930

Specialia

EXPERIE~TIA 32/7

Protein and lipid composition of lipoprotein fractions of A-positive pig serum. A activity of Iipids and nonIipids prepared from those lipoprotein fractions Fraction

Protein Lipids (mg/100 ml original serum)

Protein (% of the respective lipoprotein class)

Approximate A activity in lipids in nonlipids (% of original serum)

A-positive serum VLDL LDL HDL Lipoprotein-free residue

5,120 4.6 53.0 75.8 4,871

-10 24 49 --

20 5 5 10 --

296.5 41.5 169.0 80.0 --

in t h e lipid-free p r o t e i n . No J a c t i v i t y was f o u n d in t h e a p o p r o t e i n s of t h e l i p o p r o t e i n fractions. A similar s t u d y was c a r r i e d o u t o n A a c t i v e pig s e r u m . A m o n g 23 m i n i a t u r e pigs ( G 6 t t i n g e n bred), 16 (i.e. 70%) t u r n e d o u t t o h a v e A p o s i t i v e s e r u m as e x a m i n e d b y agg l u t i n a t i o n - i n h i b i t i o n t e s t s in t h e h o m o l o g o u s A s y s t e m . All of t h e s e A p o s i t i v e a n i m a l s c a r r i e d t h e A s u b s t a n c e in t h e t o t a l lipid f r a c t i o n of s e r u m , while t h e lipid-free resid u e p r e c i p i t a t e d b y t h e lipid e x t r a c t i o n p r o c e d u r e was A a c t i v e i n 2 a n i m a l s only. A m o n g 6 A p o s t i v e pigs, 5 a n i m a l s c a r r i e d also t h e A s u b s t a n c e o n t h e i r e r y t h r o c y t e m e m b r a n e s . T h i s cellular A a c t i v i t y was f o u n d in t h e t o t a l lipids e x t r a c t e d f r o m t h e s t r o m a , while t h e n o n l i p i d residue was h i g h l y A a c t i v e in one case o n l y ; w e a k a c t i v ities were o b s e r v e d in t h e r e m a i n i n g 4 cases. T h e d i s t r i b u t i o n of A a c t i v i t y o n v a r i o u s f r a c t i o n s of s e r u m was s t u d i e d in a pig w h o s e s e r u m was A a c t i v e in b o t h t h e t o t a l lipids a n d tile n o n l i p i d residue o b t a i n e d a f t e r lipid e x t r a c t i o n of s e r u m . S e m i q u a n t i t a t i v e assays of A a c t i v i t y were p e r f o r m e d b y a g g l u t i n a t i o n - i n h i b i t i o n t e s t s a n d c a l c u l a t e d o n t h e b a s i s of t h e v o l u m e of o r i g i n a l s e r u m f r o m w h i c h t h e r e s p e c t i v e s a m p l e was d e r i v e d . Compared with the quantitative hemolysis-inhibition t e s t s of b o v i n e J a c t i v i t y d e s c r i b e d eralier ~, t h e a g g l u t i n a t i o n - i n h i b i t i o n t e s t s of p o r c i n e A a c t i v i t y gives r e l a t i v e ly r o u g h results. W e f o u n d a b o u t 2 0 % of t h e o r i g i n a l t o t a l s e r u m A a c t i v i t y t o b e p r e s e n t i n t h e t o t a l lipids, a n d 80~ in t h e n o n - l i p i d fraction.

80 0 0 0 80

T h e s e r u m was f r a c t i o n a t e d b y u l t r a c e n t r i f u g a l flotat i o n a t d i f f e r e n t d e n s i t i e s as d e s c r i b e d p r e v i o u s l y 1~ for b o v i n e serum. 4 f r a c t i o n s were o b t a i n e d b y t h i s p r o c e d u r e : V L D L , L D L , H D L , a n d l i p o p r o t e i n - f r e e residue. T h e s e f r a c t i o n s were c h a r a c t e r i z e d b y t h e i r p r o t e i n a n d lipid c o n t e n t s . T h e v a l u e s s h o w n in t h e T a b l e are in agreem e n t w i t h t h e d a t a o b t a i n e d in pig s e r u m b y JANADO e t al. 12 L i p i d s were e x t r a c t e d f r o m all f r a c t i o n s o b t a i n e d . L i p i d a n d n o n - l i p i d f r a c t i o n s were c h e c k e d for Ac a c t i v i t y . As s h o w n in t h e Table, no A a c t i v i t y was d e t e c t e d in t h e a p o p r o t e i n s of a n y l i p o p r o t e i n class, t h u s b e i n g in agreem e n t w i t h t h e d i s t r i b u t i o n of b o v i n e J a c t i v i t y . I n cont r a s t t o b o v i n e J a c t i v i t y , t h e m a j o r lipidic A a c t i v i t y ( r o u g h l y 50%) is c a r r i e d w i t h t h e H D L class of s e r u m , w h i l e t h e V L D L a n d L D L classes c o n t a i n r o u g h l y 2 5 % each. T h e a b o v e r e s u l t s show t h a t t h e d i s t r i b u t i o n of procine A a c t i v i t y on lipid a n d n o n l i p i d f r a c t i o n s of s e r u m a n d e r y t h r o c y t e m e m b r a n e s , a n d t h a t of t h e lipidic A a c t i v i t y o n v a r i o u s s e r u m l i p o p r o t e i n classes, is in s h a r p c o n t r a s t to t h e d i s t r i b u t i o n of b o v i n e J a c t i v i t y on t h e r e s p e c t i v e fractions.

11 J. SCHR6FFEL, O. W. THIELE and J. KocH, Eur. J. Biochem. 22, 294 (1971). 12 Iv[.JANADO,W. G. MARTINand W. H. COOK, Can. J. Biochem. dd, 1201 (1966).

Preparation of Specific Antiserum against R a n a e s c u l e n t a pre-cz Lens Crystallin S. K. BRAHMA

and Vv'. J. VAN

I)OORENMAALIZN

1

Department o/ Medical Anatomy and Embryology, The State University, Janskerkho/ 3A, Utrecht (The Netherlands), 26 Ja~uary 1976. Summary. Specific a n t i s e r u m a g a i n s t Rana esculenta lens pre-~ c r y s t a l l i n was p r e p a r e d in a r a b b i t b y i n j e c t i n g a n t i g e n - a n t i b o d y p r e c i p i t a t e of t h i s c r y s t a l l i n o b t a i n e d f r o m i m m u n o e l e c t r o p h o r e s i s of esculenta t o t a l soluble lens p r o t e i n s against homologous antiserum. M a j o r h e t e r o g e n e o u s soluble c r y s t a l l i n s f r o m v e r t e b r a t e lens are n o w classified i n t o ~-, 8-, Y-, a n d d-crys t a l l i n s ; VAN DAM 2 o b s e r v e d in b o v i n e lens a n o t h e r crys t a l l i n fraction, w h i c h m i g r a t e d f a s t e r t h a n e - c r y s t a l l i n d u r i n g e l e c t r o p h o r e s i s b u t was e l u t e d w i t h a p e a k w h i c h also c o n t a i n e d some/~-, a n d 7 - c r y s t a l l i n s d u r i n g gel filtrat i o n w i t h S e p h a d e x G 200. A c c o r d i n g to SWANBOR• 3, t h i s pre-~ f r a c t i o n is r e s t r i c t e d t o m a m m a l s only, b u t l a t e r t h i s f r a c t i o n was also f o u n d t o b e p r e s e n t in some a n u r a n a n d u r o d e l e a m p h i b i a n s 4-6.

BOURS a n d BRAHMA 7 r e p o r t e d t h a t t h e e l u t i o n profiles of t h e p r e - e c r y s t a l l i n f r o m b o v i n e a n d Rana esculenta lens were i d e n t i c a I in gel p e r m e a t i o n c h r o m a t o g r a p h y w i t h S e p h a r o s e 6 B . VAN DEN BROEK, LEGET a n d BLOEMENDAL s i s o l a t e d p r e - a c r y s t a l l i n f r o m b o v i n e lens b y c h r o m a t o g r a p h y a n d p r e p a r a t i v e isoelectric-focusing. They found that at an alkaline pH this fraction had r e l a t i v e l y h i g h m o b i l i t y a n d t h e m o l e c u l a r w e i g h t was a b o u t 14,500. A m i n o acid a n a l y s i s s h o w e d no r e l a t i o n s h i p w i t h t h e a-crystallin, so t h e y n a m e d t h i s f r a c t i o n as

15.7. 1976

Speeialia

F(ast) M ( i g r a t i n g ) or F M - c r y s t a l l i n to d i s t i n g u i s h it f r o m a n o t h e r r a t h e r h i g h m o l e c u l a r w e i g h t f r a c t i o n in c o w lens, w h i c h e m e r g e d in f r o n t of t h e c~-crystallin f r o m D E A E c o l u m n c h r o m a t o g r a p h y a n d w a s also called p r e - a ~. I n t h i s c o m m u n i c a t i o n , we u s e d t h e t e r m pre-er c r y s t a l lin t o d e s i g n a t e t h e f r a c t i o n w h i c h s h o w e d h i g h e s t m o b i l i t y d u r i n g e l e c t r o p h o r e s i s in a l k a l i n e buffer. Since n o t h i n g is k n o w n a b o u t t h i s f r a c t i o n in a m p h i b i a n lens e x c e p t i t s p r e s e n c e in s o m e species, we d e c i d e d t o p r e p a r e a n t i b o d i e s a g a i n s t t h i s c r y s t a l l i n f r a c t i o n to s t u d y its o n t o g e n y during lens development. To p r e p a r e a n t i b o d i e s , we i n j e c t e d a g a r gel a n t i g e n a n t i b o d y p r e c i p i t a t e into a r a b b i t . T h i s m e t h o d h a s b e e n s u c c e s s f u l l y u s e d b y o t h e r s w i t h d i f f e r e n t antigens*~ I t w a s n o t p o s s i b l e t o d e t e r m i n e t h e e x a c t a m o u n t of a n t i g e n i n j e c t e d i n t o "clue r a b b i t , b u t t h e r e a r e r e p o r t s s h o w i n g t h a t a few 100 tzg of a n t i g e n - a n t i b o d y c o m p l e x is s u f f i c i e n t t o p r o d u c e a n t i b o d y a0. I t h a s also b e e n s h o w n t h a t i m m u n i z a t i o n w i t h specific p r e c i p i t a t e in a g a r gel is a reliable m e t h o d for p r e p a r i n g s t r o n g a n d r e m a r k a b l y p u r e a n t i b o d y ~. 4 % l y o p h i l i z e d t o t a l s o l u b l e lens p r o t e i n s f r o m R a n a esculenta was tested a g a i n s t homologous a n t i s e r u m by m i c r o - i m m u n o e l e c t r o p h o r e s i s ~a w i t h h i g h r e s o l u t i o n b u f fer a t p H 8.9 ~. E l e c t r o p h o r e s i s w a s c a r r i e d o u t a t 4 ~ for 90 m i n w i t h a c o n s t a n t c u r r e n t of 40 m A . I m m u n o d i f f u s i o n w a s c o n t i n u e d o v e r n i g h t in a h u m i d c h a m b e r a t r o o m t e m p e r a t u r e (20 • 2~ A f t e r d i f f u s i o n , pre-r w a s obs e r v e d well s e p a r a t e d f r o m t h e ~.-crystallin a t t h e a n o d i c e n d of t h e gel ( F i g u r e A). A g a r b l o c k c o n t a i n i n g t h i s prec i p i t i n line w a s c a r e f u l l y c u t a w a y f r o m t h e gel a n d t h o r o u g h l y w a s h e d b y r e p e a t e d c h a n g e of saline for s e v e r a l d a y s o v e r a G y r o t o r y s h a k e r to r e m o v e n o n - p r e c i p i t a t e d p r o t e i n s . T h e b l o c k s were t h e n b r o c k e n u p in a h o m o g e n i z e r in 1 m l s a l i n e a n d e m u l s i f i e d w i t h 1 m l c o m p l e t e F r e u n d ' s a d j u v a n t (Difco). T h e c o m p l e x m i x t u r e w a s t h e n i n j e c t e d i n t r a d e r m a l l y a t 4 d i f f e r e n t sites o n t h e b a c k of a y o u n g r a b b i t . T h e i n j e c t i o n w a s r e p e a t e d 3 t i m e s a t 3 w e e k i n t e r v a l s . F o r e a c h i n j e c t i o n we u s e d 14 a n t i g e n - a n t i b o d y p r e c i p i t a t e s . 14 d a y s a f t e r t h e 3rd injection, the rabbit was bled from the ear vein a n d the s e r u m was tested against R. esculenla total soluble lens p r o t e i n b y i m m u n o e l e c t r o p h o r e s i s . T h e slides f r o m w h i c h t h e a n t i g e n - a n t i b o d y p r e c i p i t a t e s were u s e d were w a s h e d , d r i e d a n d s t a i n e d w i t h C o o m a s s i e B r i l l i a n t B l u e R - 2 5 0 ~v to c h e c k c o n t a m i n a t i o n .

931

T h e r a b b i t a n t i p r e - a a n t i s e r u m s h o w e d a single precip i t i n line a g a i n s t R . e s c u l e n t a t o t a l s o l u b l e lens p r o t e i n ( F i g u r e B) a n d did n o t s h o w a n y c h a n g e in its m o b i l i t y w h e n c o m p a r e d w i t h t h e pre-~ f r o m a n t i b o d i e s a g a i n s t R . e s c u l e n t a t o t a l s o l u b l e l e n s p r o t e i n s ( F i g u r e A). W e failed t o g e t a n y i m m u n o l o g i c a l r e a c t i o n w h e n t h i s a n t i pre-e a n t i s e r u m was tested a g a i n s t bovine lens b y OUCI~TERLONY'S ~s d o u b l e d i f f u s i o n e x p e r i m e n t . I t t h u s a p p e a r s t h a t t h e pre-c~ c r y s t a l l i n s f r o m a m p h i b i a n a n d b o v i n e l e n s are n o t a n t i g e n i c a l l y r e l a t e d . W o r k is in p r o g r e s s on t h e o n t o g e n y of t h e p r e - a crystallin by indirect immunofluorescence staining method.

1 Acknowledgments. We thank Mr. A. WACHTERfor the excellent technicaI help, Mr. Tm HULSKESand Mr. A. M. v. EOERAATfor the illustrations and finally Dr. W. E. HAWKINSfor valuable discussion. 2 A. F. VAN DA~, Biochim. biophys. Acta 12/, 183 (1966). a p. L. SWANBORN,Expl Eye Res. 5, 302 (1966). J. C. C2k~PBELL, R. M. CI~AYTONand D. E. S. TRIJMA>r, Expl Eye Res. 7, 4 (1968). a S. K. BRAHMAand W. J. VAN DOOREN~aAALEN,Expl Eye Res. 8, 168 (1969). 6 D. S. IVIcDEvI'rT and C. R. COLLIER, Expl Eye Res. 2/, 1 (1975). 7 3. Bol0~s and S. K. BRAHMA, Expl Eye IRes. 16, 131 (1973). 8 W. G. M. VAN DEN BROEK, J. N. LEOET and H. BLOEMENDAL, Biochim. biophys. Acts 370, 278 (1973). 9 j. A. JEDZANIAK,J. H. KINOSmTA, E. M. YAw~s, L. O. HOCKER and G. B. B~VEDiK, Invest. Ophthal. ~1,905 (1972). 10 H. SMITH, R. C. GALLOt~ and B. T. TOZER, Immunology 7, 111 (1964). 1~ R. B. GOUDtE, C. H. W. HORNE and P. C. WILKINSON, Lancet 2B, 1224 (1966}. 12 C. A. SHIVERS and J. M. JAMES, Immunology 13, 547 (1967). 1~ A. N. MALAVIYA,Indian J. ned. Res. 59, 1279 (1971). la p. NANSEN, T. I2LAGSTAD and K. B. PEI)ERSE~r Acta path. microbiol, scand. 79B, 459 (1971). 15 J. J. SCI-IIEDECrOER,Int. Arch. Allergy appl. Immun. 7, 103 (1955). ~6 T. ARONSSON and A. GRO~WALL, Scand. J. clin. Lab. Invest. 9, 338 (1957). zv B. W ~ K ~ , Scand. J. Immun. 2, suppl. 15 (1973}. is O. OUC~TERgO~Y, Acta path. microbiol, stand. 32, 231 (1953).

ImmunoeIeetrophoresisof R. esculenta total soluble lens proteins vs. A) homologous antiserum from R. esculent~ and B) antiserum to the antigen-antibody precipitate of the pre-~ crystallin from R. esculenta lens. Electrophoresis was carried out for 90 rain at 4~ using 1.5% Bacto agar in high resolution buffer at pH 8.9 with a constant current of 40 mA. The slides were stained with Coomassie Brilliant Blue R-250. + = anode; = cathode. The pre-c~ is indicated by the arrow'. In B) only the pre-~ line was visible when the antiserum was tested against R. esculenta total soluble lens proteins showing the specificity of the antiserum,