Download Enzymes in Milk: Their Function in the Mammary Gland, in Milk, and

Biology of Human Milk, edited by Lars A.
Hanson. Nestte Nutrition Workshop Series,
Vol. 15. Nestec Ltd., Vevey/Raven Press,
Ltd., New York © 1988.
Enzymes in Milk: Their Function in the
Mammary Gland, in Milk, and in
the Infant
Margit Hamosh
Department of Pediatrics, Georgetown University Medical Center,
Washington, D.C. 20007
Human milk, like the milk of other species, contains numerous enzymes.
Although this topic has been reviewed (1-4), the first two publications provide little information about the physiological significance of these enzymes.
Shahani et al. (2) compare the activity level of several enzymes in human
and bovine milk, drawing attention to the great differences in the activity
levels of numerous enzymes between the two species. The reader is referred
to the above-listed reviews, which provide the background for the data reviewed in this chapter.
It seems that the best way to approach a discussion of human milk enzymes
is to arbitrarily divide the enzymes into three groups: (a) those that function
in the mammary gland; (b) enzymes present in milk whose function is unknown; and (c) enzymes that might function in the infant. The latter group
would have to remain active during passage through the infant's digestive
system.
This review does not aim to list or discuss the function of all enzymes in
human milk; rather, specific enzymes are selected for discussion of their
physiological role as components of human milk (Table 1).
MILK ENZYMES ACTIVE MAINLY IN THE MAMMARY GLAND
Although the physiology of lactation has been studied in experimental
animals (in vivo and in vitro, in tissue explants or cell cultures), very little
is known about the physiology of lactation in humans. Prepartum mammary
secretions and postpartum colostrum and milk can be used as "windows"
through which one might obtain information on the function of the human
mammary gland in the perinatal period (Table 2).
The presence of enzymes in postpartum milk, as opposed to their absence
45
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ENZYMES IN MILK
TABLE 1. General functions of human milk enzymes
Function
Enzyme
Protection (bactericidal)
Digestion
Repair
Transport (metal carrier)
Biosynthesis of milk components
Pathogenic
a
Lysozyme
Peroxidase
Lipase (LPL, BSSL)a
Amylase
Lipase (BSSL)
Sulfhydryl oxidase (SOX)
Glutathione peroxidase
Alkaline phosphatase
Xanthine oxidase
Phosphoglucomutase
Lactose synthetase
Fatty acid synthetase
Thioesterase
Glucuronidase
Lipase (LPL, BSSL)
(LPL) lipoprotein lipase; (BSSL) bile salt-stimulated lipase.
in prepartum secretions, might indicate low levels in the mammary gland
before delivery and marked increase in activity after parturition. An example
is lipoprotein lipase, the enzyme that controls the uptake of lipoprotein fatty
acids from the circulation into the mammary gland. This enzyme plays a
key role in the delivery of long-chain fatty acids (7), phospholipids, and
cholesterol (14) to the lactating mammary gland. Its absence from prepartum
mammary secretions (4) suggests low activity in the mammary gland, an
assumption confirmed by low concentrations of fat (1 g/dl) in these secretions
(15). A sharp rise in enzyme activity after birth is paralleled by an increase
in milk fat concentrations to 3 to 4 g/dl (15,16). A second lipase, the bile
TABLE 2. Milk enzymes that function in the mammary gland
Enzyme
Phosphoglucomutase
(PGM)
Galactosyltransferase
Lipoprotein lipase
Antiproteases
7-Glutamyltransferase
Xanthine oxidase
Fatty acid synthetase
Thioesterase
Function
Galactose synthesis
Lactose synthesis
Regulates transfer of triglyceride,
cholesterol, and phospholipid from
blood to milk
Protection of mammary gland from
proteolysis (leucocytes, lysosomes)?
Endo and exocytosis of proteins?
Secretion of milk fat droplets?
Lipogenesis
Ref.
5
6
7,8
9, 10
11
12
13
ENZYMES IN MILK
47
TABLE 3. De novo synthesis of fatty acids in the lactating mammary gland
Product: Fatty acids
(mol % fatty acids synthesized)
Enzymes
Species
8:0
10:0
12:0
14:0
16:0
18:0
FAS"
(ixg/106 cells)
Thiosterase
(n.g/106 cells)
Ref.
Human
Rat
4.9
5.7
18.3
29.0
36.3
29.0
22.1
29.0
13.1
15.0
1.8
0.8
2.6
20.0
0.03
0.50
13
25
a
(FAS) fatty acid synthetase.
salt-stimulated lipase of human milk, is present in early prepartum secretions
(4). This enzyme is known to function in the intestine of the newborn, where
it hydrolyzes dietary fat in the presence of bile salts (17,18). It remains to
be determined whether this enzyme might also function in the mammary
gland before and after parturition, possibly in the intracellular metabolism
of fat. The question is, How does the enzyme, which has an obligatory
dependence on bile salts (19,20), act in their absence? Although milk contains
bile salts (21), the concentration is several orders of magnitude lower than
in the intestine. It could, however, be that (a) bile salt concentrations are
higher in the mammary gland than in milk, or (b) that specific compartmentalization of bile salts and lipase might affect their interaction in the cell.
On the other hand, higher protein and enzyme concentrations in precolostrum and colostrum, compared to mature milk, might be the result of
incomplete tight junctions and of the small volumes secreted before the
second or third day postpartum. This is true for amylase (4,22), lysozyme,
lactoferrin (23,24), and other proteins, such as IgA (23,24).
Mammary secretory cells, present in human milk throughout lactation,
can also be used to learn about the function of the human mammary gland
during lactation, for example, recent studies on lipogenesis in the lactating
human mammary gland. Mammary secretory cells isolated from human milk,
were used in these studies (13). The data show that the human mammary
gland contains the two enzymes necessary for lipogenesis—fatty acid synthetase and thioesterase II—and that it synthesizes the same type of fatty
acids (i.e., C 8 -Ci 6 ), as do other mammalian species (25). These data are
summarized in Table 3. It is possible that the lipogenic system is adaptive
and can be repressed by maternal diets of high fat content or stimulated by
high-carbohydrate-low-fat diets (26).
In general, the enzymes in human milk seem to have a more highly organized tertiary structure than the same enzymes from other sources. This
results in greater hydrophobicity of human milk enzymes, possibly accounting for the remarkable resistance of many enzymes to proteolysis and denaturation in the infant's gastrointestinal system (3,4). Indeed, there are also
48
ENZYMES IN MILK
differences in the rate of disulfide bond formation (i.e., the acquisition of
native, biologically active structure by the regeneration of disulfide bonds
of denatured, reduced polypeptides) (27), which might explain the function
of the potent sulfhydryl oxidase of human milk (28). Recent studies show
that this oxidation proceeds slower with a human milk enzyme than with
the identical enzyme (lysozyme) of hen egg white, suggesting a high-energy
barrier, which would constitute a limiting step (27). It is therefore possible
that many milk proteins (enzymes included) might depend on enzyme-catalyzed oxidation of reduced sulfhydryl bonds (29). There is recent evidence
that suggests differences in isozymes, degree of glycosylation, and enzyme
pattern between identical enzymes in the lactating mammary gland and milk
and those in other tissues (3,4).
Phosphoglucomutase
Phosphoglucomutase (PGM) catalyzes the production of glucose-1-phosphate, the first intermediary in the pathway of synthesis of the galactose
moiety of lactose. Phosphoglucomutase (a-D-glucose-l,6-diphosphate:a-Dglucose-1-phosphate phosphotransferase) is the product of three loci: PGMi,
PGM2, and PGM3. In most tissues, PGMi isozymes account for 85% to 95%
of total PGM activity, PGM2 for 2% to 5%, and PGM3 for 1% to 2%. In
erythrocytes, PGMi and PGM2 are found in equal amounts, whereas PGM3
is absent. The PGM patterns in human milk are different and independent
from those in the erythrocytes and can be explained on the basis of a distinct
PGM4 locus (5). The recent study of Cantu and Ibarra (5) is the first report
of a distinct gene for a widely distributed protein being functionally restricted
to the lactating mammary gland, since no evidence of its activity has been
found in other tissues previously studied.
Galactosyltransferase
Galactosyltransferases catalyze the synthesis of the heteropolysaccharide
moieties of complex carbohydrates. One of the best known galactosyltransferases is UDP-galactose: iV-acetylglucosamine galactosyltransferase or A
protein of the lactose synthetase system. This enzyme has been purified
from various animal and human body fluids, but with the exception of the
amino acid and carbohydrate content of the bovine milk enzyme, no information about its structure is available. Recent studies show that galactosyltransferases from human amniotic and ascites fluids have similar isoelectric focusing patterns, whereas the milk enzyme is less negatively charged
(6). The study suggests that the milk enzyme contains less sialic acid, possibly as a result of the neuraminidase activity in human milk (30). This study
ENZYMES IN MILK
49
suggests that the electrophoretic difference between the enzyme in milk and
in other body fluids is of postribosomal rather than genetic origin.
Galactosyltransferases are found in the Golgi membranes of many tissues.
The binding of the regulatory protein a-lactalbumin to galactosyltransferase
increases the latter's affinity for glucose (from a Km for glucose of 1 M to 1
HIM), thus enabling lactose synthesis at physiological glucose concentrations
(31).
Lipoprotein Lipase
Lipoprotein lipase (LPL) regulates the uptake of circulating triglyceride
fatty acids, cholesterol, and phospholipids by the lactating mammary gland
(7,32,33). LPL has a central role in providing the lipid constituents of milk.
In the mammary gland, LPL is located both in the endothelium (its site of
activity) and in the alveolar cells, the site of its synthesis. The extreme
variation in enzyme levels in the milk of different species (34) suggests either
species differences in the mechanism of milk secretion or a possible relationship between damage to the mammary cells and the state of development
of the offspring (i.e., the pressure applied to the mammary gland during
sucking) (34). Since expression of milk with the aid of a breast pump might
cause some rupture of alveolar cells, we have compared the levels of LPL
activity in milk expressed from one breast by pumping and in milk that drips
spontaneously from the opposite breast (35). Our study shows that LPL
levels were lower in pumped than in drip milk. This would suggest that
leakage of enzyme from ruptured cells is probably not the mechanism of its
release into milk.
Recent studies of a patient with familial LPL deficiency (type I hyperlipoproteinemia) show that LPL is absent from milk throughout lactation,
suggesting that it is absent also from the mammary gland (36). The authors
suggest that a common or closely related genetic locus might be implicated
in the normal synthesis of LPL in different tissues. The study further highlights the key role of LPL in the control of milk fat content and composition.
Thus, milk fat concentration was significantly lower in the patient when
compared to normal lactating women; furthermore, its composition also differed, the milk containing higher amounts of lauric (C12:0) and myristic
(C14:0) acids and considerably less oleic (C18:1) and especially linoleic acid
(C18:2). The higher concentration of fatty acids synthesized in the breast
tissue (Table 3) is probably due to the restricted fat intake, as well as to
much restricted entry of long-chain fatty acids into the mammary epithelial
cells (only nonesterified fatty acids would reach these cells from the circulation). Since long-chain fatty acids or their derivatives inhibit fatty acid
synthetase (37), their reduced uptake results in enhanced fatty acid synthesis
in the mammary gland (36).
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ENZYMES IN MILK
LPL has no known function in milk or in the newborn but has been implicated in hydrolysis of milk triglycerides and in the release of free fatty
acids in human (38) and bovine milk (39) during storage. The increase in
milk-free fatty acid concentrations may be associated with the anti-infective
activity of human milk (40,41), a phenomenon previously thought to depend
only on bile salt-stimulated lipase activity (42).
Fatty Acid Synthetase and Thioesterase
Fatty acid synthetase and thioesterase catalyze de novo synthesis of fatty
acids and have recently been described for the first time in human mammary
gland secretory cells (Table 3) (13). Indirect evidence suggests that these
enzymes may be regulated by the cellular concentration of long-chain fatty
acids (26,36).
-y-Glutamyltransferase
•y-Glutamyltransferase activity is high in human colostrum, and although
activity decreases thereafter, considerable amounts of enzyme are present
in transitional and mature milk (11,43). The enzyme catalyzes the transfer
of the -y-glutamyl group, the receptors differing according to the source of
•y-glutamyltransferase (the renal enzyme utilizes different amino acid and
peptide receptors from the milk enzyme). In addition to kidney, appreciable
amounts of 7-glutamyltransferase are present in liver, pancreas, prostate,
and breast cyst fluid (43). It has been suggested that the enzyme is localized
in the Golgi apparatus and that it plays a role in the endo- and/or exocytotic
transport of proteins (11). Indeed, in milk the enzyme is also associated with
the membrane fraction of skim milk (44). The high levels of 7-glutamyltransferase in the serum of newborn infants have been attributed to absorption
of the intact enzyme from breast milk (43).
Xanthine Oxidase
Xanthine oxidase is a major component of the milk fat globule membrane
in bovine milk (1,2). It has been suggested that this enzyme has a function
in the secretion of milk fat droplets, possibly by changing the fluidity of the
plasma membrane by peroxidizing membrane-associated lipids (12). The
very low levels of xanthine oxidase in human milk suggest major differences
in the composition of the milk fat globule membrane and possibly in the
mechanism of secretion of fat globules into the milk of these two species.
In addition to its function in the mammary gland, the enzyme might also act
as a metal carrier (Table 4) thereby having also a function in the newborn.
ENZYMES IN MILK
51
TABLE 4. Milk enzymes as metal carriers
Glutathione peroxidase: selenium
30% of milk Se
Alkaline phosphatase: zinc and magnesium
20% of milk Zn (four atoms per molecule, two
essential for activity and two for structure)
Mg: two atoms per molecule
Xanthine oxidase: iron and molibdenum
eight atoms Fe, two atoms Mb per molecule
" Adapted from refs. 45, 46, and 54.
Xanthine oxidase has specific binding sites for iron (eight atoms per molecule), which are important for its enzymatic activity, and for molybdenum
(two atoms per molecule) (45,46).
MILK ENZYMES WITHOUT WELL-DEFINED FUNCTION
Although there are many enzymes in human milk whose function is unknown, only two are discussed here.
Lactate Dehydrogenase
Similar to many enzymes in milk, lactate dehydrogenase (LD) activity is
highest in colostrum and decreases as a function of lactation. Recent studies
suggest that in addition to changes in enzyme concentration, there is also a
change in isozyme pattern from an LD-5 maximum in colostrum to LD-1
maximum in transitional milk (47).
The LD molecule consists of four subunits of two different types, designated H (heart muscle) and M (skeletal muscle). Five different combinations of these subunits are possible, corresponding to LD-1 to LD-5. Cardiac
muscle is richest in LD-1 and liver in LD-5. The patterns for colostrum and
transitional milk differ from that of maternal serum for which LD-2 and LD3 are the main isozymes. The change in the isozyme spectrum of the milk
enzyme during the early stages of lactation is especially interesting in view
of the fact that the LDH isozyme pattern of each organ is considered to be
unique.
Plasminogen Activator
Plasminogen activator is another enzyme without a known function. Although the enzyme was first reported in milk in 1952 (48), it has only recently
been characterized (49,50). The purified enzyme has a molecular weight of
86,000 and was shown to be antigenically different from urokinase, a well-
52
ENZYMES IN MILK
characterized plasminogen activator isolated from urine. Inhibition of activity by di-isopropylfluorophosphate (DFP) indicates that serine is at the
active site, as in urokinase.
MILK ENZYMES IMPORTANT IN NEONATAL DEVELOPMENT
Proteolytic Enzymes and Antiproteases in Human Milk
Human milk contains both proteolytic enzymes and protease inhibiting
activity: The net proteolytic activity will therefore depend on the quantitative
interaction between the two proteins (Table 5). Recent studies indicate caseinolytic activity and elastase-like activity to be present, but no trypsin and
chymotrypsin-like activity was detected (9). It is possible that leukocytes
are the source of the elastase-like activity in human milk. Although the
presence of protease inhibitors in human milk was reported about 30 years
ago (3,4), their nature and amounts have been investigated only recently.
These studies show that al-antichymotrypsin (3,10) and al-antitrypsin are
the main protease inhibitors in human milk.
These studies also show that one-third of colostrum specimens studied (18
of a total of 53 samples) had no detectable protease inhibiting activity despite
the presence of immunoreactive protease inhibitors; furthermore, caseinolytic activity was present in all of these colostrum specimens.
The physiological function of protease inhibitors in human milk is not clear
TABLE 5. Milk enzymes that function in the infant
Enzyme
Protease
Antiproteases
a-Amylase
BSSL
Sulfhydryl oxidase
Lysozyme
Peroxidase
Glutathione peroxidase
p-Glucuronidase
Function
Ref.
Hydrolysis of milk proteins
Protect bioactive proteins (enzymes,
immunoglobulins) from hydrolysis in
milk and in the intestine of the
newborn
Facilitates digestion of
polysaccharides (in milk, formula,
and beikost) by the infant
Hydrolysis of fat in the intestine of the
newborn; bactericidal activity
Catalyzes oxidation of SH groups:
possible role in maintaining
structure and function of proteins
containing disulfide bonds
Bactericidal
Bactericidal; present in leukocytes
Selenium delivery to the infant
Breast-milk jaundice?
2, 9,48
2, 10
22
19, 20, 35, 40
28
51,52
53
54
55
ENZYMES IN MILK
53
at present. We may postulate that they may protect the mammary gland
from local proteolytic activity by leukocytic and lysosomal proteases during
different stages of differentiation and lactogenesis or during pathogenic conditions such as mastitis; they may prevent the proteolytic breakdown of other
enzymes and proteins in milk (and may thus be important in milk banking);
and they may affect the absorption of milk proteins (immunoglobulins) in
the newborn. Furthermore, the presence of antiproteases would facilitate
the delivery of compensatory digestive enzymes (lipase and a-amylase) in
an active form from milk to the infant. It has been suggested that the antitryptic and antichymotryptic activity of human milk may prevent the absorption of endogenous and bacterial proteases in infants and thereby contribute to the passive protection of extraintestinal organs such as the liver
(57). The high concentration of antiproteases in colostrum coincides with
the period of greatest transfer of nonimmunoglobulin protein from the intestine to the systemic circulation of the newborn (56).
Recent studies have determined the primary structure of human p-casein
(58) and have suggested that 7-casein is a product of endogenous human
milk proteolytic activity (59), as was previously reported for the origin of
7-casein in bovine milk. Small peptides (three to eight amino acids) derived
from casein, such as the casomorphines, have specific physiologic activity.
Morphiceptin, valmuceptin, and other derivatives are potent and specific
agonists of |x-opiate receptors (60). Lindstrom et al. (61) suggest that certain
cases of postpartum psychosis may be associated with the presence of these
peptides in blood and cerebrospinal fluid (CSF). Whether they affect infant
behavior is not known at present.
Sulfhydryl Oxidase
Sulfhydryl oxidase is another enzyme present in human milk (28) and in
the milk of other species (3,4) that might function in milk and in the gastrointestinal system of the newborn infant. This enzyme is present in colostrum as well as in mature milk. Sulfhydryl oxidase catalyzes the oxidation
of sulfhydryl groups, using O2 as an oxidant and producing equimolar quantities of H 2 O 2 and the corresponding disulfide. The enzyme has broad specificity, acting on both small thiol compounds and protein, and might be
essential for the activity of proteins whose structure and function depend
on intact disulfide bonds (29). The role of this enzyme could involve maintenance of structural and functional integrity of milk proteins, enzymes, and
immunoglobulins. Furthermore, recent reports that this enzyme is stable at
low pH (28) suggest that it might retain activity during passage through the
stomach and might function in the intestine of the newborn, where it could
be instrumental in the uptake of macromolecules by altering the physical
state of the intestinal mucus diffusion barrier (28). Recent studies show that
54
ENZYMES IN MILK
TABLE 6. Some characteristics of milk enzymes active in the infant
Enzyme
Characteristic
Amylase
BSSL
Distribution in milk
Aqueous phase
Aqueous phase
Effect of milk storage
(-20°C, -70°C)
Stability to low pH
(passage through
stomach)
Presence in preterm (PT)
and term (T) milk
Pattern through
lactation:
precolostrum,
colostrum, milk
pH optimum
Evidence of activity in
intestine
Presence in milk of other
species
Stable
Stable
Sulfhydryl
oxidase
Skim milk
membrane
Stable
pH > 3.0
pH > 3.0
pH > 3.0
Equal activity PT
andT
Colostrum greater
than milk
Equal activity PT
andT
Colostrum equal
to milk
?
6.5-7.5
7.4-8.5
7.0-7.5
Yes
Yes
Yes
??
Primates and
carnivores
?
Rat, cow,
rabbit
sulfhydryl oxidase is present in skim milk membranes and that it is resistant
to storage at -20°C (44) (Table 6).
a-Amylase
The newborn infant has adequate levels of lactase, an intestinal brush
border enzyme that hydrolyzes lactose, the main carbohydrate of human
milk; however, a-amylase, the chief polysaccharide-digesting enzyme is not
developed at birth, even in full-term infants. a-Amylase in the duodenum
amounts to only 0.2% to 0.5% of the adult level (62). Human milk contains
1.2 to 1.5 g/dl oligosaccharides ranging in chain length from penta- to tetradecasaccharides (63). In addition, many breast-fed infants receive glucose
polymers or starch from infant formulas or beikost, given often as supplements. For the digestion of glucose polymers, the infant depends on salivary
amylase (62), glucoamylase (64), and the amylase of human milk (22). Recent
studies show that amylase is present in milk secreted by mothers of preterm
and term infants (22). Enzyme levels remain high throughout lactation, and
activity is stable during storage for months at either -20°C or -70°C (4).
a-Amylase is resistant to low pH values (down to 3.0-3.5) (22) and maintains
activity in the newborn's intestine (65) (Table 6).
During the past 50 years, there has been an increasing tendency to intro-
ENZYMES IN MILK
55
duce solid foods into the infant's diet as early as the first month of life. Such
early addition of starch might be well tolerated in breast-fed infants because
of high intestinal levels of a-amylase provided by human milk. Indeed, this
assumption is supported by observations in Egypt of a lack of digestive
disturbances in breast-fed infants offered starch-containing foods early, a
practice that occurs particularly among the poor (2).
Bile Salt-Stimulated Lipase
Because of very low pancreatic lipase, the newborn infant depends mainly
on lingual, gastric, and human milk bile salt-stimulated lipase (BSSL) for
the digestion of dietary fat (66-69).
Hydrolysis of milk fat involves first the penetration of lingual (66) and
gastric (67) Upases into the milk fat globule and partial hydrolysis of the core
triglyceride (68). This process is then completed in the intestine by the BSSL
of human milk. The combined activity of these two enzymes can lead to
complete hydrolysis of dietary fat and can thus effectively substitute for low
levels of pancreatic lipase.
The properties of human milk BSSL are fully compatible with activity in
the intestine of the newborn (Table 6). The enzyme is stable at low pH and
thus not inactivated in the stomach and remains active in the intestine for
at least 2 hr (20). Its lack of substrate specificity and its ability to hydrolyze
triglycerides completely to free fatty acids and glycerol suggest efficient
hydrolysis of triglycerides and various esters (such as retinyl palmitate, the
main component of vitamin A) (18). BSSL is present in precolostrum and
maintains high activity in milk throughout lactation (4,35). Activity is stable
during storage at either — 20°C or -70°C, indicating that banked milk maintains its fat-digesting potential (70). As mentioned earlier, the enzyme might
contribute to the antiprotozoan (Giardia lamblia) activity of human milk
(40,42).
Interaction between lactoferrin (present in high concentrations in human
milk) and BSSL has recently been reported (71). This interaction leads to a
1.4-fold increase in hydrolytic activity, suggesting that lactoferrin might be
the milk factor previously thought to stimulate milk BSSL activity (72).
Contrary to earlier reports (18), we have recently found high BSSL activity
in the milk of carnivores (both terrestrial and aquatic) (73,74), indicating that
the enzyme is not limited entirely to primate milk. This will enable studies
on the biology of the enzyme not only in milk but also in the mammary gland.
Glucuronidase
Breast-fed infants have a higher incidence of jaundice than formula-fed
infants (75). Breast-milk jaundice, due to prolonged nonconjugated hyper-
56
ENZYMES IN MILK
bilirubinemia, was attributed initially to inhibition of hepatic UDP-glucuronyltransferase by the steroid pregnane-3-7-20-p-diol (76) and, subsequently, to high levels of free fatty acids in jaundice-inducing milks (77).
However, no association was found between milk lipase levels and free fatty
acid concentrations (78). A recent study shows an association between high
P-glucuronidase activity in milk and breast-milk jaundice (55). Glucuronidase cleaves glucuronic acid from bilirubin glucuronide, liberating unconjugated bilirubin, which is more easily absorbed from the intestine than the
conjugate.
Lysozyme
Lysozyme catalyzes the hydrolysis of the (1-4) linkage between N-acetylglucosamine and Af-acetylmuramic acid in bacterial cell walls. The enzyme lyses mostly gram-positive and a few gram-negative bacteria; it is a
major component of the human milk whey fraction and has been shown to
play a role in the antibacterial activity of human milk. The lysozyme of
human milk is composed of 130 amino acid residues and has a molecular
weight of 14,400 daltons. Although its sequence exhibits considerable homology with the lysozyme of chicken egg white (51), recent studies show a
marked difference in the tertiary structure of the two proteins, resulting in
greater organization and hydrophobicity of the human milk protein (52).
Lysozyme and ot-lactalbumin of human milk seem to be derived from a
common ancestor molecule on the basis of identical amino acids in 49 positions (79).
Although not related to the topic of enzymes in human milk, it is worth
noting that another antimicrobial agent in human milk, lactoferrin, was recently shown to be highly resistant to proteolysis both in its iron-free (apolactoferrin) and iron-containing forms (80,81). Because native milk lactoferrin is largely free of iron, it can withold iron from, and thus, retard, the
in vitro growth of microorganisms (82). The marked susceptibility of bovine
milk lactoferrin to proteolysis led investigators to suggest that the unusual
resistance of human apolactoferrin to proteolysis may reflect an evolutionary
development designed to permit its survival in the intestine of the infant (80).
Peroxidase
•<
Peroxidase in human milk was earlier considered to be lactoperoxidase
(83), but a recent study established that the activity in milk is derived from
milk leukocytes and is thus a myeloperoxidase (53). The distinction between
a true secretory peroxidase (lactoperoxidase) and a peroxidase derived from
leukocytes (myeloperoxidase) is important, because the two enzymes have
different structures (single polypeptide chains of molecular weight 77,000-
ENZYMES IN MILK
57
79,000 versus two subunits with a molecular weight of 118,000 and 144,000,
respectively). Both enzymes catalyze the oxidation of thiocyanate ions to
products with bacteriostatic activity; however, only myeloperoxidase catalyzes the oxidation of the chloride ion; the products of the latter reaction
only have bactericidal activity. It is interesting that bovine milk and human
saliva have potent lactoperoxidase activity. The difference in the nature and
source of human milk peroxidase reported by the two investigators can be
attributed to the different methods used, the second group (53) employing
more sensitive assay techniques.
Alkaline Phosphatase
Alkaline phosphatase is located on the luminal surface of the epithelial
cells of the ducts and acinar glands. The enzyme is released into milk as
part of the plasma membrane during the formation of milk fat globules. The
high levels of the enzyme in colostrum and intermediate milk may be due
to the sudden activation of the milk secretory mechanism. Although the
enzyme was purified from bovine milk about ten years ago (4), it has only
recently been characterized in human milk (84,85). The conclusions reached
by two groups of investigators differ as to the nature of the enzyme in milk.
Whereas one group (84) suggests that functional, antigenic, and structural
analysis indicate that the milk enzyme is the same protein species as that
of adult human liver, the data reported by the second group (85) suggest
that the milk enzyme is a mixture of isozymes similar to bone and liver
alkaline phosphatase.
Alkaline phosphatase is a metal-carrying enzyme (Table 4) (46), and it
contains four atoms of zinc per molecule, two essential for its enzymatic
activity and two fulfilling a structural role. In addition to zinc it also contains
two magnesium atoms.
Much remains to be learned about the function of many milk enzymes. It
is important to know their origin, mechanism of secretion into milk, compartmentalization among the various milk fractions as well as whether their
activity changes as a function of length of lactation and pregnancy.
ACKNOWLEDGMENTS
Supported by NIH Grant HD-20833. The expert secretarial help of Ms.
Barbara Runner is gratefully acknowledged.
REFERENCES
1. Jenness R. The composition of human milk. Semin Perinatol 1979;31:225-39.
2. Shahani KM, Kwan AJ, Friend BA. Role and significance of enzymes in human milk. Am
J Clin Nutr 1980;33:1861-8.
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ENZYMES IN MILK
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