Milk Biosynthesis
Total Page：16
File Type：pdf, Size：1020Kb
FairLife: Cold Filtration • https://www.youtube.com/watch?v=YgHFaNVofY8 Milk Biosynthesis Part 5: Lactose, Mineral, and Vitamin Secretion Lactose is… • The major carbohydrate in milk • Present in milk of most mammals • A readily digestible source of glucose for the neonate • Digested by lactase enzyme in neonate • Unique to the mammary gland • The major osmoregulator of milk (draws water into gland) Lactose Concentration • Least variable milk component • High lactose concentration will not exist • More lactose = more water coming into alveolar lumen keeping lactose concentration fairly constant • Low lactose concentration will occur during involution and some illnesses • Remember milk is the only source of water for a neonate so lactose is very important!! What is lactose? • Over 60% of whole body glucose use in the lactating dairy cow is for lactose synthesis • Lactose = 1 molecule glucose + 1 molecule galactose • Glucose alone is the primary substrate for lactose synthesis • One glucose is converted to UDP-glucose, which is converted to one UDP-galactose • Another glucose is used for lactose synthesis without modification • 2 glucoses are required for each lactose molecule synthesized Glucose • Glucose passes across the Golgi membrane into the Golgi lumen by a glucose transporter (GLUT 1) • Presence of GLUT 1 on the Golgi membrane is specific to the mammary epithelial cell • Glucose transport does not require energy, but it is affected by glucose levels in cytoplasm Lactose Synthesis • UDP-galactose is actively transported into the Golgi lumen (rate limiting step) • UDP-glucose is not transported into the Golgi • Lactose is a non-permeable disaccharide and can’t diffuse out of the Golgi membrane or out of secretory vesicles' membrane • UDP generated from lactose synthesis could be inhibitory to lactose synthesis if it accumulated in the Golgi lumen • However, UDP is rapidly hydrolyzed into UMP and inorganic P by nucleoside diphosphatase (NDPase) • UMP is actively removed from the Golgi, while the inorganic P diffuses out of the Golgi • Lactose synthesis reaction is one-way • Lactose is not hydrolyzed to form glucose and galactose Lactose Synthase Complex • What forms lactose • Lactose synthase enzyme composed of 2 subunits: • Galactosyl transferase (GT) found in several tissues (only on inner surface of Golgi) • α-lactalbumin (a-LA)specifically catalyzes transfer of galactose to glucose • Lactose and α-lactalbumin are co-secreted via Golgi secretory vesicles • α-lactalbumin is limiting to lactose synthesis Galactosyltransferase • Normally involved in glycosylation of proteins in cells (adds galactose to oligosaccharides) • Enzymatic subunit of lactose synthase • Proteolytically clipped removing the cytoplasmic and transmembrane domains • Found in most tissues of the body • Only on the inner surface of the Golgi apparatus • Unique among all glycosyltransferases in that its substrate specificity can be modified by addition of α-lactalbumin α-lactalbumin • Makes up 25% of whey protein • Not active by itself, but is necessary for lactose synthesis • Synthesized in RER and passes to the Golgi where it interacts with GT • Alters sugar-binding properties of GT so that lactose is produced instead of the normal function of GT • Lactose can’t diffuse out of the Golgi or secretory vesicles, so water is drawn into the vesicles to balance the osmotic pressure α-lactalbumin • Secretory vesicles fuse with the apical surface of the mammary cell and dump the soluble a-LA into the lumen, but much of the GT stays bound to the apical membrane • GT can be found on milk fat membranes • Why would this be the case? • Rate of lactose synthesis is dependent on a-LA to GT ratio • Maximal lactose synthesis activity observed at an a-LA to GT molar ratio of 500 to 1 • Molar ratios of a-LA to GT > 500 to 1 did not increase lactose synthesis • The actual molar ratio of a-LA to GT in the Golgi of the mammary gland is not known Abbreviations: GT = galactosyltransferase a-LA = a- lactalbumin NDPase = nucleotide diphosphatase; Pi = inorganic phosphate PPi = inorganic diphosphate UDP = uridine diphosphate UDP-galactose = Uridine diphosphate galactose UDP-glucose = uridine diphosphate glucose UMP = uridine monophosphate UTP = uridine triphosphate α-lactalbumin Deficiency in Mice Variable +/+ +/- -/- Lactose % 6.2 4.3 0.01 Fat % 28.2 29.6 45.3 Protein % 8.8 9.6 16.5 5 + is wild-type, contains α-lactalbumin gene - is α-lactalbumin gene knockout - Stacey et al. (1995) PNAS 92:2835 Lactose Synthesis • Lactose synthesis increases by 13x between parturition and peak lactation • Due to increasing amounts of GT • Formation of lactose in Golgi results in drawing water into the cell, into the Golgi, and ultimately becoming part of milk • Golgi is involved in processing of milk proteins, synthesis of lactose, and the osmotic draw for water! • Mammary-specific genes turn on when milk synthesis is active • α-lactalbumin is key regulatory component of lactose synthase enzyme • Synthesis turned on by prolactin • Inhibited by progesterone Is the Reverse True? • If no α-lactalbumin means no milk production, can we just make animals produce more α-lactalbumin to get higher milk production? Swine Study • Copies of bovine α-lactalbumin gene into swine embyros • Resulting transgenic offspring were normal in all ways during growth and reproduction • During lactation, they overexpress total α-lactalbumin by producing the bovine α-lactalbumin protein from the inserted gene, in addition to producing their own porcine α-lactalbumin protein • Transgenic sows were matched with their non-transgenic siblings. • Milk production was significantly greater in the first parity sows overexpressing α-lactalbumin, compared to their sibling controls • Differences in milk production were greatest on days 3, 6, and 9 of lactation • But by day 12, production differences were not significantly different in sows overexpressing α- lactalbumin compared with their controls • Results suggest that overexpression of α-lactalbumin in first parity sows increases milk production early in lactation Bifidus Factor • Other carbohydrates are found in milk in low concentrations (e.g. free glucose and free galactose) • Some complex oligosaccharides are also present (e.g. bifidis factor in human milk) • Likely important in helping establish the microflora of neonate intestine • Stimulates growth of Lactobacillus bifidus (aka bifidobacterium) • Works to keep the environment of the gastrointestinal tract at a higher acidity level • Pathogenic organisms like less acidic environments • Other species may have ways of developing neonate intestinal tract, but nothing similar to this mechanism has yet been discovered Lactose Intolerance • Lactase is required to breakdown ingested lactose • Lacking in the intestinal lining of those affected after they stop drinking milk as main food source (beginning at about 5 years old) • After this happens, more lactase cannot be created • If milk consumption resumes, lactose is not used so it draws in water and causes diarrhea • Can eat: yogurt, kefir, aged cheeses (harder = less lactose), lactase-fortified dairy products (e.g. Lactaid) Galactosemia • Lactose is broken down by lactase into glucose & galactose • Enzymes needed for further metabolism of galactose missing • Leads to toxic levels of galactose 1-phosphate in various tissues • Without treatment, mortality in infants is ~ 75% • Inherited in an autosomal recessive manner • Heterozygotes are carriers, but show no symptoms Think about this… • Lactase is an enzyme that potentially would be very useful to people who are lactose intolerant. Suppose someone produced a transgenic sow that expressed lactase only in its mammary gland and only during lactation, with the intent of isolating that lactase from the milk and using it for lactose intolerant people. What would you expect to be the consequences of expression of such a transgene to the sow's production of milk and milk composition? Vitamins and Minerals in Milk • Minerals: Calcium, phosphorous, iodine, magnesium, zinc, potassium, sodium, selenium, iron • Fat-soluble vitamins: A, D, E, and K • Water-soluble vitamins: thiamin, riboflavin, niacin, pantothenic acid, pyridoxine, cobalamin, vitamin C, and folate Macro- vs Micro- Minerals • Macrominerals: macrominerals include calcium, potassium, sodium, magnesium, phosphorus, chloride and sulfur • Microminerals: chromium, copper, iron, fluorine, iodine, manganese, molybdenum, selenium, cobalt and zinc • Principal source of zinc vegetarians Calcium • Bound to casein micelles • Eat your Calcium in milk is 1000 times its cheese, concentration in mammary alveolar cells please! • Must be actively transported also Buffers/Osmotic Regulators • Buffers in milk (normal pH 6.6-6.8): citrates, phosphates, bicarbonate, protein • Osmotic regulators: LACTOSE, Na, Cl, K (because those minerals are mainly in ionic form) Questions?.