RAPID COMMUNICATION
ZIPGRAM
EVIDENCE FOR THE DISSIMILARITY OF ACROSIN AND PANCREATIC TRYPSIN AS REVEALED BY A SENSITIVE IMMUNOASSAY
GERALD J. ALLEN (1) Department of Animal Husbandry, University of Bristol , Langford House, Langford, B r i s t o l , BS18 7DU, England ABSTRACT Common antigenic determinants between two species of pancreatic trypsin and twelve species of acrosin were studied using a highly s e n s i t i v e , enzyme-1 inked immunoassay. No common antigenic determinants between trypsin and acrosin could be detected. Acrosin has been shown t o share many c h a r a c t e r i s t i c s of bovine pancreatic trypsin (Zaneveld e t a l . , '72) including s i m i l a r s u b s t r a t e s p e c i f i c i t i e s , pH optima and i n h i b i t o r spectra.
Like trypsin, acrosin s p e c i f i c a l l y cleaves bonds
with lysine and arginine (Polakoski, '73; Polakoski and McRorie, '73) and i s i n h i b i t e d by DFP and TLCK which i s indicative of s e r i n e and h i s t i d i n e residues
respectively in the a c t i v e s i t e (Shaw, '70). L i t t l e , however, has been published concerning the antigenic nature o f acrosin.
Zaneveld e t a l . ( ' 7 3 ) demonstrated t h a t human acrosin and pancreatic
trypsin a r e immunologically d i s s i m i l a r .
Similarly, Garner e t a l . ( ' 7 5 ) found
no cross-reaction between bovine acrosin and pancreatic trypsin while Morton ( ' 7 5 , ' 7 6 ) could find no cross-reacting, p r e c i p i t a t i n g antibodies between bovine pancreatic trypsin and highly purified acrosin from bull ram and boar. In c o n t r a s t , Stambaugh and Smith ( ' 7 4 ) claim t o have demonstrated common a n t i genic determinants between acrosins of r a b b i t , bull and ram and bovine pancrea t i c trypsin. However, a l l of these workers have used immunodiffusion a s an immunoassay
185
which i s r e l a t i v e l y i n s e n s i t i v e a s compared w i t h complement f i x a t i o n , passive haemagglutination, radioimmunoassay, e t c . , needing the formation of a large antibody-antigen complex before the reaction can be visualized. I t was therefore decided t o assay f o r common antigenic determinants between two types of pancreatic trypsin and acrosin from twelve species of mammalian spermatozoa using a highly s e n s i t i v e immunoassay; t h a t of labelled a n t i body fragments t o pancreatic trypsin coupled w i t h microscopic observations. MATERIALS AND METHODS Antibodies t o bovine pancreatic trypsin were raised in rabbits by weekly intramuscular i n j e c t i o n s of 5 mg. of bovine pancreatic trypsin (Sigma, Type XII) dissolved in 2.5 ml of phosphate buffered s a l i n e pH 7.4 (PBS) and emulsified in 2.5 ml of Freunds Complete Adjuvant (Difco). Antibodies t o pig pancreatic trypsin were raised in a similar manner in sheep by weekly intramuscular i n j e c t i o n s of 10 mg of hog pancreatic trypsin (Sigma, Type IX). When the presence of antibodies was confirmed by p r e c i p i t i n l i n e formation in immunodiffusion (Ouchterlony, '58), blood was collected from the animals a t slaughter o r by venipuncture from l i v i n g animals.
The blood was allowed t o
c l o t a t room temperature and then the c l o t was shrunk by storage a t 4' C f o r 3-4 hr.
Finally, the c l o t was removed from the serum by centrifugation a t
2,500 g f o r 20 min. The IgG component of the antiserum was isolated by ion-exchange chromatography using diethylaminoethyl ce lulose (DEAE, Whatman) , the IgG being eluted with 0.02 M phosphate buffer pH 7.4.
Contaminating IgM was removed by
gel f i l t r a t i o n on Sephadex 6200 equ l i b r a t e d w i t h 0.1M Tris-HC1 buffer pH 7.4 containing 1M NaC1. The s p e c i f i c a n t i - t r y p s i n antibodies were then isolated by a f f i n i t y chromatography according t o the method of Porath e t a l . ( ' 6 7 ) a s modified by Newby
186
e t a l . ( ' 7 4 ) . The ligand was e i t h e r hog o r bovine pancreatic trypsin a s appropriate, and s p e c i f i c IgG antibodies were euluted by 3.5 M KSCn pH 7.4. Univalent Fab fragments of the s p e c i f i c IgG molecules were then prepared according t o the method of Porter ( ' 5 8 , '59) using papain digestion. This method was modified i n t h a t 0.1M N-ethylmaleimide was added t o stop the reaction i n stead o f d i a l y s i s against water.
Undigested IgG was removed by Sephadex 6100,
and the Fab and Fc fragments were then separated using carboxymethyl c e l l u l o s e (CMC, Whatman) ion-exchange chromatography in a c e t a t e buffer pH 5.5 with a
l i n e a r buffer gradient from 0.01 t o 0.9 M.
Divalent Fab was produced by pepsin
digestion using the method described by Williams and Chase ( ' 6 7 ) .
The s p e c i f i c Fab fragments were then labelled with horse-radish peroxidase (HRP, Sigma, Type VI) using the method of Avrameas ( ' 6 9 ) . These conjugates were then used t o assay f o r common antigenic determinants between themselves and a v a r i e t y of species of acrosin.
Bull, boar, ram, horse,
dog, r a b b i t and human spermatozoa were collected by e j a c u l a t i o n , while r a b b i t , r a t , mouse, guinea p i g , gerbil and chimpanzee spermatozoa were collected by reverse flushing of the vas deferens immediately a f t e r slaughter.
Spermatozoa
were washed three times i n Hanks balanced s a l t solution (Hanks BSS, Flow) by centrifugation a t 1,500 g f o r 7 mins.and allowed t o a i r dry on acetone-cleaned microscope s l i d e s .
The spermatozoa smears were then fixed i n absolute ethanol
f o r 30 mins. and stored a t -20'
C u n t i l use.
The fixed spermatozoa smears were then stained w i t h t h e HRP-antibody frag-
ment conjugates f o r 30 mins. a s f o r immunocytochemical techniques using the method of Avrameas ( ' 6 9 ) followed by 3-3' diaminobenzidene tetrahydrochloride
(DAB, Sigma).
Smears were observed using a Leitz Ortholux microscope.
The
presence of brown deposits over the acrosome region was a demonstration of the presence of antigen-antibody complexes, and thus of common antigenic deter187
minants between bovine o r hog pancreatic trypsin and the species of acrosin examined.
The use of such an immunoassay has several advantages over pre-
viously employed techniques.
I t allows the use of univalent Fab fragments,
thus overcoming any c r i t i c i s m t h a t interaction between antigen and antibody i s of a non-antigen s p e c i f i c type such a s through the Fc fragment which has been shown t o occur (Allen, '76a).
Thirdly, this technique allows investigation
of the antigenic determinant of acrosin while the enzyme i s s t i l l i n situ w i t h i n the sperm head, overcoming c r i t i c i s m t h a t the e x t r a c t i v e procedures
necessary f o r other immunoassays may a1 t e r the a n t i g e n i c i t y of acrosin. RESULTS AND DISCUSSION The r e s u l t s of the use of this technique show t h a t there i s no interaction between labelled antibody fragments t o bovine o r ovine pancreatic trypsin and acrosomal enzymes of guinea p i g , mouse, r a t , g e r b i l , r a b b i t , b u l l , boar, ram, dog, horse, chimpanzee o r man, indicating a lack of common antigenic determinants.
I t could be argued t h a t the negative
r e s u l t s of t h i s study a r e due t o other f a c t o r s , such a s f a i l u r e of HRP-labelled Fab fragments t o penetrate the acrosome, o r t h a t the spermatozoa1 preparation procedures caused acrosomal disruption w i t h a consequent loss of acrosomal contents.
However, other immunocytochemical observations indicate these a r e
unlikely.
The observations of Morton ( ' 7 5 ) t h a t HRP-labelled Fab fragments
t o ovine acrosin and hyaluronidase will form an immune complex w i t h the respective enzymes within the sperm head, and the similar observations f o r ovine and bovine hyaluronidase (Allen, '76b) suggest t h a t i t i s not the i n a b i l i t y of the labelled immunoglobulin fragments t o penetrate t h a t accounts f o r the negative r e s u l t s .
Similarly, since spermatozoa t r e a t e d in an identical manner
t o those i n this study will s t a i n immunochemically f o r hyaluronidase (Allen, '76b) and f o r immunoglobulin receptor sites on the acrosome (Allen, '76a), i t seems unlikely t h a t the washing procedures used caused acrosomal disruption 188
o r loss of acrosomal contents. I t m i g h t be argued t h a t antibodies t o trypsin may cross-react w i t h acrosin b u t not with i t s zymogen, proacrosin, which has been shown t o be present i n
epididymal (Meizel and Huang-Yang, '73) and ejaculated spermatozoa (Schleuning e t a l . , '76).
Thus i f a l l the acrosin was present a s the inactive precursor,
no interaction w i t h antibody fragments t o trypsin would occur.
However, the
r e s u l t s of Morton ( ' 7 5 ) show t h i s not t o be the case a s antibodies t o highly a c t i v e acrosin preparations interacted with the enzyme in ejaculated spermatozoa, indicating t h a t e i t h e r a c t i v e acrosin is present o r t h a t acrosin and proacrosin share common antigenic determinants. I t therefore appears t h a t pancreatic trypsin and acrosin a r e immunol o g i c a l l y d i s s i m i l a r , thus confirming and considerably extending the findings of Zaneveld e t a l . ( ' 7 3 ) , Garner e t a l . ( ' 7 5 ) and Morton ( ' 7 6 ) .
These findings
c o n f l i c t with those of Stambaugh and Smith ( ' 7 4 ) who found common antigenic determinants between bovine pancreatic trypsin and acrosin extracted from r a b b i t , bull and ram. matic a c t i v i t y ,
However, since these authors f a i l e d t o s t a i n f o r enzy-
there i s no d i r e c t evidence t h a t the antibodies i n the pre-
c i p i t i n l i n e s they observed were cross-reacting w i t h acrosin; i t i s possible t h a t they were due t o a contaminant. I t therefore appears t h a t although trypsin and acrosin share many chara c t e r i s t i c s , and can be considered t o have s i m i l a r a c t i v e sites, the remainder of the molecule has considerable differences in i t s antigenic nature
Thus
this enzyme probably deserves the unique name of acrosin.
LITERATURE CITED Allen, G. J. 1976a Interaction of immunoglobulins w i t h the mammalian acrosome, in preparation. Allen, G. J. 1976b Studies on the sperm acrosome. Ph.D. t h e s i s , Bristol University.
189
Avrameas, S. 1969 Coupling of enzymes t o p r o t e i n s w i t h glutaraldehyde. Use of the conjugates f o r the d e t e c t i o n of antigens and a n t i b o d i e s . Immunochemistry, 6: 43-52. Garner. D. L . , M. P. Easton. M. E. Munson. and M. A. Doane 1975 Immunofluorescent l o c a l i z a t i o n of bovine acrosin. J . Exp. Zool. , 191: 127-132. Meizel, S . , Y. H.J. Huang-Yang 1973 An i n a c t i v e form of a t r y p s i n l i k e enzyme i n r a b b i t testes and epididymal sperm. Anat. Rec. 175: 387-388. Morton, D. B. 1975 Acrosomal enzymes: immunochemical l o c a l i z a t i o n of acrosin and hyaluronidase i n r a t spermatozoa. J . Reprod. Fert., 45: 375-378. Morton, D. B. 1976 Lysosomal enzymes i n mammalian spermatozoa. In: Lysosomes i n Biology and Pathology. J. T. Dingle and R. T. Dean, eds. North Holland. Newby, T. J . , F. J . Bourne, J. W. Chidlow, M. J. S t e e l 1974 A simple method f o r the preparation of antiserum t o bovine immunoglobul i n s using immunosorbents. Res. Vet. S c i . , 17: 39-41. Ouchterlony, 0. 1958 Diffusion-in-qel methods f o r immunochemical a n a l y s i s . In: Progress i n Allergy. Vol. V. P. Kallos, ed. Karger and Basel, New York. pp. 1-78. Polakoski, K. L. 1973 P r o p e r t i e s and functions of enzymes and i n h i b i t o r s involved i n mammalian f e r t i l i z a t i o n . Diss. Abstr. I n t . , 33B: 5138-5139. Polakoski, K. L. and R. A. McRorie 1973 Boar acrosin 2. C l a s s i f i c a t i o n , i n h i b i t i o n and s p e c i f i c i t y s t u d i e s of a proteinase from sperm acrosomes. J . Biol. Chem. , 248: 8183-8188. Porath, J . , R. Axen, S. Ernback 1967 Chemical coupling of p r o t e i n s t o agarose. Nature, (Lond.) 215: 1491. P o r t e r , R. R. 1958 Separation and i s o l a t i o n of f r a c t i o n s of r a b b i t gammaglobul i n containing the antibody and a n t i g e n i c binding sites. Nature, (Lond. ) 182: 670-671. P o r t e r , R. R. 1959 The hydrolysis of r a b b i t gamma-globulin and a n t i bodies w i t h c r y s t a l l i n e papain. Bichem. J . , 73: 119-126. Schleuning, W. D., R. Hell, H. F r i t z 1976 Multiple forms of human acrosin: i s o l a t i o n and p r o p e r t i e s . Hoppe-Seyler's Z. physiol. Chem. , 357: 855-865. Shaw, E. 1970 S e l e c t i v e chemical modification o f p r o t e i n s . Physiol. Rev., 50: 244-296. Stambaugh, R . , M. Smith 1974 Amino acid content o f r a b b i t acrosomal proteinase and i t s s i m i l a r i t y t o human t r y p s i n . Science, 186: 745-746. Williams, C. A., M. W. Chase 1967 Methods i n immunology and immunochemistry. Vol. 1. Academic Press, New York and London, p . 422. Zaneveld, L. J. D . , K. L. Polakoski, and W. L. Williams 1972 Prope r t i e s of a p r o t e o l y t i c enzyme from r a b b i t sperm acrosomes. Biol. Reprod., 6: 30-39. Zaneveld, L. J. D., G. F. B. Schumacher, J. Travis 1973 Human sperm acrosomal proteinase: antibody i n h i b i t i o n and immunologic dissimi l a r i t y t o human pancreatic t r y p s i n . F e r t i l . S t e r i l . , 24: 479-484. REFERENCES
1 Present address: Research Dept. , G.D. S e a r l e and Co. , Ltd. , P.O. Box 53, Lane End Road, High Wycombe, Bucks., England
ZIPGRAM
EVIDENCE FOR THE DISSIMILARITY OF ACROSIN AND PANCREATIC TRYPSIN AS REVEALED BY A SENSITIVE IMMUNOASSAY
GERALD J. ALLEN (1) Department of Animal Husbandry, University of Bristol , Langford House, Langford, B r i s t o l , BS18 7DU, England ABSTRACT Common antigenic determinants between two species of pancreatic trypsin and twelve species of acrosin were studied using a highly s e n s i t i v e , enzyme-1 inked immunoassay. No common antigenic determinants between trypsin and acrosin could be detected. Acrosin has been shown t o share many c h a r a c t e r i s t i c s of bovine pancreatic trypsin (Zaneveld e t a l . , '72) including s i m i l a r s u b s t r a t e s p e c i f i c i t i e s , pH optima and i n h i b i t o r spectra.
Like trypsin, acrosin s p e c i f i c a l l y cleaves bonds
with lysine and arginine (Polakoski, '73; Polakoski and McRorie, '73) and i s i n h i b i t e d by DFP and TLCK which i s indicative of s e r i n e and h i s t i d i n e residues
respectively in the a c t i v e s i t e (Shaw, '70). L i t t l e , however, has been published concerning the antigenic nature o f acrosin.
Zaneveld e t a l . ( ' 7 3 ) demonstrated t h a t human acrosin and pancreatic
trypsin a r e immunologically d i s s i m i l a r .
Similarly, Garner e t a l . ( ' 7 5 ) found
no cross-reaction between bovine acrosin and pancreatic trypsin while Morton ( ' 7 5 , ' 7 6 ) could find no cross-reacting, p r e c i p i t a t i n g antibodies between bovine pancreatic trypsin and highly purified acrosin from bull ram and boar. In c o n t r a s t , Stambaugh and Smith ( ' 7 4 ) claim t o have demonstrated common a n t i genic determinants between acrosins of r a b b i t , bull and ram and bovine pancrea t i c trypsin. However, a l l of these workers have used immunodiffusion a s an immunoassay
185
which i s r e l a t i v e l y i n s e n s i t i v e a s compared w i t h complement f i x a t i o n , passive haemagglutination, radioimmunoassay, e t c . , needing the formation of a large antibody-antigen complex before the reaction can be visualized. I t was therefore decided t o assay f o r common antigenic determinants between two types of pancreatic trypsin and acrosin from twelve species of mammalian spermatozoa using a highly s e n s i t i v e immunoassay; t h a t of labelled a n t i body fragments t o pancreatic trypsin coupled w i t h microscopic observations. MATERIALS AND METHODS Antibodies t o bovine pancreatic trypsin were raised in rabbits by weekly intramuscular i n j e c t i o n s of 5 mg. of bovine pancreatic trypsin (Sigma, Type XII) dissolved in 2.5 ml of phosphate buffered s a l i n e pH 7.4 (PBS) and emulsified in 2.5 ml of Freunds Complete Adjuvant (Difco). Antibodies t o pig pancreatic trypsin were raised in a similar manner in sheep by weekly intramuscular i n j e c t i o n s of 10 mg of hog pancreatic trypsin (Sigma, Type IX). When the presence of antibodies was confirmed by p r e c i p i t i n l i n e formation in immunodiffusion (Ouchterlony, '58), blood was collected from the animals a t slaughter o r by venipuncture from l i v i n g animals.
The blood was allowed t o
c l o t a t room temperature and then the c l o t was shrunk by storage a t 4' C f o r 3-4 hr.
Finally, the c l o t was removed from the serum by centrifugation a t
2,500 g f o r 20 min. The IgG component of the antiserum was isolated by ion-exchange chromatography using diethylaminoethyl ce lulose (DEAE, Whatman) , the IgG being eluted with 0.02 M phosphate buffer pH 7.4.
Contaminating IgM was removed by
gel f i l t r a t i o n on Sephadex 6200 equ l i b r a t e d w i t h 0.1M Tris-HC1 buffer pH 7.4 containing 1M NaC1. The s p e c i f i c a n t i - t r y p s i n antibodies were then isolated by a f f i n i t y chromatography according t o the method of Porath e t a l . ( ' 6 7 ) a s modified by Newby
186
e t a l . ( ' 7 4 ) . The ligand was e i t h e r hog o r bovine pancreatic trypsin a s appropriate, and s p e c i f i c IgG antibodies were euluted by 3.5 M KSCn pH 7.4. Univalent Fab fragments of the s p e c i f i c IgG molecules were then prepared according t o the method of Porter ( ' 5 8 , '59) using papain digestion. This method was modified i n t h a t 0.1M N-ethylmaleimide was added t o stop the reaction i n stead o f d i a l y s i s against water.
Undigested IgG was removed by Sephadex 6100,
and the Fab and Fc fragments were then separated using carboxymethyl c e l l u l o s e (CMC, Whatman) ion-exchange chromatography in a c e t a t e buffer pH 5.5 with a
l i n e a r buffer gradient from 0.01 t o 0.9 M.
Divalent Fab was produced by pepsin
digestion using the method described by Williams and Chase ( ' 6 7 ) .
The s p e c i f i c Fab fragments were then labelled with horse-radish peroxidase (HRP, Sigma, Type VI) using the method of Avrameas ( ' 6 9 ) . These conjugates were then used t o assay f o r common antigenic determinants between themselves and a v a r i e t y of species of acrosin.
Bull, boar, ram, horse,
dog, r a b b i t and human spermatozoa were collected by e j a c u l a t i o n , while r a b b i t , r a t , mouse, guinea p i g , gerbil and chimpanzee spermatozoa were collected by reverse flushing of the vas deferens immediately a f t e r slaughter.
Spermatozoa
were washed three times i n Hanks balanced s a l t solution (Hanks BSS, Flow) by centrifugation a t 1,500 g f o r 7 mins.and allowed t o a i r dry on acetone-cleaned microscope s l i d e s .
The spermatozoa smears were then fixed i n absolute ethanol
f o r 30 mins. and stored a t -20'
C u n t i l use.
The fixed spermatozoa smears were then stained w i t h t h e HRP-antibody frag-
ment conjugates f o r 30 mins. a s f o r immunocytochemical techniques using the method of Avrameas ( ' 6 9 ) followed by 3-3' diaminobenzidene tetrahydrochloride
(DAB, Sigma).
Smears were observed using a Leitz Ortholux microscope.
The
presence of brown deposits over the acrosome region was a demonstration of the presence of antigen-antibody complexes, and thus of common antigenic deter187
minants between bovine o r hog pancreatic trypsin and the species of acrosin examined.
The use of such an immunoassay has several advantages over pre-
viously employed techniques.
I t allows the use of univalent Fab fragments,
thus overcoming any c r i t i c i s m t h a t interaction between antigen and antibody i s of a non-antigen s p e c i f i c type such a s through the Fc fragment which has been shown t o occur (Allen, '76a).
Thirdly, this technique allows investigation
of the antigenic determinant of acrosin while the enzyme i s s t i l l i n situ w i t h i n the sperm head, overcoming c r i t i c i s m t h a t the e x t r a c t i v e procedures
necessary f o r other immunoassays may a1 t e r the a n t i g e n i c i t y of acrosin. RESULTS AND DISCUSSION The r e s u l t s of the use of this technique show t h a t there i s no interaction between labelled antibody fragments t o bovine o r ovine pancreatic trypsin and acrosomal enzymes of guinea p i g , mouse, r a t , g e r b i l , r a b b i t , b u l l , boar, ram, dog, horse, chimpanzee o r man, indicating a lack of common antigenic determinants.
I t could be argued t h a t the negative
r e s u l t s of t h i s study a r e due t o other f a c t o r s , such a s f a i l u r e of HRP-labelled Fab fragments t o penetrate the acrosome, o r t h a t the spermatozoa1 preparation procedures caused acrosomal disruption w i t h a consequent loss of acrosomal contents.
However, other immunocytochemical observations indicate these a r e
unlikely.
The observations of Morton ( ' 7 5 ) t h a t HRP-labelled Fab fragments
t o ovine acrosin and hyaluronidase will form an immune complex w i t h the respective enzymes within the sperm head, and the similar observations f o r ovine and bovine hyaluronidase (Allen, '76b) suggest t h a t i t i s not the i n a b i l i t y of the labelled immunoglobulin fragments t o penetrate t h a t accounts f o r the negative r e s u l t s .
Similarly, since spermatozoa t r e a t e d in an identical manner
t o those i n this study will s t a i n immunochemically f o r hyaluronidase (Allen, '76b) and f o r immunoglobulin receptor sites on the acrosome (Allen, '76a), i t seems unlikely t h a t the washing procedures used caused acrosomal disruption 188
o r loss of acrosomal contents. I t m i g h t be argued t h a t antibodies t o trypsin may cross-react w i t h acrosin b u t not with i t s zymogen, proacrosin, which has been shown t o be present i n
epididymal (Meizel and Huang-Yang, '73) and ejaculated spermatozoa (Schleuning e t a l . , '76).
Thus i f a l l the acrosin was present a s the inactive precursor,
no interaction w i t h antibody fragments t o trypsin would occur.
However, the
r e s u l t s of Morton ( ' 7 5 ) show t h i s not t o be the case a s antibodies t o highly a c t i v e acrosin preparations interacted with the enzyme in ejaculated spermatozoa, indicating t h a t e i t h e r a c t i v e acrosin is present o r t h a t acrosin and proacrosin share common antigenic determinants. I t therefore appears t h a t pancreatic trypsin and acrosin a r e immunol o g i c a l l y d i s s i m i l a r , thus confirming and considerably extending the findings of Zaneveld e t a l . ( ' 7 3 ) , Garner e t a l . ( ' 7 5 ) and Morton ( ' 7 6 ) .
These findings
c o n f l i c t with those of Stambaugh and Smith ( ' 7 4 ) who found common antigenic determinants between bovine pancreatic trypsin and acrosin extracted from r a b b i t , bull and ram. matic a c t i v i t y ,
However, since these authors f a i l e d t o s t a i n f o r enzy-
there i s no d i r e c t evidence t h a t the antibodies i n the pre-
c i p i t i n l i n e s they observed were cross-reacting w i t h acrosin; i t i s possible t h a t they were due t o a contaminant. I t therefore appears t h a t although trypsin and acrosin share many chara c t e r i s t i c s , and can be considered t o have s i m i l a r a c t i v e sites, the remainder of the molecule has considerable differences in i t s antigenic nature
Thus
this enzyme probably deserves the unique name of acrosin.
LITERATURE CITED Allen, G. J. 1976a Interaction of immunoglobulins w i t h the mammalian acrosome, in preparation. Allen, G. J. 1976b Studies on the sperm acrosome. Ph.D. t h e s i s , Bristol University.
189
Avrameas, S. 1969 Coupling of enzymes t o p r o t e i n s w i t h glutaraldehyde. Use of the conjugates f o r the d e t e c t i o n of antigens and a n t i b o d i e s . Immunochemistry, 6: 43-52. Garner. D. L . , M. P. Easton. M. E. Munson. and M. A. Doane 1975 Immunofluorescent l o c a l i z a t i o n of bovine acrosin. J . Exp. Zool. , 191: 127-132. Meizel, S . , Y. H.J. Huang-Yang 1973 An i n a c t i v e form of a t r y p s i n l i k e enzyme i n r a b b i t testes and epididymal sperm. Anat. Rec. 175: 387-388. Morton, D. B. 1975 Acrosomal enzymes: immunochemical l o c a l i z a t i o n of acrosin and hyaluronidase i n r a t spermatozoa. J . Reprod. Fert., 45: 375-378. Morton, D. B. 1976 Lysosomal enzymes i n mammalian spermatozoa. In: Lysosomes i n Biology and Pathology. J. T. Dingle and R. T. Dean, eds. North Holland. Newby, T. J . , F. J . Bourne, J. W. Chidlow, M. J. S t e e l 1974 A simple method f o r the preparation of antiserum t o bovine immunoglobul i n s using immunosorbents. Res. Vet. S c i . , 17: 39-41. Ouchterlony, 0. 1958 Diffusion-in-qel methods f o r immunochemical a n a l y s i s . In: Progress i n Allergy. Vol. V. P. Kallos, ed. Karger and Basel, New York. pp. 1-78. Polakoski, K. L. 1973 P r o p e r t i e s and functions of enzymes and i n h i b i t o r s involved i n mammalian f e r t i l i z a t i o n . Diss. Abstr. I n t . , 33B: 5138-5139. Polakoski, K. L. and R. A. McRorie 1973 Boar acrosin 2. C l a s s i f i c a t i o n , i n h i b i t i o n and s p e c i f i c i t y s t u d i e s of a proteinase from sperm acrosomes. J . Biol. Chem. , 248: 8183-8188. Porath, J . , R. Axen, S. Ernback 1967 Chemical coupling of p r o t e i n s t o agarose. Nature, (Lond.) 215: 1491. P o r t e r , R. R. 1958 Separation and i s o l a t i o n of f r a c t i o n s of r a b b i t gammaglobul i n containing the antibody and a n t i g e n i c binding sites. Nature, (Lond. ) 182: 670-671. P o r t e r , R. R. 1959 The hydrolysis of r a b b i t gamma-globulin and a n t i bodies w i t h c r y s t a l l i n e papain. Bichem. J . , 73: 119-126. Schleuning, W. D., R. Hell, H. F r i t z 1976 Multiple forms of human acrosin: i s o l a t i o n and p r o p e r t i e s . Hoppe-Seyler's Z. physiol. Chem. , 357: 855-865. Shaw, E. 1970 S e l e c t i v e chemical modification o f p r o t e i n s . Physiol. Rev., 50: 244-296. Stambaugh, R . , M. Smith 1974 Amino acid content o f r a b b i t acrosomal proteinase and i t s s i m i l a r i t y t o human t r y p s i n . Science, 186: 745-746. Williams, C. A., M. W. Chase 1967 Methods i n immunology and immunochemistry. Vol. 1. Academic Press, New York and London, p . 422. Zaneveld, L. J. D . , K. L. Polakoski, and W. L. Williams 1972 Prope r t i e s of a p r o t e o l y t i c enzyme from r a b b i t sperm acrosomes. Biol. Reprod., 6: 30-39. Zaneveld, L. J. D., G. F. B. Schumacher, J. Travis 1973 Human sperm acrosomal proteinase: antibody i n h i b i t i o n and immunologic dissimi l a r i t y t o human pancreatic t r y p s i n . F e r t i l . S t e r i l . , 24: 479-484. REFERENCES
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