable. Line m 6 t h o d e de purification est d6crite. La m a s s e m o l 6 c u l a i r e estim6e p a r filtration sur Bioqel et p a r 61ectrophor~se sur qel de polya c r y l a m i d e en p r 6 s e n c e de SDS est d ' e n v i r o n 15.500. L'activit6 prot6olytique et l'activit6 est& rolytique de la s 6 r y l p r o t 6 a s e sont 6 q a l e m e n t sensibles & l'inldbiteur. L'inhibition est non comp6titive en utilisant c o m m e s u b s t r a t soft razocoll, soft le Z-tyrosine p-nitroph6nylester. En r e v a n c h e , l'inhibiteur n ' a a u c u n e action sur l'activit6 prot6olytique ou est~rolytique de la s 6 r y l p r o t 6 a s e excr6t6e par B. subfilis a p r b s la fin de la c r o i s s a n c e exponentielle. Un inhibiteur prot6ique t h e r m o s t a b l e , similaire & celui d e B. subtilis, a a u s s i 6t6 mis e n 6vidence darts les cellules de Bacillus m e q a t e r i u m .

A specific inhibitor of intracellular serylprot e a s e from Bacillus subtilis h a s b e e n isolated from both qrowinq a n d s p o r u l a t i n g cells. Like other p r o t e a s e inhibitors isolated from e u k a r y o tic cells, the inhibitor from B. subtilis is a therm o s t a b l e protein. A purification m e t h o d is described. The m o l e c u l a r weiqht e s t i m a t e d b y Bioqel filtration a n d SDS qel electrophoresis is a b o u t 15,500. Both proteolytic a n d esterolytic activities of intracellular p r o t e a s e a r e e q u a l l y sensitive to inhibition. With azocoll or Z-tyrosine p - n i t r o p h e n y l e s t e r a s substrates, noncompetitive inhibition p a t t e r n s a r e o b s e r v e d . The inhibitor h a s no effect on the proteolytic or esterolyric activities of the extracellular serylprotease. A similar t h e r m o s t a b l e inhibitor is also p r e s e n t in Bacillus m e q a t e r i u m .

Introduction.

cult to imagi.ne that such a protease exists in the cytoplasm of differentiating cells without any regulation of its activity. As previously reported [9] a protein ~rhieh specifically inhibits the intracellular serylprotease has been characterized in B. subtilis and B. megaterium. In this paper, the method of purification .an'd some properties of this inhibitor are described.

Proteins with the ability~4o inhibit proteolytic enzymes have been isolated from a wide variety of euearyotie cells [1]. Some have been shown to be involved in the regul.ation of biologically important proteolytic processes, (e.g. blood clotting [1], digestion in mam,ma~s [1], a~ti~a¢ion of prechitin synthase in yeast [2]). An intraeellular serylprotease has been ehara.oterized in several species of Bacilli [3, 4, 5, 6, 7]. According to Gheng an,d Aronson [8], this ,enzyme ,could be involved in protein turnover during sporu~ation. It is diffi.

To whom all correspondence should be addressed.

Key words : Protease, protease inhibitor, B. subtilis, sporulatiom

Chemicals. Azocoll and deoxyribonuclease I were purchased from Calbiochem ; N-Z-L-tyrosine-p-nitrophenylester and ribonuclease B from Sigma ; Soybean

386

J. Millet and J. Grdgoire.

trypsin inhibitor and chymotrypsin from Worthington ; pronase E from Merck ; cytochronie c (horse heart), ovalbumin and chymotrypsin:ogen A f r o m B o e h r i n g e r ; b o v i n e a l b u m i n ( f r a c t i o n V) from Armour; acrylamide, N,N'-bisacrylamide, N,N,N',N'detramethylethylenediamine and Coom a s s i e b r i l l i a n t b l u e G250 f r o m E a s t m a n O r g a n i c C h e m i c a l s ; D E A E - S e p h a d e x A-25 a n d G.75 f r o m P h a r m a c i a ; B i o - G e l s P60 a n d P30 f r o m B i o - R a d Laborato~ries ; G o o m a s s i e b r i l l i a n t b l u e R250 f r o m Cola~b.

Material and Methods. 1. Bacterial strains : Mutant 512 derived f r o m B. subtilts Marburg was used in this study. This m u t a n t is deficient in the synthesis of extracellular metalloprotease b u t production of other extracellular enzymes and sporulation are n o r m a l [10]. The wild type strain MA [11] of B. megaterium was also used. 2. Media and Growth : B. subtilis was grown in modified Difco n u t r i e n t b r o t h [12]. B. megaterium was grown in a complete m e d i u m containing (per liter) : KH~PO~ : 3 g ; Na2HPO~, 12 H20 : 6 g ; NH~C1 : 2 g ; MgCl~, 6 H 2 0 : 0 . 1 g ; MnC12, 4 H : O : 0.01 g ; CaCl~, 2 H 2 0 : 0.147 g ; glucose : 1 g ; Yeast extract : 2 g ; pH was a d j u s t e d at 6.8. Cultures were incubated at 30°C in a 20 liter Biolafitte f e r m e n t o r or in toxin flasks on a shaker (200 ml of m e d i u m in 2 1 flask). Growth was m o n i t o r e d by m e a s u r i n g the O.D. at 570 nm. The time at w h i c h exponential growth ceases is termed to ; t , t~ etc.., are t i m e s in h o u r s a f t e r to. The cells were h a r vested b y e e n t r i f u g a t i o n at 4°C. In order to eliminate c o n t a m i n a t i n g extracellular proteases, bacteria were first washed w i t h a buffer containing 20 mM Tris-HC1 and 1 M KC1 at pH 7.3, t h e n washed twice w i t h the same buffer devoid of KCI. Cell pellets used f o r protease study were w a s h e d with buffers also containing 2 mM CaCI.~ to preserve protease activity. Those extracted for i n h i b i t o r studies were washed w i t h o u t CaCI~ to reduce the activity of the intraeellular protease. All w a s h i n g s and centrifugations were p e r f o r m e d at 4°C. 3. Preparation o[ proteolytic enzymes : The intracellular serylprotease f r o m B. subtilis or f r o m B. megaterium was p r e p a r e d as previously described [3, 4]. Cells were harvested at t~-tz.6. Routinely, the enzyme p r e p a r a t i o n obtained after a m m o n i u m sulfate precip i t a t i o n and dialysis was used. The extraeellular serylprotease f r o m B. subtilis was p r e p a r e d previously described [12]. Since strain 512 is devoid of metalloprotease, the serylprotease extract used was t h a t obtained after the DEAE cellulose c h r o m a t o g r a p h y step w h i c h e l i m i n a t e s the esterase [12]. 4. Enzymatic assays : The proteolytie activity of t h e intraeellular and extraeellular serytproteases was assayed w i t h azoeoll as substrate [13] : 30 nag of azocoll in 6 ml of 0.2 M Tris-HC1 buffer, pH 7.3 containing 2 mM CaCI~. I n c u b a t i o n was carried out at 30°C w i t h gentle agitation. One u n i t of activity (UA) was equivalent to the h y d r o l y s i s of 1 gg of azocoll per min at 30°C. BIOCHIMIE, 1979, 61, n ° 3.

The esterolytic activity was d e t e r m i n e d using N-Z-L-tyrosine-p-nitrophenylester as substrate at a final concentration of 45 jxM in 0.1 M Tris-HC1 buffer, pH 8.2 containing 2 mM CaCI~. One unit of activity was equivalent to the h y d r o l y s i s of 1 ~tmole of substrate per m i n at 30°C. P r o t e i n concentrations were measured by the method of Lowry et al. [14], w i t h bovine serum a l b u m i n as a standard. 5. Assay of inhibitory activity : I n h i b i t o r y activity was expressed, according to Betz et at. [15], as the a m o u n t of enzyme activity inh;bited per ml of inhib i t o r y solution : XUA - YUA I n h i b i t o r y activity : ml of i n h i b i t o r solution where XUA ---- protease activity w i t h o u t inhibitor. YUA : protease activity with inhibitor. Only values of YUA between 30 per cent and 60 per cent of XUA were used for calculation. Routinely 25 to 30 u n i t s of intracellular serylprotease were used for i n h i b i t o r y activity. The proteolytic enzyme was incubated w i t h the i n h i b i t o r solution for 5 rain at 30°C in 1 ml of 0.2 M Tris-HCl buffer, pH 7.3 containing 2 mM CaC12. The volume was then b r o u g h t to 6 n d with the same buffer, 30 mg of azocoll were added and the r e m a i n i n g proteolytie activity determined. The specific activity of the i n h i b i t o r is expressed in units per mg of protein as m e a s u r e d e i t h e r by the method of Lowry et al. [14] or by a method using Coomassie blue [16]. 6. Polyacrylamide gel electrophoresis : Polyacryla m i d e gel eleetrophoresis was p e r f o r m e d as described by Ornstein [17]. R u n n i n g gels of 7 or 14 cm (7 per cent acrylamide) were used. Eleetrophoresis was carried out by r u n n i n g each gel at 2.5 mA for 1.5 h in 5 mM Trisglyeine buffer, pH 8.4 at 4°C. To localize the inhibitor a f t e r electrophoresis, the gels were frozen, and cut into 2 m m slices, which were then crushed in 0.2 M TrisHC1 buffer, pH 7.3 containing 2 mM CaC12. Polyaerylamide gel electrophoresis in the presence of sodium dodecyl sulfate was p e r f o r m e d according to the method of Laemmli [18] in 10 per cent p o l y a c r y l a m i d e gels.

Results. ]. PURIFICATION OF B. s u b t i l i s INHIBITOR FROM SPORULATING CFALLS : T h e c e l l s w e r e h a r v e s t e d at tl, w a s h e d a n d k e p t frozen. The p u r i f i c a t i o n p r o c e d u r e d e s c r i b e d bel o w w a s p e r f o r m e d o n 6.3 g of b a c t e r i a ( d r y weight). 1.1. S t e p 1 - C r u d e b o i l e d e x t r a c t : Cells w e r e s u s p e n d e d i n 0.2 M Tris-HC1 b u f f e r , p H 7.3 (10 m g d r y w e i g h t / m l ) . T h e y w e r e d i s r u p t e d at 4°C b y s o n i c a t i o n i n a I ~ r a n s o n S o n i f i e r (Model 12B) until t h e o p t i c a l d e n s i t y d r o p p e d to 25 p e r c e n t of its i n i t i a l v a l u e . T h e s a m p l e w a s c e n t r i f u g e d at 27,000 × g f o r 30 r a i n ; t h e s u p e r n a t a n t w a s k e p t

Protease

from B a c i l l u s

inhibitor

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Characterization of an inhibitor of the intracellular protease from Bacillus subtilis.

BIOCHIMIE, 1979, 61, 385-391. Characterization of an inhibitor of the intracellular protease from Bacillus subtilis. Jacqueline M I L L E T ~ and J o...
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