15.9. 1976
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Purification and Partial C h a r a c t e r i z a t i o n of L y s o z y m e f r o m M o u s e S m a l l Intestine T. L. PEETERS a n d G. R. VAI~TI~AI~PEN~
Laboratorium voor gastrointestinale /ysiopathologie, Departement Medische Navorsing, Katholieke Universiteit te Leuven, A. Z. St. Ra/agl, Leuven (Belgium), 20 February 7976. Summary. L y s o z y m e was isolated f r o m t h e small i n t e s t i n e of mice b y c o m b i n e d i o n - e x c h a n g e a n d molecular sieve c h r o m a t o g r a p h y . This l y s o z y m e differs f r o m t h a t isolated f r o m t h e urine of mice w i t h m o n o c y t o m a in a m i n o acid composition, a n d m i g r a t i o n r a t e in cellulose a c e t a t e electrophoresis. As i n t e s t i n a l l y s o z y m e originates a t least in p a r t f r o m t h e P a n e t h cell, our results p o i n t t o w a r d s t h e existence of i s o z y m e s of l y s o z y m e s in mice. I n a v i a n egg-whites t w o m a r k e d l y d i f f e r e n t f o r m s of l y s o z y m e ( ' c h i c k - t y p e ' a n d ' g o o s e - t y p e ' ) h a v e b e e n described ~-~. M a m m a l i a n l y s o z y m e s belong to t h e 'chickt y p e ' lysozyme, a n d a l t h o u g h it h a s b e e n s u g g e s t e d t h a t g r a n u l o c y t i c a n d m a c r o p h a g e i s o z y m e s of l y s o z y m e e x i s t in m a n 5, it is generally a s s u m e d t h a t w i t h i n one m a m m a l i a n species l y s o z y m e s f r o m d i f f e r e n t sources h a v e identical s t r u c t u r e s 6. The p r e s e n c e of l y s o z y m e in ~the P a n e t h cells is n o w welt d o c u m e n t e d T - H a n d i n t e s t i n a l l y s o z y m e is, a t least in p a r L p r o d u c e d b y t h e P a n e t h cell. D u r i n g our i m m u n o h i s t o c h e m i c a l s t u d i e s 1~ l y s o z y m e was isolated f r o m m o u s e small intestine. The c h a r a c t e r i s tics of t h i s lysozyme, d e s c r i b e d in t h i s p a p e r , differ f r o m t h o s e r e p o r t e d for m o u s e m o n o c y t i c l y s o z y m e 1~. These results suggest t h a t l y s o z y m e i s o z y m e s exist in mice. Materials and methods. Purification. Mice of t h e NM]~I strain, a p p r o x i m a t e l y 6 weeks old, were killed b y a s h a r p blow on t h e head. The s m a l l i n t e s t i n e was q u i c k l y removed, homogenized, and the homogenate centrifuged at 100,000 g for 60 m i n a t 4~ L y s o z y m e was isolated f r o m t h e s u p e r n a t a n s b y i o n - e x c h a n g e a n d molecular sieve c h r o m a t o g r a p h y according to s t a n d a r d p r o c e d u r e s . C h a r a c t e r i z a t i o n . A m i n o acid analyses were p e r f o r m e d on a T e c h n i c o n Model 100 a m i n o acid analyzer, following h y d r o l y s i s of t h e s a m p l e s for 8, 15 a n d 24 h in 6 N HC1 u n d e r v a c u u m a t 110~ The UV~spectra of h e n ' s eggw h i t e l y s o z y m e (HEL) a n d m o u s e i n t e s t i n a l l y s o z y m e (MIL) in 0.1 M HC1 a n d 0.1 M N a O H were r e c o r d e d on a U n i c a m SP-800. The m o l e c u l a r w e i g h t was e s t i m a t e d b y c o m p a r i n g t h e m i g r a t i o n r a t e s of H E L a n d M I L on t h i n layers of S e p h a d e x G-50 (grade superfine). L y s o z y m e s were localized b y s p r a y i n g t h e p l a t e s w i t h a Micrococcus lysodeikticus s u s p e n s i o n of 10 mg/ml. Cellulose a c e t a t e electr0Phoresis was carried o u t w i t h t h e Microzone equipm e n t . ( B e c k m a n I n s t r u m e n t s Inc., Palo Alto, Calif., USA) a t t w o d i f f e r e n t p H - v a l u e s . Results. Purification. A s u p e r n a t a n s o b t a i n e d f r o m t h e h o m o g e n a t e of t h e small i n t e s t i n e of 20 mice, c o n t a i n i n g a p p r o x i m a t e l y 7.5 m g l y s o z y m e was loaded on a c o l u m n of a m b e r l i t e CG-50, e q u i l i b r a t e d w i t h 0.2 2kI p h o s p h a t e b u f f e r p H 6.5. A s t e p w i s e g r a d i e n t of NaC1 f r o m 0.0 t o 0.5 3 / / w a s applied in 5 s t e p s of 0.1 iV/, t o elute a b s o r b e d proteins. L y s o z y m e a c t i v i t y was Fluted a t 0.3 M and c o n c e n t r a t i o n of this eluate was a c h i e v e d b y a m m o n i u m sulfate p r e c i p i t a t i o n (90% s a t u r a t i o n a t 25~ The p r e c i p i t a t e w a s collected b y c e n t r i f u g a t i o n a n d c h r o m a t o g r a p h e d on S e p h a d e x G-50. T h e overall yield a t t h i s p o i n t was 30%, especially due to a low r e c o v e r y on a m b e r l i t e (44% only). Therefore, t h e l y s o z y m e c o n t a i n i n g fractions of several s u c h p r e p a r a t i o n s w e r e c o m b i n e d , again prec i p i t a t e d b y a m m o n i u m sulfate a n d r e c h r o m a t o g r a p h e d on S e p h a d e x G - 7 5 yielding a n a l m o s t s y m m e t r i c a l p e a k w i t h c o n s t a n t specific a c t i v i t y . The c e n t e r fractions w e r e d e s a l t e d on S e p h a d e x G-10 w i t h 0.l N acetic acid as t h e eluate, and lyophilized. C h a r a c t e r i z a t i o n . The p u r i f i e d e n z y m e was dissolved into 2 buffer solutions (acetate buffer 0.1 M, p H 5.5 and
Amino acid composition of mouse intestinal lysozyme (15 h) amino acid residues per molecule Amino acid
M1L ~
HEL
Human milk is
Aspartic acid Threonine Serine Glutanlic acid Proline Glycine Alanine Valine Cystine (half) Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Lysine Histidine Arginine
21 7 8 13 5 11 13 6 8 2 7 8 5 3 9b 13 2 13
21 7 10 5 2 12 12 6 8 2 6 8 3 3 6 6 1 11
18 5 6 9 2 11 14 9 8 2 5 8 6 2 5 5 1 14
9Assuming 2 histidine residues per nmlecule. Deternfined spectrophotometrically is.
b a r b i t a l buffer 0.1 M , p H 8.6) a n d s u b j e c t e d t o cellulose a c e t a t e e l e c t r o p h o r e s i s a t b o t h p H values. A t e a c h p H , t h e e n z y m e m o v e d as a single b a n d , b u t t h e progression of M I L was s l i g h t l y faster t h a n t h a t of H E L . A t p H 8.6, M I L was f o u n d a t 2.0 c m f r o m t h e a p p l i c a t i o n p o i n t in t h e c a t h o d i c direction, a f t e r e l e c t r o p h o r e s i s for 20 min a t 250 V, w h e r e a s H E L was f o u n d a t 1.8 c m f r o m t h e
1 Acknowledgments. Supported by grant No. 20159 from the 'Fonds voor Wetensehappelijk Geneeskundig Onderzoek, Brussel, Belgiuin'. We should like to thank Mrs. PAREIN Ior skillful technical assistance. 2 F. J. MORGANand N. ARNt~EIM,in Lysozyme (Eds. F. F. OSSERMAN, 1~. g. CANFIELD and S. BEYCHOK;Academic Press, New York 1973), p. 81. 3 N. ARNHEIM and R. STELLER; Arch. Biochem. Biophys. 141, 656 (1970). 4 N. ARNHEIM, ill Lysozyme (Eds. E. F. OSSERMAN,R. E. CANFIELD and S. BEYCHOK; Academic Press, New York 1973), p. 153. 5 A. MO~TON and J. JOLLES, FEBS Lett. d, 337 (1969). 6 E. F. OSSERMAN,New Engl. J. Med. 292, 424 (1975). R. J. DECKX, G. R. VANTRAPP~N and M. M. PAREIN, BiochinI. biophys. Acts 139, 204 (1967). s y. GHOOS and G. VANTRAePEN,ttistocheln. J. 3, 175 (1971). 9 T. L. PEETERS and G. R. VANS'RAPPEN,Gut 76, 553 (1975). 10 S. L. J~]RLANDSEN,J. A. PARSONSand T. D. TAYLOR,J. Histochem. 22, 401 (1974). 11 M. KLOCKARSand E. F. OSSERMAN,J. Histochem. Cytocheln. 22, 139 (1974). le R. J. RIBLET and L. A. I-IERZENBERG,Science 168, 1595 (1970). la G. H. BEAVENand E. R. HOLIDAY,Adv. Prot. Chem. 7, 319 (1952).
1126
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a p p l i c a t i o n p o i n t . A t p H 5.5, t h e figures were 2.2 c m for M I L a n d 1.9 c m for H E L . A m i n o acid a n a l y s e s p e r f o r m e d on 2 d i f f e r e n t p r e p a r a t i o n s of MIL, yielded a l m o s t identical results. T h e s i m i l a r i t y w i t h H E L is striking, a l t h o u g h M I L is still m o r e basic t h a n H E L (Table), a n d t h e t o t a l n u m b e r of residues is higher. F r o m t h e a m i n o acid comp o s i t i o n a m o l e c u l a r w e i g h t of 17,800 is c o m p u t e d , s l i g h t l y m o r e t h a n a c c e p t e d m o l e c u l a r w e i g h t of h e n e g g - w h i t e l y s o z y m e 14,600. B o t h lysozymes h a d i d e n t i c a l m i g r a t i o n r a t e s in t h i n layer-gel c h r o m a t o g r a p h y . Discussion. I n n o r m a l mice t h e s m a l l i n t e s t i n e is a r e l a t i v e l y r i c h source of l y s o z y m e ( a b o u t 375 ~xg p e r mouse). W e decided t o isolate m o u s e i n t e s t i n a l l y s o z y m e as a p r e l i m i n a r y step in t h e s t u d y of t h e f u n c t i o n of t h e P a n e t h cell% A m u c h m o r e c o n v e n i e n t m o d e l was used b y RIBLET a n d HERZENBERG 12, w h o i s o l a t e d l y s o z y m e f r o m t h e u r i n e of mice b e a r i n g a t y p e A r e t i c u l u m sarcoma, in w h i c h t h e u r i n e c o n t a i n e d u p to 25 m g / m l . P r o b a b l y , t h i s u r i n a r y lysozyme, as well as t h a t f o u n d in some t y p e s of h u m a n l e u k a e m i a ~ is p r o d u c e d b y monocytes. A c c o r d i n g t o I~IBLET a n d HERZENBERG 12, t h e ext i n c t i o n coefficient of m o u s e l y s o z y m e is lower t h a n t h a t of egg w h i t e lysozyme, due to a lower a m o u n t of t r y p t o p h a n residues (4 a g a i n s t 6), a l t h o u g h m o r e residues of t h e less a b s o r b i n g t y r o s i n e (7 a g a i n s t 3) are p r e s e n t . T h e m o u s e i n t e s t i n a l l y s o z y m e w h i c h we isolated h a s a h i g h e x t i n c t i o n coefficient due to t h e p r e s e n c e of 9 t r y p t o p h a n residues a n d 5 t y r o s i n e residues. Mouse m o n o c y t i c lysoz y m e h a s t h e s a m e t o t a l n u m b e r of residues (130) a n d t h e s a m e n u m b e r of basic residues (18) as H E L , b u t t h e r e are 29 acidic residues a g a i n s t 26 in H E L . This m o n o c y t i c l y s o z y m e m o v e d m o r e slowly t h a n H E L in cellulose
EXPERIENTIA 32/9
a c e t a t e electrophoresis. I n c o n t r a s t , M I L h a s a t o t a l of 154 residues, 34 acidic a n d 28 basic. T h e surplus of c h a r g e d residues p r o b a b l y e x p l a i n s w h y t h i s l y s o z y m e m o v e d f a s t e r t h a n H E L in cellulose a c e t a t e electrophoresis. I t is difficult t o e s t i m a t e h o w m u c h of t h e l y s o z y m e in a n int e s t i n a l h o m o g e n a t e o r g i n a t e s f r o m t h e P a n e t h cell, a n d h o w m u c h is d e r i v e d f r o m w h i t e b l o o d cells in t h e int e s t i n a l wall. I t is possible t h a t m o n o c y t i c l y s o z y m e was e l i m i n a t e d d u r i n g t h e p u r i f i c a t i o n p r o c e d u r e (this could e x p l a i n t h e low r e c o v e r y we o b t a i n e d in t h e a m b e r l i t e eluate), or t h a t s c r a p i n g off m u c o s a f r o m muscle layers e l i m i n a t e s m o s t of t h e m o n o c y t e s . I n t h i s c o n t e x t it is w o r t h n o t i n g t h a t r a b b i t s , deficient in l y s o z y m e in s e r u m a n d m o s t o t h e r tissues still c o n t a i n 50% of t h e l y s o z y m e of n o r m a l r a b b i t s in t h e i r i n t e s t i n e ~5. T h e suggestion t h a t s t r u c t u r a l differences e x i s t b e t w e e n h u m a n g r a n u l o c y t i c a n d m a c r o p h a g e lysozymes% s h o u l d be i n v e s t i g a t e d in t h e l i g h t of o u r o b s e r v a t i o n s , as P a n e t h ceils m a y be c o n s i d e r e d to f u n c t i o n as fixed m a c r o p h a g e s 1~ F u r t h e r r e s e a r c h is n e e d e d to clarify t h e s e points, p a r t i c u l a r l y because t h e existence of isozymes in sera of p a t i e n t s w i t h i n f l a m m a t o r y bowel disease could be t h e cause of t h e d i v e r g e n t r e s u l t s o b t a i n e d b y d i f f e r e n t m e t h o d s 16-1
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1125
Purification and Partial C h a r a c t e r i z a t i o n of L y s o z y m e f r o m M o u s e S m a l l Intestine T. L. PEETERS a n d G. R. VAI~TI~AI~PEN~
Laboratorium voor gastrointestinale /ysiopathologie, Departement Medische Navorsing, Katholieke Universiteit te Leuven, A. Z. St. Ra/agl, Leuven (Belgium), 20 February 7976. Summary. L y s o z y m e was isolated f r o m t h e small i n t e s t i n e of mice b y c o m b i n e d i o n - e x c h a n g e a n d molecular sieve c h r o m a t o g r a p h y . This l y s o z y m e differs f r o m t h a t isolated f r o m t h e urine of mice w i t h m o n o c y t o m a in a m i n o acid composition, a n d m i g r a t i o n r a t e in cellulose a c e t a t e electrophoresis. As i n t e s t i n a l l y s o z y m e originates a t least in p a r t f r o m t h e P a n e t h cell, our results p o i n t t o w a r d s t h e existence of i s o z y m e s of l y s o z y m e s in mice. I n a v i a n egg-whites t w o m a r k e d l y d i f f e r e n t f o r m s of l y s o z y m e ( ' c h i c k - t y p e ' a n d ' g o o s e - t y p e ' ) h a v e b e e n described ~-~. M a m m a l i a n l y s o z y m e s belong to t h e 'chickt y p e ' lysozyme, a n d a l t h o u g h it h a s b e e n s u g g e s t e d t h a t g r a n u l o c y t i c a n d m a c r o p h a g e i s o z y m e s of l y s o z y m e e x i s t in m a n 5, it is generally a s s u m e d t h a t w i t h i n one m a m m a l i a n species l y s o z y m e s f r o m d i f f e r e n t sources h a v e identical s t r u c t u r e s 6. The p r e s e n c e of l y s o z y m e in ~the P a n e t h cells is n o w welt d o c u m e n t e d T - H a n d i n t e s t i n a l l y s o z y m e is, a t least in p a r L p r o d u c e d b y t h e P a n e t h cell. D u r i n g our i m m u n o h i s t o c h e m i c a l s t u d i e s 1~ l y s o z y m e was isolated f r o m m o u s e small intestine. The c h a r a c t e r i s tics of t h i s lysozyme, d e s c r i b e d in t h i s p a p e r , differ f r o m t h o s e r e p o r t e d for m o u s e m o n o c y t i c l y s o z y m e 1~. These results suggest t h a t l y s o z y m e i s o z y m e s exist in mice. Materials and methods. Purification. Mice of t h e NM]~I strain, a p p r o x i m a t e l y 6 weeks old, were killed b y a s h a r p blow on t h e head. The s m a l l i n t e s t i n e was q u i c k l y removed, homogenized, and the homogenate centrifuged at 100,000 g for 60 m i n a t 4~ L y s o z y m e was isolated f r o m t h e s u p e r n a t a n s b y i o n - e x c h a n g e a n d molecular sieve c h r o m a t o g r a p h y according to s t a n d a r d p r o c e d u r e s . C h a r a c t e r i z a t i o n . A m i n o acid analyses were p e r f o r m e d on a T e c h n i c o n Model 100 a m i n o acid analyzer, following h y d r o l y s i s of t h e s a m p l e s for 8, 15 a n d 24 h in 6 N HC1 u n d e r v a c u u m a t 110~ The UV~spectra of h e n ' s eggw h i t e l y s o z y m e (HEL) a n d m o u s e i n t e s t i n a l l y s o z y m e (MIL) in 0.1 M HC1 a n d 0.1 M N a O H were r e c o r d e d on a U n i c a m SP-800. The m o l e c u l a r w e i g h t was e s t i m a t e d b y c o m p a r i n g t h e m i g r a t i o n r a t e s of H E L a n d M I L on t h i n layers of S e p h a d e x G-50 (grade superfine). L y s o z y m e s were localized b y s p r a y i n g t h e p l a t e s w i t h a Micrococcus lysodeikticus s u s p e n s i o n of 10 mg/ml. Cellulose a c e t a t e electr0Phoresis was carried o u t w i t h t h e Microzone equipm e n t . ( B e c k m a n I n s t r u m e n t s Inc., Palo Alto, Calif., USA) a t t w o d i f f e r e n t p H - v a l u e s . Results. Purification. A s u p e r n a t a n s o b t a i n e d f r o m t h e h o m o g e n a t e of t h e small i n t e s t i n e of 20 mice, c o n t a i n i n g a p p r o x i m a t e l y 7.5 m g l y s o z y m e was loaded on a c o l u m n of a m b e r l i t e CG-50, e q u i l i b r a t e d w i t h 0.2 2kI p h o s p h a t e b u f f e r p H 6.5. A s t e p w i s e g r a d i e n t of NaC1 f r o m 0.0 t o 0.5 3 / / w a s applied in 5 s t e p s of 0.1 iV/, t o elute a b s o r b e d proteins. L y s o z y m e a c t i v i t y was Fluted a t 0.3 M and c o n c e n t r a t i o n of this eluate was a c h i e v e d b y a m m o n i u m sulfate p r e c i p i t a t i o n (90% s a t u r a t i o n a t 25~ The p r e c i p i t a t e w a s collected b y c e n t r i f u g a t i o n a n d c h r o m a t o g r a p h e d on S e p h a d e x G-50. T h e overall yield a t t h i s p o i n t was 30%, especially due to a low r e c o v e r y on a m b e r l i t e (44% only). Therefore, t h e l y s o z y m e c o n t a i n i n g fractions of several s u c h p r e p a r a t i o n s w e r e c o m b i n e d , again prec i p i t a t e d b y a m m o n i u m sulfate a n d r e c h r o m a t o g r a p h e d on S e p h a d e x G - 7 5 yielding a n a l m o s t s y m m e t r i c a l p e a k w i t h c o n s t a n t specific a c t i v i t y . The c e n t e r fractions w e r e d e s a l t e d on S e p h a d e x G-10 w i t h 0.l N acetic acid as t h e eluate, and lyophilized. C h a r a c t e r i z a t i o n . The p u r i f i e d e n z y m e was dissolved into 2 buffer solutions (acetate buffer 0.1 M, p H 5.5 and
Amino acid composition of mouse intestinal lysozyme (15 h) amino acid residues per molecule Amino acid
M1L ~
HEL
Human milk is
Aspartic acid Threonine Serine Glutanlic acid Proline Glycine Alanine Valine Cystine (half) Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Lysine Histidine Arginine
21 7 8 13 5 11 13 6 8 2 7 8 5 3 9b 13 2 13
21 7 10 5 2 12 12 6 8 2 6 8 3 3 6 6 1 11
18 5 6 9 2 11 14 9 8 2 5 8 6 2 5 5 1 14
9Assuming 2 histidine residues per nmlecule. Deternfined spectrophotometrically is.
b a r b i t a l buffer 0.1 M , p H 8.6) a n d s u b j e c t e d t o cellulose a c e t a t e e l e c t r o p h o r e s i s a t b o t h p H values. A t e a c h p H , t h e e n z y m e m o v e d as a single b a n d , b u t t h e progression of M I L was s l i g h t l y faster t h a n t h a t of H E L . A t p H 8.6, M I L was f o u n d a t 2.0 c m f r o m t h e a p p l i c a t i o n p o i n t in t h e c a t h o d i c direction, a f t e r e l e c t r o p h o r e s i s for 20 min a t 250 V, w h e r e a s H E L was f o u n d a t 1.8 c m f r o m t h e
1 Acknowledgments. Supported by grant No. 20159 from the 'Fonds voor Wetensehappelijk Geneeskundig Onderzoek, Brussel, Belgiuin'. We should like to thank Mrs. PAREIN Ior skillful technical assistance. 2 F. J. MORGANand N. ARNt~EIM,in Lysozyme (Eds. F. F. OSSERMAN, 1~. g. CANFIELD and S. BEYCHOK;Academic Press, New York 1973), p. 81. 3 N. ARNHEIM and R. STELLER; Arch. Biochem. Biophys. 141, 656 (1970). 4 N. ARNHEIM, ill Lysozyme (Eds. E. F. OSSERMAN,R. E. CANFIELD and S. BEYCHOK; Academic Press, New York 1973), p. 153. 5 A. MO~TON and J. JOLLES, FEBS Lett. d, 337 (1969). 6 E. F. OSSERMAN,New Engl. J. Med. 292, 424 (1975). R. J. DECKX, G. R. VANTRAPP~N and M. M. PAREIN, BiochinI. biophys. Acts 139, 204 (1967). s y. GHOOS and G. VANTRAePEN,ttistocheln. J. 3, 175 (1971). 9 T. L. PEETERS and G. R. VANS'RAPPEN,Gut 76, 553 (1975). 10 S. L. J~]RLANDSEN,J. A. PARSONSand T. D. TAYLOR,J. Histochem. 22, 401 (1974). 11 M. KLOCKARSand E. F. OSSERMAN,J. Histochem. Cytocheln. 22, 139 (1974). le R. J. RIBLET and L. A. I-IERZENBERG,Science 168, 1595 (1970). la G. H. BEAVENand E. R. HOLIDAY,Adv. Prot. Chem. 7, 319 (1952).
1126
Specialia
a p p l i c a t i o n p o i n t . A t p H 5.5, t h e figures were 2.2 c m for M I L a n d 1.9 c m for H E L . A m i n o acid a n a l y s e s p e r f o r m e d on 2 d i f f e r e n t p r e p a r a t i o n s of MIL, yielded a l m o s t identical results. T h e s i m i l a r i t y w i t h H E L is striking, a l t h o u g h M I L is still m o r e basic t h a n H E L (Table), a n d t h e t o t a l n u m b e r of residues is higher. F r o m t h e a m i n o acid comp o s i t i o n a m o l e c u l a r w e i g h t of 17,800 is c o m p u t e d , s l i g h t l y m o r e t h a n a c c e p t e d m o l e c u l a r w e i g h t of h e n e g g - w h i t e l y s o z y m e 14,600. B o t h lysozymes h a d i d e n t i c a l m i g r a t i o n r a t e s in t h i n layer-gel c h r o m a t o g r a p h y . Discussion. I n n o r m a l mice t h e s m a l l i n t e s t i n e is a r e l a t i v e l y r i c h source of l y s o z y m e ( a b o u t 375 ~xg p e r mouse). W e decided t o isolate m o u s e i n t e s t i n a l l y s o z y m e as a p r e l i m i n a r y step in t h e s t u d y of t h e f u n c t i o n of t h e P a n e t h cell% A m u c h m o r e c o n v e n i e n t m o d e l was used b y RIBLET a n d HERZENBERG 12, w h o i s o l a t e d l y s o z y m e f r o m t h e u r i n e of mice b e a r i n g a t y p e A r e t i c u l u m sarcoma, in w h i c h t h e u r i n e c o n t a i n e d u p to 25 m g / m l . P r o b a b l y , t h i s u r i n a r y lysozyme, as well as t h a t f o u n d in some t y p e s of h u m a n l e u k a e m i a ~ is p r o d u c e d b y monocytes. A c c o r d i n g t o I~IBLET a n d HERZENBERG 12, t h e ext i n c t i o n coefficient of m o u s e l y s o z y m e is lower t h a n t h a t of egg w h i t e lysozyme, due to a lower a m o u n t of t r y p t o p h a n residues (4 a g a i n s t 6), a l t h o u g h m o r e residues of t h e less a b s o r b i n g t y r o s i n e (7 a g a i n s t 3) are p r e s e n t . T h e m o u s e i n t e s t i n a l l y s o z y m e w h i c h we isolated h a s a h i g h e x t i n c t i o n coefficient due to t h e p r e s e n c e of 9 t r y p t o p h a n residues a n d 5 t y r o s i n e residues. Mouse m o n o c y t i c lysoz y m e h a s t h e s a m e t o t a l n u m b e r of residues (130) a n d t h e s a m e n u m b e r of basic residues (18) as H E L , b u t t h e r e are 29 acidic residues a g a i n s t 26 in H E L . This m o n o c y t i c l y s o z y m e m o v e d m o r e slowly t h a n H E L in cellulose
EXPERIENTIA 32/9
a c e t a t e electrophoresis. I n c o n t r a s t , M I L h a s a t o t a l of 154 residues, 34 acidic a n d 28 basic. T h e surplus of c h a r g e d residues p r o b a b l y e x p l a i n s w h y t h i s l y s o z y m e m o v e d f a s t e r t h a n H E L in cellulose a c e t a t e electrophoresis. I t is difficult t o e s t i m a t e h o w m u c h of t h e l y s o z y m e in a n int e s t i n a l h o m o g e n a t e o r g i n a t e s f r o m t h e P a n e t h cell, a n d h o w m u c h is d e r i v e d f r o m w h i t e b l o o d cells in t h e int e s t i n a l wall. I t is possible t h a t m o n o c y t i c l y s o z y m e was e l i m i n a t e d d u r i n g t h e p u r i f i c a t i o n p r o c e d u r e (this could e x p l a i n t h e low r e c o v e r y we o b t a i n e d in t h e a m b e r l i t e eluate), or t h a t s c r a p i n g off m u c o s a f r o m muscle layers e l i m i n a t e s m o s t of t h e m o n o c y t e s . I n t h i s c o n t e x t it is w o r t h n o t i n g t h a t r a b b i t s , deficient in l y s o z y m e in s e r u m a n d m o s t o t h e r tissues still c o n t a i n 50% of t h e l y s o z y m e of n o r m a l r a b b i t s in t h e i r i n t e s t i n e ~5. T h e suggestion t h a t s t r u c t u r a l differences e x i s t b e t w e e n h u m a n g r a n u l o c y t i c a n d m a c r o p h a g e lysozymes% s h o u l d be i n v e s t i g a t e d in t h e l i g h t of o u r o b s e r v a t i o n s , as P a n e t h ceils m a y be c o n s i d e r e d to f u n c t i o n as fixed m a c r o p h a g e s 1~ F u r t h e r r e s e a r c h is n e e d e d to clarify t h e s e points, p a r t i c u l a r l y because t h e existence of isozymes in sera of p a t i e n t s w i t h i n f l a m m a t o r y bowel disease could be t h e cause of t h e d i v e r g e n t r e s u l t s o b t a i n e d b y d i f f e r e n t m e t h o d s 16-1
