Lipolytic Activity with the Membrane Fraction of Bovine Skim Milk * EDNA SUSTEK, C. W. DILL, and S. A. HERLICK Department of Animal Science Texas A&M University College Station 77843 ABSTRACT
fat globule. Since the fraction contains the dense material derived from the cytoplasm of the secretory cell (16, 17), it likely contains a variety of enzymes.
Colloidal phosphate-free skim milk was subjected to gel filtration on Sepharose 4B. Lipolytic activity was observed in the membrane material eluted in the void volume fraction and in the protein fraction representing a broad range of molecular weights.
EXPERIMENTAL PROCEDURE
INTRODUCTION
Bovine milk lipase has been the object of reviews by Shahani (12, 13) and Jensen (6) which summarize the nature and properties of the enzyme(s). Nelson and Jezeski (8) demonstrated lipolytic activity in separator slime. Chandan and Shahani (1) and Richter and Randolph (11) subsequently purified an 8000 molecular weight lipase from this source. Fox and Tarassuk (4) purified a lipase with a molecular weight of 210,000 from renneted casein. Shahani and Chandan (14) and Downey and Andrews (2, 3) demonstrated an association of milk lipase with the milk proteins which would produce a variety of molecular weights. A specific source of milk lipase has not been elucidated (6). Shahani (12, 13) reported a similarity between bovine milk lipase and pancreatic lipase, but thorough compatative studies have not been made. Gaffney and Harper (5) described lipolytic activity in the somatic cells from milk separator slime but suggested this lipase was different from milk lipase. Wooding (16, 17) described the vesiculation of the plasmalemma from the initial fat globule membrane and its subsequent release into the plasma. Activity of several enzymes has been observed in these plasma membrane fragments of skim milk (10). The need to investigate lipolytic activity in the fraction containing plasma membrane fragments is emphasized by the association of this material with the milk
Received February 24, 1975. 1Technical Article No. 11724 of the Texas Agricultural Experiment Station.
Skim milk was obtained by separating mixed whole milk from the university dairy herd with a cold milk separator. It was stored at 5 +- 1 C until used. The skim milk had a milk fat content ranging from .02 to .04%. Three milliliter aliquots of skim milk were resolved on Sepharose 4B to obtain the microsomal fraction of Plantz and Patton (10). A jacketed column 2.5 x 100 cm was packed with siliconized glass beads to a height of 72 cm and Sepharose 4B was slurried to a total column height of 82 cm. The column was eluted with .5% NaCl at a constant head pressure of 43 cm to maintain a flow rate of 28.8 ml/h. A Blue M Constant-Flow Portable Cooling Unit stabilized the temperature of the column at 5 C. Lipolytic activity was measured with .01 N NaOH b y the pH-stat (Metrohm-Herisau) method of Parry et al. (9). Assays were at 37 C under a nitrogen atmosphere. One milliliter aliquots of enzyme source were assayed, and one unit of enzyme activity is expressed as the micromoles of base per minute per milliliter of enzyme source required to maintain the reaction mixture at pH 9.0. RESULTS AND DISCUSSION
Plantz and Patton (10) used Sepharose 4B to resolve skim milk membrane fragments from the classical milk proteins. The membrane material almost exclusively eluted from the column in the void volume; however, it was n o t resolved completely from large molecular weight proteins or protein micelles. The versene procedure of Morr et al. (7) was used to disaggregate casein micelles in skim milk. Fig. 1 includes the elution pattern on Sepharose 4B for 3 ml of colloidal phosphate-free skim milk prepared by the procedure of Morr et al. (7).
1519
1520
SUSTEK ET AL.
REFERENCES
1.00
,40C
= a
ii
.2oc
il
il
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i:
,,//
...'.
o .b.s lb
=60
..... =so
a00
ELUTION VOLUME
a~0
400
{m|l
FIG. 1. Elution diagram of protein ( - - - ) lipolytic activity (--) from Sepharose 4B.
and
This procedure improved the resolution of the membrane fragments from the milk proteins as compared to the results by Plahtz and Patton
O0). Lipolytic activity was observed in the void volume fraction eluted both from skim milk and from colloidal phosphate-free skim milk (Fig. 1). If these membrane fragments are a part of the initial fat globule membrane as suggested by Wooding (16, 17), then they may represent the source of entry of lipase into milk. The lipolytic activity in the membrane fraction is probably due to the membrane lipase reported by Tarassuk and Frankel (15). Lipase activity also was observed among the skim milk proteins representing a broad spectrum of molecular weight species. Estimation of molecular weights from Kay values associated with the Sepharose 4B column show the other lipases within the elution volume corresponded to an approximate molecular weight range of 40,000 to 400,000. This range would include the molecular weights reported by Downey and Andrews (3). Further work is needed to determine similarities between the purified lipases (1, 11), the plasma membrane lipase, and the skim milk protein fractions possessing lipolytic activity.
Journal of Dairy Science Vol. 58, No. 10
1 Chandan, R. C., and K. M. Shahani. 1963. Purification and characterization of milk [ipase. I. Purification. J. Dairy Sci. 46:275. 2 Downey, W. K., and P. Andrews. 1966. Studies on the properties of cow's milk tributyrinases and their interaction with milk proteins. Biochem. J. 101:651. 3 Downey, W. K., and P. Andrews. 1969. Evidence for the presence of several lipases in cow's milk. Biochem. J. 112:559. 4 Fox, P. F., and N. P. Tarassuk. 1968. Bovine milk lipase. I. Isolation from skimmilk. J. Dairy Sci. 51:826. 5 Gaffney, P. J., and W. J. Harper. 1965. Lipase activity in somatic cells from separator slime. J. Dairy Sci. 48:613. 6 Jensen, R. G. 1971. Lipolytic enzymes. Prog. Chem. Fat. 11:349. 7 Morr, C. V., R. V. Josephson, R. Jenness, and P. B. Manning. 1971. Composition and properties of suhmicdiar casein complexes in colloidal phosphate-free skimmilk. J. Dairy Sci. 54:1555. 8 Nelson, H. G., and J. J. Jezeski. 1955. Milk lipase. I. The lipolytic activity of separator slime. J. Dairy Sci. 38:479. 9 Parry, R. M., Jr., R. C. Chandan, and K. M. Shahani. 1966. Rapid and sensitive assay for milk lipase. J. Dairy Sci. 49:356. 10 Plantz, E., and S. Patton. 1973. Plasma membrane fragments in bovine and caprine skim milks. Biochim. Biophys. Acta 291 : 51. 11 Richter, R. L., and H. E. Randolph. 1971. Purification and properties of a bovine milk lipase. J. Dairy Sci. 54:1275. 12 Shahani, K. M. 1966. Milk enzymes: Their role and significance. J. Dairy Sci. 49:907. 13 Shahani, K. M. 1970. Isolation, characterization and significance of lysozyme and lipase in Milk. XVIII Int. Dairy Congr. 1 E:61. 14 Shahani, K. M., and R. C. Chandan. 1965. Activity of purified milk lipase in the presence of milk constituents. Arch. Biochem. Biophys. 111:257. 15 Tarassuk, N. P., and E. N. Frankel. 1957. The specificity of milk lipase. IV. Partition of the lipase system in milk. J. Dairy Sci. 40:418. 16 Wooding, F. B. P. 1971. The structure of the milk fat globule membrane. J. Ultrastruct. Res. 37:388. 17 Wooding, F. B. P. 1972. Milk microsomes, viruses, and the milk fat globule membrane. Experentia 28:1077.
fat globule. Since the fraction contains the dense material derived from the cytoplasm of the secretory cell (16, 17), it likely contains a variety of enzymes.
Colloidal phosphate-free skim milk was subjected to gel filtration on Sepharose 4B. Lipolytic activity was observed in the membrane material eluted in the void volume fraction and in the protein fraction representing a broad range of molecular weights.
EXPERIMENTAL PROCEDURE
INTRODUCTION
Bovine milk lipase has been the object of reviews by Shahani (12, 13) and Jensen (6) which summarize the nature and properties of the enzyme(s). Nelson and Jezeski (8) demonstrated lipolytic activity in separator slime. Chandan and Shahani (1) and Richter and Randolph (11) subsequently purified an 8000 molecular weight lipase from this source. Fox and Tarassuk (4) purified a lipase with a molecular weight of 210,000 from renneted casein. Shahani and Chandan (14) and Downey and Andrews (2, 3) demonstrated an association of milk lipase with the milk proteins which would produce a variety of molecular weights. A specific source of milk lipase has not been elucidated (6). Shahani (12, 13) reported a similarity between bovine milk lipase and pancreatic lipase, but thorough compatative studies have not been made. Gaffney and Harper (5) described lipolytic activity in the somatic cells from milk separator slime but suggested this lipase was different from milk lipase. Wooding (16, 17) described the vesiculation of the plasmalemma from the initial fat globule membrane and its subsequent release into the plasma. Activity of several enzymes has been observed in these plasma membrane fragments of skim milk (10). The need to investigate lipolytic activity in the fraction containing plasma membrane fragments is emphasized by the association of this material with the milk
Received February 24, 1975. 1Technical Article No. 11724 of the Texas Agricultural Experiment Station.
Skim milk was obtained by separating mixed whole milk from the university dairy herd with a cold milk separator. It was stored at 5 +- 1 C until used. The skim milk had a milk fat content ranging from .02 to .04%. Three milliliter aliquots of skim milk were resolved on Sepharose 4B to obtain the microsomal fraction of Plantz and Patton (10). A jacketed column 2.5 x 100 cm was packed with siliconized glass beads to a height of 72 cm and Sepharose 4B was slurried to a total column height of 82 cm. The column was eluted with .5% NaCl at a constant head pressure of 43 cm to maintain a flow rate of 28.8 ml/h. A Blue M Constant-Flow Portable Cooling Unit stabilized the temperature of the column at 5 C. Lipolytic activity was measured with .01 N NaOH b y the pH-stat (Metrohm-Herisau) method of Parry et al. (9). Assays were at 37 C under a nitrogen atmosphere. One milliliter aliquots of enzyme source were assayed, and one unit of enzyme activity is expressed as the micromoles of base per minute per milliliter of enzyme source required to maintain the reaction mixture at pH 9.0. RESULTS AND DISCUSSION
Plantz and Patton (10) used Sepharose 4B to resolve skim milk membrane fragments from the classical milk proteins. The membrane material almost exclusively eluted from the column in the void volume; however, it was n o t resolved completely from large molecular weight proteins or protein micelles. The versene procedure of Morr et al. (7) was used to disaggregate casein micelles in skim milk. Fig. 1 includes the elution pattern on Sepharose 4B for 3 ml of colloidal phosphate-free skim milk prepared by the procedure of Morr et al. (7).
1519
1520
SUSTEK ET AL.
REFERENCES
1.00
,40C
= a
ii
.2oc
il
il
_. =
i:
,,//
...'.
o .b.s lb
=60
..... =so
a00
ELUTION VOLUME
a~0
400
{m|l
FIG. 1. Elution diagram of protein ( - - - ) lipolytic activity (--) from Sepharose 4B.
and
This procedure improved the resolution of the membrane fragments from the milk proteins as compared to the results by Plahtz and Patton
O0). Lipolytic activity was observed in the void volume fraction eluted both from skim milk and from colloidal phosphate-free skim milk (Fig. 1). If these membrane fragments are a part of the initial fat globule membrane as suggested by Wooding (16, 17), then they may represent the source of entry of lipase into milk. The lipolytic activity in the membrane fraction is probably due to the membrane lipase reported by Tarassuk and Frankel (15). Lipase activity also was observed among the skim milk proteins representing a broad spectrum of molecular weight species. Estimation of molecular weights from Kay values associated with the Sepharose 4B column show the other lipases within the elution volume corresponded to an approximate molecular weight range of 40,000 to 400,000. This range would include the molecular weights reported by Downey and Andrews (3). Further work is needed to determine similarities between the purified lipases (1, 11), the plasma membrane lipase, and the skim milk protein fractions possessing lipolytic activity.
Journal of Dairy Science Vol. 58, No. 10
1 Chandan, R. C., and K. M. Shahani. 1963. Purification and characterization of milk [ipase. I. Purification. J. Dairy Sci. 46:275. 2 Downey, W. K., and P. Andrews. 1966. Studies on the properties of cow's milk tributyrinases and their interaction with milk proteins. Biochem. J. 101:651. 3 Downey, W. K., and P. Andrews. 1969. Evidence for the presence of several lipases in cow's milk. Biochem. J. 112:559. 4 Fox, P. F., and N. P. Tarassuk. 1968. Bovine milk lipase. I. Isolation from skimmilk. J. Dairy Sci. 51:826. 5 Gaffney, P. J., and W. J. Harper. 1965. Lipase activity in somatic cells from separator slime. J. Dairy Sci. 48:613. 6 Jensen, R. G. 1971. Lipolytic enzymes. Prog. Chem. Fat. 11:349. 7 Morr, C. V., R. V. Josephson, R. Jenness, and P. B. Manning. 1971. Composition and properties of suhmicdiar casein complexes in colloidal phosphate-free skimmilk. J. Dairy Sci. 54:1555. 8 Nelson, H. G., and J. J. Jezeski. 1955. Milk lipase. I. The lipolytic activity of separator slime. J. Dairy Sci. 38:479. 9 Parry, R. M., Jr., R. C. Chandan, and K. M. Shahani. 1966. Rapid and sensitive assay for milk lipase. J. Dairy Sci. 49:356. 10 Plantz, E., and S. Patton. 1973. Plasma membrane fragments in bovine and caprine skim milks. Biochim. Biophys. Acta 291 : 51. 11 Richter, R. L., and H. E. Randolph. 1971. Purification and properties of a bovine milk lipase. J. Dairy Sci. 54:1275. 12 Shahani, K. M. 1966. Milk enzymes: Their role and significance. J. Dairy Sci. 49:907. 13 Shahani, K. M. 1970. Isolation, characterization and significance of lysozyme and lipase in Milk. XVIII Int. Dairy Congr. 1 E:61. 14 Shahani, K. M., and R. C. Chandan. 1965. Activity of purified milk lipase in the presence of milk constituents. Arch. Biochem. Biophys. 111:257. 15 Tarassuk, N. P., and E. N. Frankel. 1957. The specificity of milk lipase. IV. Partition of the lipase system in milk. J. Dairy Sci. 40:418. 16 Wooding, F. B. P. 1971. The structure of the milk fat globule membrane. J. Ultrastruct. Res. 37:388. 17 Wooding, F. B. P. 1972. Milk microsomes, viruses, and the milk fat globule membrane. Experentia 28:1077.
