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Membrane of Mammary Gland. II. 5′-Nucleotidase Activity of Bovine Mammary Plasma Membranes
TECHNICAL
NOTES
Membranes of Mammary Gland. II. 5"-Nucleotidase Activity of Bovine Mammary Membranes Plasma Abstract
The 5'-nucleotidase activity of a plasmamembrane rich fraction from bovine mammary gland was characterized. I n the absence of added metal ions a broad plateau of maximum activity was observed between p H 7.8 and 9.8. I n the presence of 5 m~ Mg ÷+, the enzyme was activated and displayed p H optima at 7.8 and 9.5. Michaelis "constants were 4.0 and 1.1 m ~ for eytidine-5"-monophosphate and adenosine-5"-monophosphate ( A M P ) . W i t h A M P as substrate the activabion energy was 10,300 cal/mole. A t 5 m ~ relative rates of hydrolysis of 5"-monophosphates were cytidine ~ uridine ~ inosine ~ guanosine ~ adenosine ~ deoxyguanosine xanthine ~ deoxycytidine. The membrane fraction also contained large amounts of adenosine triphosphatase as well as some nonspecific phosphatase activity.
Introduction
The enzyme 5"-nncleotidase (5'-ribonucleotidase phosphohydrolase, EC 3.1.3.5) has been identified and studied in a number of animal and microbial tissues and cells. This enzyme has recently been shown to be present nearly exclusively in the plasma membrane of cells and it has been widely used as a marker enzyme for identification of plasma membranes in cell fractionation studies (2, 5, 6, 15). Patton and Trams (13) recently reported detecbion of 5"-nucleotidase activity in milk fat globule membranes and t:Iuang and I(eenan (8) isolated and characterized the 5'-nucleotidases of fat globule membranes. Because of its utility as a marker enzyme, it was desirable to characterize the 5"-nucleotidase activity of plasma membrane fraction from bovine mammary gland. Experimental Procedures
Mammary tissue, obtained from lactating Holstein cows at slaughter, was held on ice during transportation to the laboratory, where subsequent operations were performed at 0 to 4 C. The tissue was trimmed, minced, and Received for publication July 21, 1971.
rinsed with .25 ~ sucrose and homogenized ill .25 M sucrose (4 ml/g) with a Polytron homogenizer (Brinkman, Westbury, New York). A smooth microsomal fraction was obtained from this homogenate as described by Dallner et al. (4) except that 1.35 M sucrose replaced 1.3 ~r sucrose in the density gradient. The smooth mierosomal fraction was collected, suspended in .05 ~ Tris-HC1, p i t 7.5, and used as the enzyme source. 5'-Nucleotidase was assayed as described by Emmelot et al. (6) under conditions of linear kinetics for enzyme concentration and reaction time. The standard reaction mixture conrained 5 m ~ MgC12, 5 m ~ Tris-HC1, p i t 7.8, and 5 mx~ of the appropriate substrate in a final volume of 2 ml. Incubations were at 37 C with agitation and reactions were terminated by addition of 2 ml of 10% trichloroacetic acid. The isolated fraction was also assayed for glucose-6-phosphatase (17) and succinic reductase (14) activities. Inorganic phosphate was determined by the method of Fiske and Subbarow (7). Protein was measured with the Folin phenol reagent (12) with crystalline bovine serum albumin as standard. Substrates were obtained from Sigma, St. Louis, Missouri. Results and Discussion
The average 5"-nucleotidase specific activity of smooth microsome fractions was approximately 25 t~moles of inorganic phosphate liberated hour milligram protein with A M P as substrate. This represented a 10- to 15fold enrichment relative ot the total homogenate. The glucose-6-phosphatase activity of this fraction was approximately .6 times that of the homogenate, indicating the presence of only small amounts of endoplasmic reticulum. Mitochondrial contamination was negligible as judged by low levels of succinate reductase activity. When examined in the electron microscope, the fraction was enriched in plasma membrane. Other smooth membranes, presumably derived from the smooth endoplasmie retieulum-Golgi complex were also present. Since only low levels of 5'-nucleotidase are present in mammary gland endoplasmic reticulum and Golgi membranes (9), the 5'nuclcotidase activity of this fraction can be assumed to be localized in the plasma membrane.
862
JOURNAL
OF
TABLE 1. Substrate specificity of bovine mammary plasma membranes, a
Substrate b
100.0 69.7 62.0 59.6 52.4 16.8 34.7 16.4 13.3 5.3 52.9 17.8 0.5
a Reaction mixtures contained 5 m ~ substrate, 5 m ~ MgC12, 50 m ~ Tris-HC1, p i t 7.8, and constant amounts of enzyme protein in a final volume of 2.0 ml. b Abbreviations are: MP, the 5'-monophosphates of bhe ribonucleosides of adenine, A; cytosine, C; guanine, G; uracil, U, inosine, I ; and xanthine, X. d Refers to the 2-deoxyribose isomer.
When tested with various substrates at 5 m~, the highest rate of hydrolysis was observed with CMP (abbreviations in Table 1). Other nucleotide 5"-monophosphates actively hydrolyzed included UMP, IMP, GMP, and AMP. Xanthine 5'-monophosphate as well as the 2" and 3" mixed monophosphates of guanosine and cytosine were hydrolyzed to only a limited extent. Some activity was also observed with the 2-deoxyribose analogs of CMP and GMP. Adenosine triphosphatase activity was also present in the active fraeVion (Table 1). This activity is characteristic of plasma membranes (6). fl-Glycerophosphate was also hydrolyzed to a limited degree, indicating the presence of some nonspecific phosphatase activity in this fraction. Figure 1 shows the effect of p H on bhe activity of plasma membrane 5'-nueleotidase. I n the absence of added metal ions a broad plateau of maximum activity was observed between p H 7.8 and 9.8. In the presence of 5 m~ Mg ÷+, the enzyme was activated and displayed two distinct p H optima, a~ 7.8 and
863
SCIENCE
• 5mM
~L E
~
Relative acbivity
(%) C:MP UMP IMP GMP AMP XMP dCMP dGMP Cytidine 2' + 3' monophosphate Guanosine 2" + 3' mGnophosphate Adenosine triphosphate fl-Glycerophosphate Glneose-6-phosphate
DAIRY
0.16
Mg CL 2
oN
I-- 0 . 1 2 Z o 0.08 .J (9 ~. 0.04 n 0
I
I
I
I
I
I
5
6
7
8
9
I0
pH
II
Fro. 1. Effect of pH on the activity of bovine mammary plasma membrane 5'-nucleotidase. The reaction mixture contained 5 rn~ AMP and 5 m~ Tris buffer. 9.5. This double p H optimum profile closely resembles those observed for the 5'-nucleotidases of human liver (16), rat liver plasma membranes (15), and bull seminal plasma (10). Similar profiles were observed with 5"-nucleotidases purified from fat globule membranes (8). Levin and Bodansky (10) have presented a detailed theoretical explanation to account for bhis double p H optimum phenomenon. Straight-line relationships were obtained when reciprocal reaction velocity was plotted against reciprocal substrate concentration according to Lineweaver and Burk (11). Apparent Michaelis constants (Kin) were 4.0 and 1.1 m ~ for CiVIP and A_~IP. The corresponding maximum velocities were 16.5 and 2].5 ~M of inorganic phosphate liberated per hour milligram protein. These Kra values were similar to those for fat globule membrane 5"nucleotidases (8). However, these Km values are higher, by more than an order of magnitude, than those for 5"-nucleotidases from other mammalian sources (3, 10, ]5, 16, 18). The reason for this great difference in substrate binding ability comparing the bovine mammary gland enzyme to those from other sources is not apparent. The energy of activation was calculated between 28 and 69 C, with AMP as substrate in reaction mixtures containing 5 mM MgC12, in accordance with the Arrhenius equation (1). The value obtained was 10,300 cal/mole. This is in the range of values reported for 5'nucleotidases from fat globule membranes (8) and bull seminal plasma (10). Results obtained in this study demonstrate that bovine mammary gland plasma membranes contain true 5'-nucleotidase activity. Further, JOUR::AL OF DAIRY SCIENCE ~OL. 55, NO. 6
864
TECHNICAL NOTES
these results c a n serve as a g u i d e f o r m o n i t o r i n g s u b c e l l u l a r fraetionabions b y a s s a y f o r 5 ~nucleotidase. W e consider t h e overall similarities between the p l a s m a m e m b r a n e a n d fat globule m e m b r a n e 5"-nueleotidases as f u r t h e r evidence f o r direct d e r i v a t i o n o f the f a t globule membrane from plasma membrane.
Acknowledgements
(7) (8)
(9)
Supported in part by a g r a n t from the National Science Foundation (GB-25110). Purdue University AES Journal P a p e r 4483.
¢. M. HUANG and T. W. KEENAN, Department of Animal Sciences, Purdue University,
(10)
Lafayette, Indiana 47907
References (1) Bodansky, O. 1939. The energy of activation of the hydrolysis of sodium fl-glycerophosphate by bone phosphatase at optimal pH. J. Biol. Chem., 129: 197. (2) Bosmann, H. B., and G. Z. Pike. 1971. Membrane marker enzymes : Isolation, purification, and properties of 5'-nueleotidase from r a t cerebellum. Biochim. Biophys. Acta, 227: 402. (3) Cheetham, R. D., D. James MorrO, and W. N. ¥unghans. 1970. Isolation of a Golgi apparatus-rich fraction from r a t liver. II. Enzymatic characterization and comparison with other cell fractions. J. Cell Biol., 44: 492. (4) Dallner, G., P. Siekevitz, and G. E. Palade. 1966. Biogenesis of endoplasmic reticuIum membranes. Structural and chemical differentiation in developing r a t hepatoeyte. J. Cell Biol., 30: 73. (5) Emmelot, P., and C. J. Bos. 1966. Studies on plasma membranes. I l L Mg+*-ATPase, (Na+-K+-Mg÷÷)-ATPase and 5'-nueleotidase activity of plasma membranes isolated from r a t liver. Biochim. Biophys. Acta, 120:369. (6) Emmelot, P., C. J. Bos, E. L. Benedetti, and P. Rumke. 1964. Studies on plasma membranes. I. Chemical composition and enzyme content of plasma membranes
JOURNAL OF DAIRY SCIENCE VOL. 55, NO. 6
(11) (12)
(13)
(14)
(15)
(16) (17) (18)
isolated from r a t liver. Bioehim. Biophys. Acta, 90: 126. Fiske, C. H., and Y. Subbarow. 1925. The colorimetrie determination of phosphorus. J. Biol. Chem., 66: 375. Huang, C. M., and T. W. Keenan. 1971. Characterization of 5'-nu¢leotidase of milk f a t globule membrane. Abstr. J. Dairy Sci., 54 : 770. Keenan, T. W., C. M. Huang, and D. J. Morr& 1971. Membranes of mammary gland. I I I . Lipid composition of Golgi apparatus from r a t mammary gland. J. Dairy Sci., 55: 51. Levin, S. J., and O. Bodansky. 1966. The double p H optimum of 5"-nucleotidase of bull seminal plasma. J. Biol. Chem., 241: 51. Lineweaver, H., and D. Burk. 1934. The determination of enzyme dissociation constants. J. Amer. Chem. Soe., 56: 658. Lowry, O. H., N. J. Rosebrough, A~ L. F a r r , and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265. Patton, S., and E. G. Trams. 1971. The presence of plasma membrane enzymes on the surface of bovine milk f a t globules. F E B S Letters, 14: 230. Pennington, R. J. 1961. Biochemistry of dystrophic muscle. Mitochondrial suceinatetetrazolium reductase and adenosine triphosphatase. Biochem. J., 80: 649. Song, C. S., a n d O. Bodansky. 1967. Subeelluiar localization and properties of 5'nueleotidase in the rat liver. J. Biol. Chem., 242 : 694. Song, C. S., and O. Bodansky. 1966. Puri~cation of 5'-nucleotidase from human liver. Bioehem: J., 101: 5c. Swanson, M. A. 1955. Glucose-6-phosphatase from liver. Methods Enzymol., 2:541. Widnell, C. C., and J. C. Unkeless. 1968. P a r t i a l purification of a lipoproteln with 5'-nueleotidase activity from membranes of r a t liver cells. Proc. Nat. Acad. Sei., 61 : 1050.
NOTES
Membranes of Mammary Gland. II. 5"-Nucleotidase Activity of Bovine Mammary Membranes Plasma Abstract
The 5'-nucleotidase activity of a plasmamembrane rich fraction from bovine mammary gland was characterized. I n the absence of added metal ions a broad plateau of maximum activity was observed between p H 7.8 and 9.8. I n the presence of 5 m~ Mg ÷+, the enzyme was activated and displayed p H optima at 7.8 and 9.5. Michaelis "constants were 4.0 and 1.1 m ~ for eytidine-5"-monophosphate and adenosine-5"-monophosphate ( A M P ) . W i t h A M P as substrate the activabion energy was 10,300 cal/mole. A t 5 m ~ relative rates of hydrolysis of 5"-monophosphates were cytidine ~ uridine ~ inosine ~ guanosine ~ adenosine ~ deoxyguanosine xanthine ~ deoxycytidine. The membrane fraction also contained large amounts of adenosine triphosphatase as well as some nonspecific phosphatase activity.
Introduction
The enzyme 5"-nncleotidase (5'-ribonucleotidase phosphohydrolase, EC 3.1.3.5) has been identified and studied in a number of animal and microbial tissues and cells. This enzyme has recently been shown to be present nearly exclusively in the plasma membrane of cells and it has been widely used as a marker enzyme for identification of plasma membranes in cell fractionation studies (2, 5, 6, 15). Patton and Trams (13) recently reported detecbion of 5"-nucleotidase activity in milk fat globule membranes and t:Iuang and I(eenan (8) isolated and characterized the 5'-nucleotidases of fat globule membranes. Because of its utility as a marker enzyme, it was desirable to characterize the 5"-nucleotidase activity of plasma membrane fraction from bovine mammary gland. Experimental Procedures
Mammary tissue, obtained from lactating Holstein cows at slaughter, was held on ice during transportation to the laboratory, where subsequent operations were performed at 0 to 4 C. The tissue was trimmed, minced, and Received for publication July 21, 1971.
rinsed with .25 ~ sucrose and homogenized ill .25 M sucrose (4 ml/g) with a Polytron homogenizer (Brinkman, Westbury, New York). A smooth microsomal fraction was obtained from this homogenate as described by Dallner et al. (4) except that 1.35 M sucrose replaced 1.3 ~r sucrose in the density gradient. The smooth mierosomal fraction was collected, suspended in .05 ~ Tris-HC1, p i t 7.5, and used as the enzyme source. 5'-Nucleotidase was assayed as described by Emmelot et al. (6) under conditions of linear kinetics for enzyme concentration and reaction time. The standard reaction mixture conrained 5 m ~ MgC12, 5 m ~ Tris-HC1, p i t 7.8, and 5 mx~ of the appropriate substrate in a final volume of 2 ml. Incubations were at 37 C with agitation and reactions were terminated by addition of 2 ml of 10% trichloroacetic acid. The isolated fraction was also assayed for glucose-6-phosphatase (17) and succinic reductase (14) activities. Inorganic phosphate was determined by the method of Fiske and Subbarow (7). Protein was measured with the Folin phenol reagent (12) with crystalline bovine serum albumin as standard. Substrates were obtained from Sigma, St. Louis, Missouri. Results and Discussion
The average 5"-nucleotidase specific activity of smooth microsome fractions was approximately 25 t~moles of inorganic phosphate liberated hour milligram protein with A M P as substrate. This represented a 10- to 15fold enrichment relative ot the total homogenate. The glucose-6-phosphatase activity of this fraction was approximately .6 times that of the homogenate, indicating the presence of only small amounts of endoplasmic reticulum. Mitochondrial contamination was negligible as judged by low levels of succinate reductase activity. When examined in the electron microscope, the fraction was enriched in plasma membrane. Other smooth membranes, presumably derived from the smooth endoplasmie retieulum-Golgi complex were also present. Since only low levels of 5'-nucleotidase are present in mammary gland endoplasmic reticulum and Golgi membranes (9), the 5'nuclcotidase activity of this fraction can be assumed to be localized in the plasma membrane.
862
JOURNAL
OF
TABLE 1. Substrate specificity of bovine mammary plasma membranes, a
Substrate b
100.0 69.7 62.0 59.6 52.4 16.8 34.7 16.4 13.3 5.3 52.9 17.8 0.5
a Reaction mixtures contained 5 m ~ substrate, 5 m ~ MgC12, 50 m ~ Tris-HC1, p i t 7.8, and constant amounts of enzyme protein in a final volume of 2.0 ml. b Abbreviations are: MP, the 5'-monophosphates of bhe ribonucleosides of adenine, A; cytosine, C; guanine, G; uracil, U, inosine, I ; and xanthine, X. d Refers to the 2-deoxyribose isomer.
When tested with various substrates at 5 m~, the highest rate of hydrolysis was observed with CMP (abbreviations in Table 1). Other nucleotide 5"-monophosphates actively hydrolyzed included UMP, IMP, GMP, and AMP. Xanthine 5'-monophosphate as well as the 2" and 3" mixed monophosphates of guanosine and cytosine were hydrolyzed to only a limited extent. Some activity was also observed with the 2-deoxyribose analogs of CMP and GMP. Adenosine triphosphatase activity was also present in the active fraeVion (Table 1). This activity is characteristic of plasma membranes (6). fl-Glycerophosphate was also hydrolyzed to a limited degree, indicating the presence of some nonspecific phosphatase activity in this fraction. Figure 1 shows the effect of p H on bhe activity of plasma membrane 5'-nueleotidase. I n the absence of added metal ions a broad plateau of maximum activity was observed between p H 7.8 and 9.8. In the presence of 5 m~ Mg ÷+, the enzyme was activated and displayed two distinct p H optima, a~ 7.8 and
863
SCIENCE
• 5mM
~L E
~
Relative acbivity
(%) C:MP UMP IMP GMP AMP XMP dCMP dGMP Cytidine 2' + 3' monophosphate Guanosine 2" + 3' mGnophosphate Adenosine triphosphate fl-Glycerophosphate Glneose-6-phosphate
DAIRY
0.16
Mg CL 2
oN
I-- 0 . 1 2 Z o 0.08 .J (9 ~. 0.04 n 0
I
I
I
I
I
I
5
6
7
8
9
I0
pH
II
Fro. 1. Effect of pH on the activity of bovine mammary plasma membrane 5'-nucleotidase. The reaction mixture contained 5 rn~ AMP and 5 m~ Tris buffer. 9.5. This double p H optimum profile closely resembles those observed for the 5'-nucleotidases of human liver (16), rat liver plasma membranes (15), and bull seminal plasma (10). Similar profiles were observed with 5"-nucleotidases purified from fat globule membranes (8). Levin and Bodansky (10) have presented a detailed theoretical explanation to account for bhis double p H optimum phenomenon. Straight-line relationships were obtained when reciprocal reaction velocity was plotted against reciprocal substrate concentration according to Lineweaver and Burk (11). Apparent Michaelis constants (Kin) were 4.0 and 1.1 m ~ for CiVIP and A_~IP. The corresponding maximum velocities were 16.5 and 2].5 ~M of inorganic phosphate liberated per hour milligram protein. These Kra values were similar to those for fat globule membrane 5"nucleotidases (8). However, these Km values are higher, by more than an order of magnitude, than those for 5"-nucleotidases from other mammalian sources (3, 10, ]5, 16, 18). The reason for this great difference in substrate binding ability comparing the bovine mammary gland enzyme to those from other sources is not apparent. The energy of activation was calculated between 28 and 69 C, with AMP as substrate in reaction mixtures containing 5 mM MgC12, in accordance with the Arrhenius equation (1). The value obtained was 10,300 cal/mole. This is in the range of values reported for 5'nucleotidases from fat globule membranes (8) and bull seminal plasma (10). Results obtained in this study demonstrate that bovine mammary gland plasma membranes contain true 5'-nucleotidase activity. Further, JOUR::AL OF DAIRY SCIENCE ~OL. 55, NO. 6
864
TECHNICAL NOTES
these results c a n serve as a g u i d e f o r m o n i t o r i n g s u b c e l l u l a r fraetionabions b y a s s a y f o r 5 ~nucleotidase. W e consider t h e overall similarities between the p l a s m a m e m b r a n e a n d fat globule m e m b r a n e 5"-nueleotidases as f u r t h e r evidence f o r direct d e r i v a t i o n o f the f a t globule membrane from plasma membrane.
Acknowledgements
(7) (8)
(9)
Supported in part by a g r a n t from the National Science Foundation (GB-25110). Purdue University AES Journal P a p e r 4483.
¢. M. HUANG and T. W. KEENAN, Department of Animal Sciences, Purdue University,
(10)
Lafayette, Indiana 47907
References (1) Bodansky, O. 1939. The energy of activation of the hydrolysis of sodium fl-glycerophosphate by bone phosphatase at optimal pH. J. Biol. Chem., 129: 197. (2) Bosmann, H. B., and G. Z. Pike. 1971. Membrane marker enzymes : Isolation, purification, and properties of 5'-nueleotidase from r a t cerebellum. Biochim. Biophys. Acta, 227: 402. (3) Cheetham, R. D., D. James MorrO, and W. N. ¥unghans. 1970. Isolation of a Golgi apparatus-rich fraction from r a t liver. II. Enzymatic characterization and comparison with other cell fractions. J. Cell Biol., 44: 492. (4) Dallner, G., P. Siekevitz, and G. E. Palade. 1966. Biogenesis of endoplasmic reticuIum membranes. Structural and chemical differentiation in developing r a t hepatoeyte. J. Cell Biol., 30: 73. (5) Emmelot, P., and C. J. Bos. 1966. Studies on plasma membranes. I l L Mg+*-ATPase, (Na+-K+-Mg÷÷)-ATPase and 5'-nueleotidase activity of plasma membranes isolated from r a t liver. Biochim. Biophys. Acta, 120:369. (6) Emmelot, P., C. J. Bos, E. L. Benedetti, and P. Rumke. 1964. Studies on plasma membranes. I. Chemical composition and enzyme content of plasma membranes
JOURNAL OF DAIRY SCIENCE VOL. 55, NO. 6
(11) (12)
(13)
(14)
(15)
(16) (17) (18)
isolated from r a t liver. Bioehim. Biophys. Acta, 90: 126. Fiske, C. H., and Y. Subbarow. 1925. The colorimetrie determination of phosphorus. J. Biol. Chem., 66: 375. Huang, C. M., and T. W. Keenan. 1971. Characterization of 5'-nu¢leotidase of milk f a t globule membrane. Abstr. J. Dairy Sci., 54 : 770. Keenan, T. W., C. M. Huang, and D. J. Morr& 1971. Membranes of mammary gland. I I I . Lipid composition of Golgi apparatus from r a t mammary gland. J. Dairy Sci., 55: 51. Levin, S. J., and O. Bodansky. 1966. The double p H optimum of 5"-nucleotidase of bull seminal plasma. J. Biol. Chem., 241: 51. Lineweaver, H., and D. Burk. 1934. The determination of enzyme dissociation constants. J. Amer. Chem. Soe., 56: 658. Lowry, O. H., N. J. Rosebrough, A~ L. F a r r , and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265. Patton, S., and E. G. Trams. 1971. The presence of plasma membrane enzymes on the surface of bovine milk f a t globules. F E B S Letters, 14: 230. Pennington, R. J. 1961. Biochemistry of dystrophic muscle. Mitochondrial suceinatetetrazolium reductase and adenosine triphosphatase. Biochem. J., 80: 649. Song, C. S., a n d O. Bodansky. 1967. Subeelluiar localization and properties of 5'nueleotidase in the rat liver. J. Biol. Chem., 242 : 694. Song, C. S., and O. Bodansky. 1966. Puri~cation of 5'-nucleotidase from human liver. Bioehem: J., 101: 5c. Swanson, M. A. 1955. Glucose-6-phosphatase from liver. Methods Enzymol., 2:541. Widnell, C. C., and J. C. Unkeless. 1968. P a r t i a l purification of a lipoproteln with 5'-nueleotidase activity from membranes of r a t liver cells. Proc. Nat. Acad. Sei., 61 : 1050.