30P RELEASE OF ENZYMES FROM BACTERIA .* D . F. Broad and J . T . Smith, Microbiology Section, Department of Pharmaceutics, School of Pharmacy, University of London, Brunswick Square, London WClN l A X , U.K. B. lactamases a r e enzymes which commonly cause c l i n i c a l s t r a i n s of bacteria t o r e s i s t the p e n i c i l l i n and cephalosporin a n t i b i o t i c s . Certain 6. lactamases can be released from Gram-negative bacteria by a variety o f techniques which d i s r u p t the outer c e l l wall b u t do not cause c e l l l y s i s n o r r e l e a s e cytoplasmic enzymes. On t h e other hand, some B. lactamases remain c e l l bound during these treatments. One such technique, osmotic shock, h a s been used t o study the r e l e a s e of a variety o f enzymes from E . c o l i . Enzymes released by t h i s procedure have been assigned t o a d i s c r e e t c e ' n u l a r location termed the periplasm (Heppel, 1971 Those enzymes which a r e retained d u r i n g osmotic shock have been assumed t o have a d i f f e r e n t c e l l u l a r location and they have been termed c e l l bound (Heppel, 1971). However, there i s no evidence t h a t enzymes e x i s t i n these two locations during normal conditions.

Neu (1968) studied the r e l e a s e of B. lactamases by osmotic shock treatment and concluded t h a t R-factor mediated B. lactamases were periplasmic whereas chromosomally mediated ones were c e l l bound. However, r e s u l t s presented here ( t a b l e ) and by Wyatt and Smith (1974) i n d i c a t e t h a t i t i s the molecular weight of the enzyme, and n o t the genetic location of i t s s t r u c t u r a l gene, which determines whether o r not a B. lactamase can be released by osmotic shock treatment; B . lactamases 4 f molecular weight g r e a t e r t h a n 30,000 not being released. B.

lactamase ,R7268 RP 1 RGN238 R22Ka

K . aerogenes 418 E . c o l i D3

*

Genetic location*

Molecular weight 20 ,500 19,500 24,000 31,800

C C

20,080 31,800

R represents R . f a c t o r a n d C represents Chromosomal f3.

Percent. Release 96.2 94.8 83.8 0 74.4 0

lactamases

When t h e r e l e a s e of the 6. lactamases mediated by R . f a c t o r s R46 a n d R55 was studied i t was found t h a t d e s p i t e having molecular weights of 44,600 and 41,200 they were released t o some e x t e n t by osmotic shock ( 1 1 . 2 % and 52.0%, r e s p e c t i v e l y ) . This apparantly anomolous behaviour can be explained by the finding t h a t these two enzymes a r e dimeric (Dale and Smith, 1976). Thus e i t h e r these enzymes in the dimeric s t a t e a r e s u f f i c i e n t l y asymmetric, o r t h e i r individual sub-units a r e s u f f i c i e n t l y small, t o pass through the c e l l wall during osmotic shock. The c e l l wall therefore appears t o a c t simply as a molecular s i e v e during osmotic shock treatment and thus l i t t l e o r no conclusions regarding c e l l u l a r location should be drawn from osmotic shock data. Dale J.W. a n d Smith J . T . (1976) Biochem. Biophys. Res. Comm. 68, 100 Heppel L . A (1971) "Structure and Function of Bioloqical Membranes " Ed. L . I . Rothfield ~ 1 2 2 3 Neu H . C . (1968) Biochem. Biophys. Res. Comm: 32, 258 Wyatt J.M. and Smith J.T. (1974) J.Bacterio1. TT7,931