Int.
3.
Biochem., 1975, Vol. 6, pp. 43 to 46.
BULL SEMINAL R. STANEK, Institute
of Animal
Physiology 277
Pergamon Press.
VESICLE
Printed in Great Britain
RIBONUCLEASE
J. MATOUSEK
AND
AS
J. DOSTAL
and Genetics, The Czechoslovak 2 I Lib&hov, Czechoslovakia
(Received24
43
Academy
of Sciences,
June I 974)
ABSTRACT I. The identity of ribonuclease Vs,, ribonuclease BS-I and ribonuclease AS is presented. 2. The proposal for its further designation as ribonuclease AS is made. 3. The molecular weight of ribonuclease AS studied by gel filtration is 22,000 in its monomer form and 44,000 in its dimer form. 4. The elution patterns of ribonuclease AS by gel filtration differ considerably according to the nH of the solution used. The most critical pH region is between pH 5.5 and pH g. I with buffer solutions of about 0.1 M.
FLORIDI & D’ALESSIO (1967) have reported the occurrence of a pyrimidine-specific ribonuclease having a molecular weight of 24,000-26,000 in bull seminal plasma. The purification of bull semen ribonuclease (BS- I ) and its physico-chemical properties are presented by D’Alessio, Floridi, Deprisco, Pignero & Leone (1972). Hosokawa & Irie (1971) have purified ribonuclease Vs, of a very similar molecular weight (25,500) from bovine seminal vesicles. The purification of aspermatogenic substance in bull seminal vesicle fluid and some of its properties similar to those of ribonuclease BS- I and ribonuclease Vsl, are presented by Dostal & Matougek (I 972, 1973). Recently, Matougek, Pavlok, Dostdl & GrozdanoviE ( I 973) reported a high ribonuclease activity of aspermatogenic subHoly & GrozdanoviE (1972) classistance. fied aspermatogenic substance as a n-pyrimidine 2’nucleotidyltransferase (cyclizing), and called it ribonuclease AS. The aspermatogenic properties of bull seminal ribonuclease B&I comparable to those of ribonuclease AS were described by Leone, Greco, Rastogi & Iela (‘973). The aim of this contribution is to compare ribonuclease AS (B&I) with ribonuclease Vs, and to suggest a common designation for this enzyme. MATERIALS
AND METHODS
PREPARATION OF RIBONUCLEA~E The isolation
of ribonuclease
AS from bull (Bos
taurucfiontosw) seminal as described by Dostll Ribonuclease Vs, seminal vesicle tissue described by Hosokawa ASSAY
vesicle fluid was performed & MatouHek (1972). was prepared from bull according to the method & Irie (197 I).
OF RIBONUCLEASE ACTIVITY
Ribonuclease activity was measured spectrophotometrically on the basis of hydrolysis of yeast RNA by the enzyme. The reaction mixture contained I ml 0.1 M Tris-HCl buffer, pH 8.0, I mg. yeast RNA (Serva) and 50 ~1 0.004% enzyme solution. The reaction mixture was incubated at 37°C for 5 min after which 6-5 ml. of methanolglacial acetic acid (I 2 : I) were added to stop the reaction and to nrecioitate unhvdrolvzed RNA. After a further in’cuba’tion for ro’min ihe samples were filtered and the amounts of the product in the supernatant were measured spectrophotometrically at 260 nm. ELECTROPHORESIS The electrophoretic mobility of ribonuclease AS and ribonuclease Vs, was comoared bv disc electrophoresis in polyac$amide gel at pH 4.3 accordin2 to instructions nrovided bv the firm Canalco Industrial Corp., Md., U.S.A.: and by starch gel electrophoresis according to the method described by Aschaffenburg ( 1966). MOLECULAR WEIGHT DETERMINATION The molecular weight of isolated ribonucleases was checked by the gel filtration technique (Andrews, 1964) on Bio-Gel P-60 in o* I M sodium acetate buffer, pH 5.5, in 0. I M Tris-HCl, pH 7.6, in 0.1 M ammonium carbonate, pH 7.9, and in 0.05 M boric acid-o.05 M potassium .h‘;droxide0.025 M sodium hydroxide, nH q-1. As reference proteins, ovalbumin, chym&yp&ogen and ribonuclease A (Koch-Light Laboratories Ltd, U.K.) were used.
STAN’K
44 A~PERMATOGENICACTIVITY TEST
The test for aspermatogenic activity was described by Dostal & Matou3ek (1972). It involved the injection of 0.02 ml of each I Y ribonuclease solution in saline into the left testis‘bf male mice. The reaction was determined histologically IO days later. The right testis of each animal was used as a control. IMMUNOLOGICALTESTS Rabbit anti-bull seminal vesicle ribonuclease AS serum and rabbit anti-pancreatic ribonuclease A serum were prepared following three immunization series (4 injections of 0.5 ml. 0*5-x.0% ribonuclease solution in one series). The injections followed at T-day intervals and the immunization series were repeated at 5- to 8-month intervals. A week after the last immunization the rabbits were bled and the serum was collected. Two rabbits were subjected to immunization by ribonuclease AS and two rabbits by pancreatic ribonuclease A (Reanal) . Double diffusion tests were carried out on the dishes in I yOagar Difco gel in 0.2 M sodium borate buffer, pH 8.6. RESULTS
AND
DISCUSSION
Bull seminal vesicles cleaned of fat and connective tissues (0.475 kg.) were used for isolation of ribonuclease Vs, according to the method described by Hosokawa & Irie (I 97 I ). The final product of separation was compared with bull seminal vesicle fluid ribonuclease AS prepared by our method (DostPl & MatouSek, 1972) following studies of enzymatic activity, physico-chemical properties and antigenic structure of both enzymes. Both ribonuclease Vs, and ribonuclease AS showed comparable enzymatic activity based on hydrolysis of yeast RNA. Comparison of the enzymes was further made using disc electrophoresis in polyacrylamide gel at pH 4.3. The method revealed identical electrophoretic mobility of both enzymes migrating as single protein band either in separate samples or in a mixture. This observation was confirmed using starch gel electrophoresis with the addition of urea as denaturing agent. Both ribonucleases also migrated to the same position on the gel as a single band. The aspermatogenic activity of the enzymes was compared by the injection of 0.02 ml. of a I o/Osolution into the left testis of male mice. On the 10th day after application the
et d.
Int. J. Biochem.
injected testes were significantly smaller (1.58 _+0.38 mg. in comparison to 3.95 + 0.28 mg. in controls). The histological observations showed aspermatogenesis and a clear reduction in the diameter of the seminiferous tubules. Using double-diffusion technique for the comparison of the antigenic structure of both enzymes with rabbit anti-bull seminal vesicle ribonuclease AS serum resulted in the formation of identical precipitation reactions. No reaction was observed after absorption of antiserum either by ribonuclease AS isolated from bull seminal vesicle fluid according to the method of Dostal & Matougek (1972) or by ribonuclease Vs, prepared from bull seminal vesicle tissue according to the method of Hosokawa & Irie ( 197 I). The identity of the antigenic structure of both ribonucleases was confirmed using rabbit anti-pancreatic ribonuclease A serum. Both enzymes again formed identical precipitation arcs on the double-diffusion plates and both enzymes also absorbed antibodies to a similar degree. The results presented in this report prove the identity of ribonucleases isolated by different techniques from bull seminal vesicle fluid and tissue. On the basis of the results presented by Leone et aZ. ( I 973) these enzymes are identical with bovine seminal ribonuclease BS-I isolated by Floridi & D’Alessio (1g67), while Leone et al. (1973) have proved the identity of ribonuclease AS and ribonuclease BS-I. Holy & GrozdanovZ (1972) classified this ribonuclease as a o-pyrimidine 2’-nucleotidyltransferase (cyclizing) with the structural requirement for substrates identical to those of pancreatic ribonuclease A. The proposal is made to call bull seminal vesicle ribonuclease isolated from any source (seminal plasma, seminal vesicle tissue, seminal vesicle fluid) according to Holy & Grozdanovic (1972) as ribonuclease AS. This designation is the shortest and simplest of those used in any other report. Furthermore it indicates the identity of substrate requirement with pancreatic ribonuclease A as well as indicating the source of the enzyme in seminal vesicles. The letters ‘AS’ represent the specific aspermatogenic properties of this ribonuclease
* 9%
RIBONUCLEASE
6 Table I.-THE
MOLECULAR
WEIGHT
OF
AS
45
RIBONUCLEASE AS DETERMINED
BY
GEL FILTRATION ON SEPHADEX h%PERBNCE
Floridi & D’Alessio (1967) Hosokawa & Irie (197 I) D’Alessio et al. (1972) Dostal & MatouHek (1972, 1973)
pH OF BUFFER
24,ooo-26,000 25,500 28,ooo-3 1,000 44,000 44,000 2g,ooo
6.5 7’5
22,000 22,000 22,000 28,000-30,000
Leone et al. ( 1973)
unknown so far in any of the other ribonucleases described. There are some discrepancies concerning the determination of the molecular weight of ribonuclease AS by gel titration (Table I). To check them the gel filtration technique on Bio-Gel P-60 was used instead of that on Sephadex. Ribonuclease AS isolated by any technique behaves as protein of different molecular weight according to the pH of the buffers used for the elution. The results of these observations are summarized in Table II. It is evident that the elution pattern of ribonuclease AS is pH-dependent. The critical region appears to be between pH 5.5 and 9.1 in buffer solutions of about 0.1 M. The ribonuclease AS dissociates to the monomer at pH 9.1 and associates to the dimer at pH 5.5. Between pH 5.5 and pH 9.1 the buffers (approx. 0.1 M) used for the elution most probably cause incomplete dissociation and ribonuclease AS behaves as a protein with molecular weight of between 22,000 and 44,000 according to the degree of dissociation. Table II.-THE
MOLECULAR WEIGHT
USED FOR ELUTION
3’5 5’5 7.6 9”
10.5 5.5 with 8 M urea
This was not considered by Floridi & D’Alessio (rg67), Hosokawa & Irie (rg7r), D’Alessio et al. ( I 972) and Leone et al. ( 1973). Therefore they reported molecular weight determinations of ribonuclease AS which were different in different reports. The dissociation theory of ribonuclease AS has been supported by determination of its molecular weight in 8 M urea-o-r M sodium acetate buffer, pH 5.5, as reported by Dostal & Matougek (r 972, 1973). In such a denaturing solvent the elution pattern of ribonuclease AS corresponds to a molecular weight of 22,000 in comparison with 44,000 in 0.1 M sodium acetate buffer, pH 5.5, without addition of urea. The dimer form of ribonuclease AS seems to be held together by relatively weak forces which can easily be dissociated by a change of pH. We suppose that the effect of pH on the dissociation and association of ribonuclease AS may affect the results of molecular weight determination by other techniques also.
MOLECULAR WEIGHT OF RIBONUCLEASEAS, DETERMINEDBY GEL FILTRATION ON BIO-GEL P-60 BUFFER
0.1 M Sodium acetate
0. I M Ammonium carbonate 0. I M Tris-HCl 0.05 M Boric acid-o-05 M potassium hydroxide0’025 M sodium hydroxide
PH
MOLECULAR wEIGHT
5’5 7’9 7.6
44,000 32,000 28,000
9”
22,000
46
STAN& et al.
REFERENCES ANDREWS,P. (1g64), ‘ Estimation of the molecular weight of proteins by Sephadex gel filtration ‘, Biochem. J., gr, 222-223. AWXAFFENBURG, R. (rg66), ‘ Modified procedure of starch gel electrophoresis for beta-casein phenotyping ‘, 3. Dairy Sci., 49, 1284-1285. D’ALESSIO,G., FLORIDI,A., DEPRISCO, R., PIGNERO, A., & LEONE, E. (rg72), ‘ Bull semen ribonucleases. I. Purification and physico-chemical properties of the major component ‘, Eur. 3. Biochxm., 26, 153-161. DOSTAL,J., & MATOUSEK, J. (rg72), ‘ Purification of aspermatogenic substance in bull seminal vesicle fluid ‘, 3. Reprod. Fert., 31, 273-274. DOSTAL, J., & MATOUSEK, J. (IgTS), ‘ Isolation and some chemical properties of aspermatogenic substance from bull seminal vesicle fluid ‘, 3. Reprod. Fert., 33, 263-274. FLORIDI,A., & D’ALESSIO,G. (rg67), ‘ Compartamento cromatografico della ribonucleasi seminale ‘, Boll. Sot. Ital. Biol. SF., 4, 32-36.
Int.
3. Biochem.
HoL-?, A., & GROZDANOVIE, J. (rg72), ‘ The substrate specificity of bovine seminal vesicle fluid ribonuclease (Ribonuclease AS) ‘, Biochim. Biophys. Acta, 277, 556-563. HOSOKAWA,S., & IRIE, M. (1g71), ‘ Purification and properties of seminal vesicle ribonucleases ‘, 3. Biochem., Tokyo, 6g, 683-697. LEONE, E., G-CO, L., RA.WOOI,R. K., & IELA, L. (rg7S), ‘ Antispermatogenic properties of bull seminal ribonuclease ‘, 3. Reprod. Fert., 34, 1g7-200.
J., PAVLOK,A., DowhI,, J., & GROZJ. (rg7$, ‘ Some biological properties of bull seminal vestcle aspermatogenic substance and its effect on mice ‘, 3. Reprod. Fert., 34, g-22.
MATOUSEK, DANOLd,
Key Word In&x: Bos taurusfrontosus, ribonuclease Vs,, ribonuclease BS-I, ribonuclease AS, pancreatic ribonuclease A, n-pyrimidine 2’-nucleotidyltransferase (cyclizing) .
3.
Biochem., 1975, Vol. 6, pp. 43 to 46.
BULL SEMINAL R. STANEK, Institute
of Animal
Physiology 277
Pergamon Press.
VESICLE
Printed in Great Britain
RIBONUCLEASE
J. MATOUSEK
AND
AS
J. DOSTAL
and Genetics, The Czechoslovak 2 I Lib&hov, Czechoslovakia
(Received24
43
Academy
of Sciences,
June I 974)
ABSTRACT I. The identity of ribonuclease Vs,, ribonuclease BS-I and ribonuclease AS is presented. 2. The proposal for its further designation as ribonuclease AS is made. 3. The molecular weight of ribonuclease AS studied by gel filtration is 22,000 in its monomer form and 44,000 in its dimer form. 4. The elution patterns of ribonuclease AS by gel filtration differ considerably according to the nH of the solution used. The most critical pH region is between pH 5.5 and pH g. I with buffer solutions of about 0.1 M.
FLORIDI & D’ALESSIO (1967) have reported the occurrence of a pyrimidine-specific ribonuclease having a molecular weight of 24,000-26,000 in bull seminal plasma. The purification of bull semen ribonuclease (BS- I ) and its physico-chemical properties are presented by D’Alessio, Floridi, Deprisco, Pignero & Leone (1972). Hosokawa & Irie (1971) have purified ribonuclease Vs, of a very similar molecular weight (25,500) from bovine seminal vesicles. The purification of aspermatogenic substance in bull seminal vesicle fluid and some of its properties similar to those of ribonuclease BS- I and ribonuclease Vsl, are presented by Dostal & Matougek (I 972, 1973). Recently, Matougek, Pavlok, Dostdl & GrozdanoviE ( I 973) reported a high ribonuclease activity of aspermatogenic subHoly & GrozdanoviE (1972) classistance. fied aspermatogenic substance as a n-pyrimidine 2’nucleotidyltransferase (cyclizing), and called it ribonuclease AS. The aspermatogenic properties of bull seminal ribonuclease B&I comparable to those of ribonuclease AS were described by Leone, Greco, Rastogi & Iela (‘973). The aim of this contribution is to compare ribonuclease AS (B&I) with ribonuclease Vs, and to suggest a common designation for this enzyme. MATERIALS
AND METHODS
PREPARATION OF RIBONUCLEA~E The isolation
of ribonuclease
AS from bull (Bos
taurucfiontosw) seminal as described by Dostll Ribonuclease Vs, seminal vesicle tissue described by Hosokawa ASSAY
vesicle fluid was performed & MatouHek (1972). was prepared from bull according to the method & Irie (197 I).
OF RIBONUCLEASE ACTIVITY
Ribonuclease activity was measured spectrophotometrically on the basis of hydrolysis of yeast RNA by the enzyme. The reaction mixture contained I ml 0.1 M Tris-HCl buffer, pH 8.0, I mg. yeast RNA (Serva) and 50 ~1 0.004% enzyme solution. The reaction mixture was incubated at 37°C for 5 min after which 6-5 ml. of methanolglacial acetic acid (I 2 : I) were added to stop the reaction and to nrecioitate unhvdrolvzed RNA. After a further in’cuba’tion for ro’min ihe samples were filtered and the amounts of the product in the supernatant were measured spectrophotometrically at 260 nm. ELECTROPHORESIS The electrophoretic mobility of ribonuclease AS and ribonuclease Vs, was comoared bv disc electrophoresis in polyac$amide gel at pH 4.3 accordin2 to instructions nrovided bv the firm Canalco Industrial Corp., Md., U.S.A.: and by starch gel electrophoresis according to the method described by Aschaffenburg ( 1966). MOLECULAR WEIGHT DETERMINATION The molecular weight of isolated ribonucleases was checked by the gel filtration technique (Andrews, 1964) on Bio-Gel P-60 in o* I M sodium acetate buffer, pH 5.5, in 0. I M Tris-HCl, pH 7.6, in 0.1 M ammonium carbonate, pH 7.9, and in 0.05 M boric acid-o.05 M potassium .h‘;droxide0.025 M sodium hydroxide, nH q-1. As reference proteins, ovalbumin, chym&yp&ogen and ribonuclease A (Koch-Light Laboratories Ltd, U.K.) were used.
STAN’K
44 A~PERMATOGENICACTIVITY TEST
The test for aspermatogenic activity was described by Dostal & Matou3ek (1972). It involved the injection of 0.02 ml of each I Y ribonuclease solution in saline into the left testis‘bf male mice. The reaction was determined histologically IO days later. The right testis of each animal was used as a control. IMMUNOLOGICALTESTS Rabbit anti-bull seminal vesicle ribonuclease AS serum and rabbit anti-pancreatic ribonuclease A serum were prepared following three immunization series (4 injections of 0.5 ml. 0*5-x.0% ribonuclease solution in one series). The injections followed at T-day intervals and the immunization series were repeated at 5- to 8-month intervals. A week after the last immunization the rabbits were bled and the serum was collected. Two rabbits were subjected to immunization by ribonuclease AS and two rabbits by pancreatic ribonuclease A (Reanal) . Double diffusion tests were carried out on the dishes in I yOagar Difco gel in 0.2 M sodium borate buffer, pH 8.6. RESULTS
AND
DISCUSSION
Bull seminal vesicles cleaned of fat and connective tissues (0.475 kg.) were used for isolation of ribonuclease Vs, according to the method described by Hosokawa & Irie (I 97 I ). The final product of separation was compared with bull seminal vesicle fluid ribonuclease AS prepared by our method (DostPl & MatouSek, 1972) following studies of enzymatic activity, physico-chemical properties and antigenic structure of both enzymes. Both ribonuclease Vs, and ribonuclease AS showed comparable enzymatic activity based on hydrolysis of yeast RNA. Comparison of the enzymes was further made using disc electrophoresis in polyacrylamide gel at pH 4.3. The method revealed identical electrophoretic mobility of both enzymes migrating as single protein band either in separate samples or in a mixture. This observation was confirmed using starch gel electrophoresis with the addition of urea as denaturing agent. Both ribonucleases also migrated to the same position on the gel as a single band. The aspermatogenic activity of the enzymes was compared by the injection of 0.02 ml. of a I o/Osolution into the left testis of male mice. On the 10th day after application the
et d.
Int. J. Biochem.
injected testes were significantly smaller (1.58 _+0.38 mg. in comparison to 3.95 + 0.28 mg. in controls). The histological observations showed aspermatogenesis and a clear reduction in the diameter of the seminiferous tubules. Using double-diffusion technique for the comparison of the antigenic structure of both enzymes with rabbit anti-bull seminal vesicle ribonuclease AS serum resulted in the formation of identical precipitation reactions. No reaction was observed after absorption of antiserum either by ribonuclease AS isolated from bull seminal vesicle fluid according to the method of Dostal & Matougek (1972) or by ribonuclease Vs, prepared from bull seminal vesicle tissue according to the method of Hosokawa & Irie ( 197 I). The identity of the antigenic structure of both ribonucleases was confirmed using rabbit anti-pancreatic ribonuclease A serum. Both enzymes again formed identical precipitation arcs on the double-diffusion plates and both enzymes also absorbed antibodies to a similar degree. The results presented in this report prove the identity of ribonucleases isolated by different techniques from bull seminal vesicle fluid and tissue. On the basis of the results presented by Leone et aZ. ( I 973) these enzymes are identical with bovine seminal ribonuclease BS-I isolated by Floridi & D’Alessio (1g67), while Leone et al. (1973) have proved the identity of ribonuclease AS and ribonuclease BS-I. Holy & GrozdanovZ (1972) classified this ribonuclease as a o-pyrimidine 2’-nucleotidyltransferase (cyclizing) with the structural requirement for substrates identical to those of pancreatic ribonuclease A. The proposal is made to call bull seminal vesicle ribonuclease isolated from any source (seminal plasma, seminal vesicle tissue, seminal vesicle fluid) according to Holy & Grozdanovic (1972) as ribonuclease AS. This designation is the shortest and simplest of those used in any other report. Furthermore it indicates the identity of substrate requirement with pancreatic ribonuclease A as well as indicating the source of the enzyme in seminal vesicles. The letters ‘AS’ represent the specific aspermatogenic properties of this ribonuclease
* 9%
RIBONUCLEASE
6 Table I.-THE
MOLECULAR
WEIGHT
OF
AS
45
RIBONUCLEASE AS DETERMINED
BY
GEL FILTRATION ON SEPHADEX h%PERBNCE
Floridi & D’Alessio (1967) Hosokawa & Irie (197 I) D’Alessio et al. (1972) Dostal & MatouHek (1972, 1973)
pH OF BUFFER
24,ooo-26,000 25,500 28,ooo-3 1,000 44,000 44,000 2g,ooo
6.5 7’5
22,000 22,000 22,000 28,000-30,000
Leone et al. ( 1973)
unknown so far in any of the other ribonucleases described. There are some discrepancies concerning the determination of the molecular weight of ribonuclease AS by gel titration (Table I). To check them the gel filtration technique on Bio-Gel P-60 was used instead of that on Sephadex. Ribonuclease AS isolated by any technique behaves as protein of different molecular weight according to the pH of the buffers used for the elution. The results of these observations are summarized in Table II. It is evident that the elution pattern of ribonuclease AS is pH-dependent. The critical region appears to be between pH 5.5 and 9.1 in buffer solutions of about 0.1 M. The ribonuclease AS dissociates to the monomer at pH 9.1 and associates to the dimer at pH 5.5. Between pH 5.5 and pH 9.1 the buffers (approx. 0.1 M) used for the elution most probably cause incomplete dissociation and ribonuclease AS behaves as a protein with molecular weight of between 22,000 and 44,000 according to the degree of dissociation. Table II.-THE
MOLECULAR WEIGHT
USED FOR ELUTION
3’5 5’5 7.6 9”
10.5 5.5 with 8 M urea
This was not considered by Floridi & D’Alessio (rg67), Hosokawa & Irie (rg7r), D’Alessio et al. ( I 972) and Leone et al. ( 1973). Therefore they reported molecular weight determinations of ribonuclease AS which were different in different reports. The dissociation theory of ribonuclease AS has been supported by determination of its molecular weight in 8 M urea-o-r M sodium acetate buffer, pH 5.5, as reported by Dostal & Matougek (r 972, 1973). In such a denaturing solvent the elution pattern of ribonuclease AS corresponds to a molecular weight of 22,000 in comparison with 44,000 in 0.1 M sodium acetate buffer, pH 5.5, without addition of urea. The dimer form of ribonuclease AS seems to be held together by relatively weak forces which can easily be dissociated by a change of pH. We suppose that the effect of pH on the dissociation and association of ribonuclease AS may affect the results of molecular weight determination by other techniques also.
MOLECULAR WEIGHT OF RIBONUCLEASEAS, DETERMINEDBY GEL FILTRATION ON BIO-GEL P-60 BUFFER
0.1 M Sodium acetate
0. I M Ammonium carbonate 0. I M Tris-HCl 0.05 M Boric acid-o-05 M potassium hydroxide0’025 M sodium hydroxide
PH
MOLECULAR wEIGHT
5’5 7’9 7.6
44,000 32,000 28,000
9”
22,000
46
STAN& et al.
REFERENCES ANDREWS,P. (1g64), ‘ Estimation of the molecular weight of proteins by Sephadex gel filtration ‘, Biochem. J., gr, 222-223. AWXAFFENBURG, R. (rg66), ‘ Modified procedure of starch gel electrophoresis for beta-casein phenotyping ‘, 3. Dairy Sci., 49, 1284-1285. D’ALESSIO,G., FLORIDI,A., DEPRISCO, R., PIGNERO, A., & LEONE, E. (rg72), ‘ Bull semen ribonucleases. I. Purification and physico-chemical properties of the major component ‘, Eur. 3. Biochxm., 26, 153-161. DOSTAL,J., & MATOUSEK, J. (rg72), ‘ Purification of aspermatogenic substance in bull seminal vesicle fluid ‘, 3. Reprod. Fert., 31, 273-274. DOSTAL, J., & MATOUSEK, J. (IgTS), ‘ Isolation and some chemical properties of aspermatogenic substance from bull seminal vesicle fluid ‘, 3. Reprod. Fert., 33, 263-274. FLORIDI,A., & D’ALESSIO,G. (rg67), ‘ Compartamento cromatografico della ribonucleasi seminale ‘, Boll. Sot. Ital. Biol. SF., 4, 32-36.
Int.
3. Biochem.
HoL-?, A., & GROZDANOVIE, J. (rg72), ‘ The substrate specificity of bovine seminal vesicle fluid ribonuclease (Ribonuclease AS) ‘, Biochim. Biophys. Acta, 277, 556-563. HOSOKAWA,S., & IRIE, M. (1g71), ‘ Purification and properties of seminal vesicle ribonucleases ‘, 3. Biochem., Tokyo, 6g, 683-697. LEONE, E., G-CO, L., RA.WOOI,R. K., & IELA, L. (rg7S), ‘ Antispermatogenic properties of bull seminal ribonuclease ‘, 3. Reprod. Fert., 34, 1g7-200.
J., PAVLOK,A., DowhI,, J., & GROZJ. (rg7$, ‘ Some biological properties of bull seminal vestcle aspermatogenic substance and its effect on mice ‘, 3. Reprod. Fert., 34, g-22.
MATOUSEK, DANOLd,
Key Word In&x: Bos taurusfrontosus, ribonuclease Vs,, ribonuclease BS-I, ribonuclease AS, pancreatic ribonuclease A, n-pyrimidine 2’-nucleotidyltransferase (cyclizing) .
