Occurrence of an unusual phosphorylated N-acetyllactosamine in horse colostrum

The colostrum of horses (thoroughbreds) was extracted and fractionated to yield Gal(beta1-4)GlcNAcalpha1-phosphate, which has not previously been detected in any mammalian milk or colostrum, as well as Neu5Ac(alpha2-3)Gal(beta1-4)Glc. The structures of these saccharides were established by NMR spectroscopy and MALDI-TOF mass spectrometry.     

Occurrence of sulfate in an asparagine-linked complex oligosaccharide of chicken adipose lipoprotein lipase

After adipocytes were labeled with Na2[35SO4], immunoadsorbed with immobilized antilipoprotein lipase, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography, a labeled band was identified at 59,700 daltons, the molecular mass of chicken lipoprotein lipase (LPL). Excess unlabeled LPL prevented the immunoadsorption of this labeled species, hence the labeled species was determined to be LPL. Digestion of LPL with endo-beta-N-acetylglucosaminidase H (Endo H) caused a shift in mobility of LPL in SDS-PAGE with no loss of radioactivity, whereas digestion with glycopeptidase F resulted in removal of 99% of the radioactivity. Adipocytes cultured with Trans35S-label and tunicamycin produced an LPL species of 52,000 daltons, but tunicamycin abolished the incorporation of 35SO4 into LPL. This established that 35SO4 was incorporated into an N-linked oligosaccharide of LPL. Endo H digestion of pulse-chase labeled LPL revealed the presence of two complex and one high mannose-type N-linked oligosaccharides. A single 35SO4-labeled tryptic peptide was isolated by reverse phase chromatography. The amino acid sequence of the peptide established that the 35SO4 oligosaccharide is conjugated at Asn-45. Behavior of the 35SO4-labeled oligosaccharide on concanavalin A-agarose, sequential exoglycosidase digestion, and chemical analysis of the 35SO4 oligosaccharide confirms that this moiety is of the complex type. Sequential exoglycosidase digestion, thin layer chromatography of the released monosaccharides, and the use of glycosylation inhibitors established that the sulfated sugar is a core N-acetylglucosamine (GlcNAc). The data show that chicken LPL contains two complex and one high mannose N-linked oligosaccharides and that 35SO4 is incorporated into LPL on a GlcNAc residue of a complex oligosaccharide located at Asn-45.     

Occurrence of tyrosine sulfate in proteins--a balance sheet. 2. Membrane proteins

1. The abundance of tyrosine sulfate in membrane proteins was quantified in four different cell lines and compared to that in soluble cellular and secreted proteins. 2. Upon metabolic labelling of HepG2, Ltk-, AtT20 and PC12 cells with [35S]sulfate or [3H]tyrosine, a fraction enriched in integral membrane proteins was found to contain small, but significant, amounts of protein-bound tyrosine sulfate (up to 2.5% of the total cellular plus secreted protein-bound tyrosine sulfate). On the other hand, the frequency of sulfation of tyrosine residues of membrane proteins was within the same order of magnitude as that of secreted proteins, indicating that the low abundance of tyrosine sulfate in membrane proteins was largely a reflection of the low abundance of these proteins themselves. Consistent with this conclusion were the results of an analysis showing that 14 out of 32 selected membrane-spanning proteins contain potential tyrosine sulfation sites. 3. In HepG2 cells, three tyrosine-sulfated integral membrane glycoproteins of molecular mass 100, 125 and 150 kDa were identified. Characterization of the 150-kDa tyrosine-sulfated membrane protein revealed that it was protected from proteolysis in intact cells, suggesting a localization in an intracellular organelle. 4. Together with the results reported in the preceding paper in this journal, our data suggest that tyrosine sulfation occurs in various classes of trans-Golgi-derived proteins, soluble as well as membrane, and extracellularly exposed as well as intracellularly retained, proteins. This suggests that tyrosine sulfation may have a variety of physiological functions, depending on the individual tyrosine-sulfated protein or protein class.     

Relationship of bacteriological status of the mammary gland to lactose level in milk yield.

The analysis of variance showed a significant (P less than or equal to 0.01) effect of the presence of pathogenioc bacteria in the udder on lactose content in milk. The relationship between lactose level and milk yield was significant (P less than or equal to 0.05).     

Evolution of milk oligosaccharides and lactose: a hypothesis

Mammalian milk or colostrum contains up to 10% of carbohydrate, of which free lactose usually constitutes more than 80%. Lactose is synthesized within lactating mammary glands from uridine diphosphate galactose (UDP-Gal) and glucose by a transgalactosylation catalysed by a complex of β4-galactosyltransferase and α-lactalbumin (α-LA). α-LA is believed to have evolved from C-type lysozyme. Mammalian milk or colostrum usually contains a variety of oligosaccharides in addition to free lactose. Each oligosaccharide has a lactose unit at its reducing end; this unit acts as a precursor that is essential for its biosynthesis. It is generally believed that milk oligosaccharides act as prebiotics and also as receptor analogues that act as anti-infection factors. We propose the following hypothesis. The proto-lacteal secretions of the primitive mammary glands of the common ancestor of mammals contained fat and protein including lysozyme, but no lactose or oligosaccharides because of the absence of α-LA. When α-LA first appeared as a result of its evolution from lysozyme, its content within the lactating mammary glands was low and lactose was therefore synthesized at a slow rate. Because of the presence of glycosyltransferases, almost all of the nascent lactose was utilized for the biosynthesis of oligosaccharides. The predominant saccharides in the proto-lacteal secretions or primitive milk produced by this common ancestor were therefore oligosaccharides rather than free lactose. Subsequent to this initial period, the oligosaccharides began to serve as anti-infection factors. They were then recruited as a significant energy source for the neonate, which was achieved by an increase in the synthesis of α-LA. This produced a concomitant increase in the concentration of lactose in the milk, and lactose therefore became an important energy source for most eutherians, whereas oligosaccharides continued to serve mainly as anti-microbial agents. Lactose, in addition, began to act as an osmoregulatory molecule, controlling the milk volume. Studies on the chemical structures of the milk oligosaccharides of a variety of mammalian species suggest that human milk or colostrum is unique in that oligosaccharides containing lacto-N-biose I (LNB) (Gal(β1 → 3)GlcNAc, type I) predominate over those containing N-acetyllactosamine (Gal(β1 → 4)GlcNAc, type II), whereas in other species only type II oligosaccharides are found or else they predominate over type I oligosaccharides. It can be hypothesized that this feature may have a selective advantage in that it may promote the growth of beneficial colonic bacteria, Bifidobacteria, in the human infant colon.     

Occurrence of tyrosine sulfate in proteins--a balance sheet. 1. Secretory and lysosomal proteins

1. The abundance of tyrosine sulfate in secretory proteins and in various classes of cellular proteins has been quantified and compared to protein-bound carbohydrate sulfate. 2. HepG2 cells and fibroblasts, two cell types showing only the constitutive pathway of secretion, and PC12 cells, which show both the constitutive and the regulated pathway of secretion, were subjected to pulse-chase and/or long-term labelling with [35S]sulfate and [3H]tyrosine, followed by analysis of proteins in the cells and medium. Under both conditions of labelling, 65-92% of the protein-bound tyrosine sulfate and 44-84% of the protein-bound carbohydrate sulfate were found to be secretory. In HepG2 cells, the frequency of sulfation of tyrosine residues, which can be determined independently from protein abundance and the rate of protein synthesis, was 8-22 times higher in proteins secreted into the medium than in cellular proteins. 3. All cell lines studied contained significant amounts, not only of carbohydrate sulfate, but also of tyrosine sulfate in specific cellular proteins. As shown for fibroblasts, these tyrosine-sulfated proteins were retained within the cells for at least 100 min of chase following a pulse with [35S]sulfate and were almost completely recovered in a light membrane fraction after subcellular fractionation. 4. Lysosomes were found to contain small, but significant, amounts of protein-bound tyrosine sulfate in addition to protein-bound carbohydrate sulfate. Protein-bound tyrosine sulfate in lysosomes reached a peak at 20 min of chase and rapidly disappeared thereafter, whereas protein-bound carbohydrate sulfate accumulated after 20 min of chase. Examination of the known sequences of eleven lysosomal enzymes revealed the presence of potential tyrosine sulfation sites in five of them. 5. Our results show that secretory proteins are the most abundant, but not exclusive, in vivo substrates for tyrosine sulfation and suggest the presence of soluble tyrosine-sulfated proteins in lysosomes and other, as yet unidentified, organelles of the secretory pathway. In the following paper in this journal we describe the abundance of tyrosine sulfate in integral membrane proteins.     

Hydrolysis of milk lactose by immobilized beta-galactosidase-hen egg white powder

Immobilized beta-galactosidase was obtained by crosslinking the enzyme with hen egg white using 2% glutaraldehyde. The gel obtained could be lyophilized to give a dry enzyme powder. The pH optimum of both the soluble and immobilized enzyme was found to be 6.8. The immobilized enzyme showed a higher K(m) for the substrates. The extent of enzyme inhibition by galactose was reduced upon immobilization. The stability towards inactivation by heat, urea, gamma irradiation, and protease treatment were enhanced. The bound enzyme as tested in a batch reactor could be used repeatedly for the hydrolysis of milk lactose. The possible application of this system for small-scale domestic use has been suggested.     

Measurement of lactose concentrations in milk of Microtus montebelli with methylamine reaction

Concentrations of lactose in milk of Microtus montebelli were measured with the method using with the methylamine reaction. In this method, a small amount of sample (0.5 g) was sufficient for assay, and reproducibility and sensitivity were excellent. This method was very useful for measuring of lactose concentrations in experimental small animals. The average lactose concentration in milk of Microtus montebelli was 1.57g/100g, considerably low in comparison with the value of ICR mice (2.70g/100g). The low concentration of lactose in milk was considered to be one of metabolic characteristics of Microtus montebelli as a herbivorous animal.     

Preparation of lactose free milk by fermentation using immobilizedSaccharomyces fragilis

Summary Saccharomyces fragilis cells (40% w/v) were immobilized in 2% Ca-alginate and were used in a batch process for the removal of lactose from milk by fermentation. Immobilized cells (10 g) could completely desugarate 100 mL of milk in 3.5 h. The immobilized preparation was used repeatedly in 15 batches without decrease in the activity.     

The sulfhydryl group microenvironment of lactose synthase from bovine milk

Galactosyltransferase from bovine milk was inactivated by a series of sulfhydryl group specific reagents of different structures and sizes. The inactivation rate constants suggest that the thiol is located in a nonpolar microenvironment. The ESR spectrum of a spin labeled galactosyltransferase showed that the sulfhydryl group is in a region of non-restricted rotation, consistent with its broad reactivity towards various thiol reagents. Galactosyltransferase immobilized onto agarose through its sulfhydryl group retained its ability to catalyze the synthesis of N-acetyllactosamine and lactose. Thus the residual activity of the sulfhydryl group modified enzyme is not due to an isozyme lacking such a group. In addition, the active thiol can not be located at the active site nor the protein-protein interaction site between galactosyltransferase and alpha-lactalbumin.