Involvement of SDS-stable higher M(r) forms of bovine normal milk alpha-lactalbumin in inducing intestinal IEC-6 cell death

Monomeric 14-kDa bovine alpha-lactalbumin was purified with a preparation of lower molecular weight whey protein concentrate from Holstein cow normal milk followed by size exclusion chromatography. The protein showed a stimulatory rather than an inhibitory effect on the proliferation of a cultured IEC-6 cell line from the rat small intestine. But incubation in 30% trifluoroethanol/acetate buffer (pH 5.5) at 37 degrees C for 5 d in a slowly rotating test tube rendered it highly cytotoxic with concomitant appearance of SDS-stable 20- and 30-kDa forms of alpha-lactalbumin on electrophoresis. Furthermore, alpha-lactalbumin obtained by a one-step purification procedure by affinity chromatography on an anti-alpha-lactalbumin antibody column from the lower molecular weight whey protein concentrate, which had been found to contain several SDS-stable higher M(r) forms of alpha-lactalbumin, exhibited potent inhibitory activity on IEC-6 cell growth. These results indicate the involvement of SDS-stable higher M(r) forms of bovine normal milk alpha-lactalbumin in inducing cell death on the intestinal IEC-6 cell line.     

Changes in alpha-lactalbumin, total lactose, UDP-galactose hydrolase and other factors in tammar wallaby (Macropus eugenii) milk during lactation

alpha-Lactalbumin was isolated from milk of M. eugenii and its concentration in milk samples taken at various times during lactation (0-40 weeks post partum) was determined by single radial immunodiffusion using rabbit antiserum to the purified protein. The alpha-lactalbumin concentration remained almost constant throughout lactation even though the concentration of total lactose (free lactose plus lactose contained in oligosaccharides) fell to zero after 34 weeks post partum. This fall in lactose was accompanied by a rise in the free galactose and glucose concentrations and marked increases in UDP-galactose hydrolase, nucleotide pyrophosphatase, alkaline phosphatase and acid beta-galactosidase activities. It is suggested that the in vitro hydrolysis of UDP-galactose was due to nucleotide pyrophosphatase and that this enzyme may also play a role in vivo late in lactation by making UDP-galactose unavailable for the synthesis of lactose. Alternatively, lactose and lactose-containing oligosaccharides might be degraded by the acid beta-galactosidase during or after secretion.     

Characteristics of a copper-dependent cross-linking reaction between two forms of cytochrome P-450 in rabbit-liver microsomal membranes.

alpha-Lactalbumin was purified to apparent homogeneity from mouse milk by combined use of gel filtration, chromatography on DEAE-cellulose and hydroxyapatite, and concanavalin A-Sepharose affinity chromatography. Mouse alpha-lactalbumin exists in several species with different charges and in two molecular-size forms. The smaller form, which constituted over 90% of total alpha-lactalbumin, included two major and two minor species, each of which showed different electrophoretic mobility on polyacrylamide-gel electrophoresis, but gave the same single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in two different buffer systems and over the range 10-15% acrylamide concentrations. The molecular weight was estimated as 14 100. The two major species of the smaller form had the same amino acid composition and contained no significant amount of carbohydrate. The larger form of alpha-lactalbumin, consisting of two species with different charges, was present in a small amount (less than 10%) in the milk and was isolated by its ability to interact with concanavalin A-Sepharose. Each of the two species also gave the same single band of apparent mol.w.t 18 500 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. However, this value may be anomalous, since this larger form appears to be glycosylated, and glycoproteins can behave anomalously on sodium dodecyl sulphate/polyacrylamide gels by binding less sodium dodecyl sulphate. All species of mouse alpha-lactalbumin from milk were active in the lactose synthase reaction and showed identical immunological properties, as determined by the mono-specific antibody prepared against the small major species. The presence of both the larger and the smaller forms, each in a percentage concentration similar to that found in milk, was also demonstrated in alpha-lactalbumin induced by hormones in organ cultureof pregnant-mouse mammary gland.     

Evolution of Pinnipedia lactation strategies: a potential role for α-lactalbumin?

Despite the considerable variation in milk composition found among mammals, a constituent common across all groups is lactose, the main sugar and osmole in most eutherians milk. Exceptions to this are the families Otariidae (fur seals and sea lions) and Odobenidae (walruses), where lactose has not been detected. We investigated the molecular basis for this by cloning alpha-lactalbumin, the modifier protein of the lactose synthase complex. A mutation was observed which, in addition to preventing lactose production, may enable otariids to maintain lactation despite the extremely long inter-suckling intervals during the mother's time at sea foraging (more than 23 days in some species).     

A multinational study of alpha-lactalbumin concentrations in human milk

Alpha-lactalbumin, a 14-kD protein, plays a central biochemical role in the mammary gland as the regulatory subunit of lactose synthase, and also plays a nutritional role for the rapidly growing neonate as the protein in highest concentration in human milk. The current study was undertaken to better characterize alpha-lactalbumin concentrations in human milk from a variety of countries. Mature human milk (lactation duration > or =1 month) was collected from at least 50 women from nine different countries on five continents. Alpha-lactalbumin concentration was determined by HPLC. The mean +/- SD for 452 samples was 2.44 +/- 0.64 g/L. The mean value of the samples from the United States was significantly higher than that from any other country, and the mean in Mexico was significantly lower than that from every country except China and Canada. Alpha-lactalbumin concentration decreased with increasing duration of lactation and was positively correlated with total nitrogen. On average, alpha-lactalbumin contributed 16% of the total nitrogen content of human milk and consequently an important part of the amino acid content.     

Purification and properties of two forms of rat alpha-lactalbumin.

Two species of alpha-lactalbumin, alpha-lactalbumin1 and alpha-lactalbumin2, were separated from rat milk and purified to homogeneity by gel filtration, followed by the DEAE-cellulose chromatography. alpha-Lactalbumin1 is a bigger molecule in contrast to other known alpha-lactalbumins, and has a molecular weight of 21,500 as determined by sedimentation equilibrium and sodium dodecyl sulfate-polyacrylamide gel analysis. alpha-Lactalbumin2 has a molecular weight of 16,000 measured by sedimentation analysis. alpha-Lactalbumin2, however, exhibits abnormally high molecular weight of 22,500 on sodium dodecyl sulfate polyacrylamide gels. Both alpha-lactalbumins are active in lactose synthase assay and are glycoproteins containing 7 to 9% carbohydrate. Antiserum raised against alpha-lactalbumin1 cannot discriminate between the two species in a radioimmunoassay.     

Redox transitions of cytochromes and pyridine nucleotides upon stimulation of an isolated rat ganglion

A fairly simple procedure for isolating lactose synthetase A protein from human milk in 36% yield is described. The A protein has a molecular weight of about 40,000. Its interaction with alpha-lactalbumin in the presence of N-acetylglucosamine has been demonstrated by affinity chromatography and gel filtration.     

Heterogeneity of human milk beta(1-4)-D-galactosyltransferase.

beta(1-4)-Galactosyltransferase from human milk (the A protein of lactose synthase) has been found to be heterogeneous when fractionated by affinity chromatography against insolubilized alpha-lactalbumin, using a linear gradient of decreasing N-acetylglucosamine concentration. Three forms were isolated. Molecular weights of the different species, as determined by sodium dodecylsulphate gel electrophoresis, were found to be 38 000, 43 000 and 50 000. The 38 000 and 50 000 species were studied for their catalytic ability to synthesize either lactose in the presence of alpha-lactalbumin, or N-acetyllactosamine in the presence and absence of the 'specifier' protein. Appreciable difference was observed between the two enzyme forms with respect to their catalysis of lactose synthesis with alpha-lactalbumins from various sources. Differences in the rate of production of N-acetyllactosamine in the presence of alpha-lactalbumin were also observed. For the lowest-molecular-weight species it was found that the inhibitory effect of alpha-lactalbumin upon N-acetyllactosamine synthesis becomes an activating effect at higher alpha-lactalbumin concentrations, while no such inversion was observed for the other species. The results suggest that the conformation at the site of association of the enzyme with the acceptor saccharide or alpha-lactalbumin has been changed, presumably by a pratial enzymic hydrolysis.     

Milk secretion in the rat: progressive changes in milk composition during lactation and weaning and the effect of diet.

1. Progressive changes in the composition of milk from rats has been studied from day 0 to 20 of lactation and for 3 days following separation of the dams and pups at day 20 post partum. 2. The changes in concentration of Na, K and lactose suggested that secretion both prepartum and following weaning occurred by a paracellular mechanism whereas a transcellular pathway existed during established lactation. 3. The concentration of total protein and casein increased gradually throughout lactation. In contrast, the concentration of serum albumin increased and transferrin decreased markedly during early lactation. The fat content of milk declined 3-fold within 5 days of birth but the concentration of Ca, Mg and inorganic P increased. The concentration of each of these milk constituents remained constant during established lactation. 4. Following weaning the pronounced decline in lactose, K and inorganic P was negatively correlated with an increase in all other milk constituents except fat. 5. Rats fed a low energy diet produced milk with a lower fat content but with an unaltered concentration of protein and carbohydrate. The growth rate of these litters was similar for the first 5 days of lactation when compared to litters from dams fed a high energy diet. The growth rate of litters thereafter and following weaning was greater for rats fed a high energy diet.     

Synthesis and evolution of concentration of beta-lactoglobulin and alpha-lactalbumin from cow and sheep colostrum and milk throughout early lactation

Synthesis of beta-lactoglobulin and alpha-lactalbumin by explants of ovine mammary gland and evolution of concentration of these proteins in cow and sheep colostrum and milk throughout early lactation have been studied. The evolution of both proteins was similar in cow and sheep species. The highest concentration was found in the first milking (19 and 2 mg/ml for beta-lactoglobulin and alpha-lactalbumin, respectively). Then, levels of beta-lactoglobulin decreased sharply and those of alpha-lactalbumin slowly during the first days of lactation, reaching stable values during the second week postpartum (4 and 1.5 mg/ml). The concentration ratio beta-lactoglobulin/alpha-lactalbumin was four times greater in colostrum than in mature milk. On the other hand, synthesis of these proteins represented about 25-30% of the synthesized total soluble proteins. The synthesis ratio beta-lactoglobulin/alpha-lactalbumin in explants obtained at 12 and 30 hours postpartum was found to be 3.5 and 1.7. These results indicate that the synthesis and secretion of beta-lactoglobulin are comparatively greater than those of alpha-lactalbumin during colostral period, suggesting that beta-lactoglobulin could have some specific function during this period.     

Purification and characterization of human serum galactosyltransferase (lactose synthetase A protein)

A galactosyltransferase, which transfers galactose from UDP-galactose to N-acetylglucosamine, was purified 286,000-fold to homogeneity with 40% yield from human plasma by repeated affinity chromatography on alpha-lactalbumin-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with molecular weight of 49,000. The enzyme is a glycoprotein with 11% by weight carbohydrate, which seems to have only asparagine-N-acetylglucosamine linkage-type carbohydrate chains. The enzyme showed characteristic changes in activity at different alpha-lactalbumin concentrations, indicating that the enzyme is the A protein of lactose synthetase. Km values for the substrates were found to be 0.056 mM for UDP-galactose, 3.2 mM for GlcNAc, and 0.44 mM for Mn2+, and in the presence of alpha-lactalbumin, 3.4 mM for Glc, and 0.20 mM for Mn2+. The activity of the enzyme was neutralized by anti-enzyme antibody, but the antibody did not neutralize the bovine milk galactosyltransferase (A protein) activity.     

Detection of a single nucleotide polymorphism in the human alpha-lactalbumin gene: implications for human milk proteins

Variability in the protein composition of breast milk has been observed in many women and is believed to be due to natural variation of the human population. Single nucleotide polymorphisms (SNPs) are present throughout the entire human genome, but the impact of this variation on human milk composition and biological activity and infant nutrition and health is unclear. The goals of this study were to characterize a variant of human alpha-lactalbumin observed in milk from a Filipino population by determining the location of the polymorphism in the amino acid and genomic sequences of alpha-lactalbumin. Milk and blood samples were collected from 20 Filipino women, and milk samples were collected from an additional 450 women from nine different countries. alpha-Lactalbumin concentration was measured by high-performance liquid chromatography (HPLC), and milk samples containing the variant form of the protein were identified with both HPLC and mass spectrometry (MS). The molecular weight of the variant form was measured by MS, and the location of the polymorphism was narrowed down by protein reduction, alkylation and trypsin digestion. Genomic DNA was isolated from whole blood, and the polymorphism location and subject genotype were determined by amplifying the entire coding sequence of human alpha-lactalbumin by PCR, followed by DNA sequencing. A variant form of alpha-lactalbumin was observed in HPLC chromatograms, and the difference in molecular weight was determined by MS (wild type=14,070 Da, variant=14,056 Da). Protein reduction and digestion narrowed the polymorphism between the 33rd and 77th amino acid of the protein. The genetic polymorphism was identified as adenine to guanine, which translates to a substitution from isoleucine to valine at amino acid 46. The frequency of variation was higher in milk from China, Japan and Philippines, which suggests that this polymorphism is most prevalent in Asia. There are SNPs in the genome for human milk proteins and their implications for protein bioactivity and infant nutrition need to be considered.     

Purification and characterization of the major whey proteins from the milks of the bottlenose dolphin (Tursiops truncatus), the Florida manatee (Trichechus manatus latirostris), and the beagle (Canis familiaris)

The major whey proteins of the milks of the dolphin, manatee, and beagle were purified by gel filtration and ion exchange chromatography and characterized and identified by molecular weight determination, amino acid analysis, N-terminal sequencing, and activity measurements. The major whey protein components from all three species were found to be monomeric beta-lactoglobulins. These proteins were all active in binding retinol. Dolphin milk contained two beta-lactoglobulins (designated 1 and 2) which showed a slight difference in molecular weight and considerably divergent N-terminal sequences, whereas the other milks only contained a single form of beta-lactoglobulin. alpha-Lactalbumins were purified from dolphin and dog milks and were active in promoting lactose synthesis by bovine galactosyltransferase. The dolphin protein had an N-terminal sequence more similar to ruminant alpha-lactalbumins than to those known from other species. Although alpha-lactalbumin activity has been detected in manatee milk at low levels, the corresponding protein was not isolated. In addition, dog milk was found to contain high levels of lysozyme (greater than 1.0 mg/ml), which were identified by activity and sequencing. The functional and evolutionary implications of these results are discussed.     

Southern elephant seal,Mirounga leonina: composition of milk during lactation

An analysis of milk constituents during various stages of lactation in the southern elephant seal Mirounga leonina was carried out. Forty-six milk samples were taken from 30 females throughout lactation during 1985, 1987, 1990 and 1991 on Stranger Point, King George Island, South Shetland Islands. Total nitrogen (TN), non-protein nitrogen (NPN), sugar, fat, ash and water were measured, and from some of these data true protein and energy content were calculated. The results showed a high degree of variation in water and fat concentrations among samples at different stages of lactation. During the first 20 days the fat content of milk increased from about 12 to approximately 52%, while water content fell from 70 to 33%. The composition of milk changes rapidly during the first days post-partum. Protein, minerals and sugar appear to remain stable after the fourth day of lactation. Milk samples contain significant levels of sugars; thin layer chromatography indicates the presence of lactose and glucose together with other unidentified components. There is evidence of a striking change in composition of the milk in the later part of lactation; the progressive increase in the fat:water ratio is abruptly reversed just prior to weaning.     

Physiological basis of longitudinal changes in human milk yield and composition

Currently in Western Australia more than 85% of women are breast-feeding after discharge from the hospital. Breast milk is the only fluid consumed by 64% of infants at 6 months of age, and 25% of infants are still breast-fed at 12 months of age. Therefore, many Western Australian mothers have optimized the art of breast-feeding and thereby provide an ideal population for studies of the physiological, pathological, and pharmacological factors affecting lactation. Reports from other countries conclude that the maximum milk yield of well-nourished women is 700-900 ml/24 h. Our studies have found the average milk yield of mothers breast-feeding single infants was in excess of 1100 ml/24 h for the first 6 months of lactation. Furthermore, mothers breast-feeding twins produced in excess of 2100 ml/24 h over this period, which demonstrates that the potential milk yield for mothers is much higher than 700-900 ml/24 h. Substantial alterations in the composition of breast milk occur at the initiation of lactation and after the cessation of suckling. During established lactation it is generally believed that there is only a slight variation in the composition of the milk. However, intensive studies have revealed acute changes in the concentrations of lactose, glucose, sodium, potassium, and chloride 5-6 days before and 6-7 days after ovulation. These findings suggest that unknown hormonal events associated with the reproductive cycle in women also influence breast milk composition.     

Composition of the milks of the bottlenose dolphin (Tursiops trucatus) and the Florida manatee (Trichechus manatus latirostris)

Milk samples from four individual bottlenose (Tursiops truncatus) and two Florida manatees (Trichechus manatus latirostris) of known lactation stages were analyzed for protein, carbohydrate and lipid composition, as well as for activity levels of alpha-lactalbumin, the regulatory protein of lactose synthase. The milk from both species had relatively high protein and lipid levels, as reported previously for other marine mammals. The major proportion of the lipid was in the form of triglycerides. Dolphin milk contained an average of 2.2% neutral sugars, which was essentially all in the form of lactose, as determined by several criteria. Manatee milk samples contained 0.6% of neutral sugars, and a larger proportion (about 2%) of amino sugars. Lactose was not detected by enzymatic assay or paper chromatography, but HPLC analysis indicated the presence of low levels of lactose together with two components that were tentatively identified as oligosaccharides. alpha-Lactalbumin activity, determined by assay with bovine galactosyltransferase, was found in both dolphin and manatee milk. The level in dolphin milk was comparable with that found in bovine and other milk, but the level in the manatee was less than 10% of that in the dolphin.     

An expression profile of human α-lactalbumin in the milk of transgenic mouse

Five female transgenic mice were produced by microinjection using a construct made up of a 7.3-kb-5′ flanking region and a 2.0-kb coding region of human α-lactalbumin, as well as a 227-bp 3′-flanking region from bovine growth hormone gene. A founder female expressed human α-lactalbumin as much as 0.3 g per liter of its milk, approximately a 3-fold increase in the total α-lactalbumin concentration of the transgenic mouse milk. Compared with the normal mice, the expression profile of the hα-Lac transgene in the transgenics is different during the lactation, showing low level in the first 3 days and becoming increased from day 4, then gradually reaching and stabilizing at the highest level from day 13. In addition, the milk yielding volume in the transgenics tended to be higher than in normal mice, suggesting higher concentrations of α-lactalbumin might boost more milk output.     

Nutrient composition and concentrations of immunoglobulins in milk of sows supplemented with L-carnitine

Recent studies have shown that L-carnitine supplementation of sows increases growth of their piglets during the suckling period. In this study, the composition of the milk of sows supplemented with L-carnitine was determined to find out whether an altered milk composition could account for the increased growth rates of the piglets. Milk of 13 control sows and 14 sows supplemented with L-carnitine (125 mg/d during pregnancy, 250 mg/d during lactation) was collected 5-8 h after birth (colostrum) and on days 10 and 20 of lactation. Concentrations of fat and lactose and the energy content in milk at day 10 and 20 did not differ between both groups of sows. Sows supplemented with L-carnitine had a higher concentration of protein in colostrum (p < 0.05) while concentrations of fat, lactose, immunoglobulins G, M and A as well as the energy content in colostrum did not differ between both groups of sows. These findings show that milk composition does not play a major role for the increased postnatal growth of piglets from sows supplemented with L-carnitine observed in recent studies.     

Hormonal induction and regulation of lactose synthetase in mouse mammary gland

The augmentation of lactose synthetase activity during late pregnancy and lactation was measured by using both a tissue-culture assay and a cell-free assay. The results indicated at least a 100-fold augmentation in specific activity between late pregnancy and lactation. The cell-free assay indicated that the activities of both subunits of this enzyme had increased to 20-30% of the value during lactation by the last day of pregnancy. The tissue-culture assay, however, showed activities only 3-4% of the maximum at the time of parturition. This suggests that not all the enzyme present in the tissue before lactation commenced was active. Since at all stages of pregnancy and lactation the B subunit, alpha-lactalbumin (which is also a milk protein), was rate-limiting, it is suggested that the rate of lactose synthesis may be linked to the rate of milk-protein synthesis. Both subunits of lactose synthetase could be induced in tissue culture by the hormones insulin+hydrocortisone+prolactin. Of the three hormones, prolactin appeared to be the ;trigger' that induced the synthesis of these proteins if the tissue had been stimulated previously by insulin+hydrocortisone.     

Fatty acids in the milk of goats after cessation of lactation

Regular hand milking of six goats was discontinued after 32-35 weeks of lactation. A few days after milking out ceased, the concentration of triglyceride in peripheral blood plasma increased. Over a period of weeks, the concentration of triglyceride in small samples of fluid taken from the teat canal fell gradually. Lipase activity of the milk fluid was temporarily reduced shortly after milking out ended, but, despite this, its concentration of free fatty acids increased. It is suggested that free fatty acids are released during clearance of milk triglyceride from residual fluid in the mammary gland after cessation of lactation.