Lactose Synthetase Research Articles

Inhibition by progesterone of the lactogenic effect of prolactin in the pseudopregnant rabbit.

The effects of progesterone on lactose synthesis activity and changes in mammary gland cells were studied in pseudopregnant rabbits simultaneously treated with prolactin. The injection of progesterone alone on Days 15 and 17 of pseudopregnancy decreased the activity of lactose synthetase (LSA) and galactosyl transferase (GTA), while the administration of prolactin for 2-4 days increased their activities. Th e simultaneous administration of progesterone and prolactin decreased the increase in LSA observed with prolactin alone by 70% on the 4th day of treatment, and decreased GTA by 30%. Progesterone completely suppressed the polyribosome profile and the ratio of endoplasmic reticulum bound polyribosomes to free polyribosomes induced by prolactin. The increase in RNA content in the mammary gland induced by prolactin was also suppressed by progesterone. The results suggest that progesterone inhibits the lactogenic action of prolactin without interfe ring with its mammogenic role.

Inactivation of Soluble Bovine Milk Galactosyltransferase (Lactose Synthetase) by Sulfhydryl Reagents and Trypsin: PROTECTION BY SUBSTRATES AND PRODUCTS

Abstract Galactosyltransferase exhibited a time-dependent loss of activity when incubated with sulfhydryl reagents p-chloromercuribenzoate and N-ethylmaleimide. Though incubations were continued for periods 20-fold greater than the half-time of inactivation, neither reagent eliminated all catalytic activity, and generally 5 to 10% of the initial activity remained. p-Chloromercuribenzoate inactivated the enzme more rapidly then N-ethylmaleimide, but the inhibition by p-chloromercuribenzoate was reversed by mercaptoethanol. Substrates and substrate analogs protected against inhibition by p-chloromercuribenzoate and N-ethylmaleimide. Ligands giving the most protection against inhibition were analogs of the nucleotide substrate and product, uridine diphosphate galactose and UDP, respectively, in the presence of manganous chloride. When incubated with 10 mm N-ethylmaleimide, galactosyltransferase exhibited an apparent rate constant of inactivation of 0.023 min-1; adding analogs of UDP at concentrations of 2 mm reduced this rate by 30% and the same analogs in the presence of manganous chloride reduced the rate constant of inactivation by 98%. Inhibition of galactosyltransferase by N-ethylmaleimide resulted in a dramatic decrease in Vmax whereas the apparent Km for UDP-galactose remained constant. A similar pattern of protection against inactivation of galactosyltransferase by trypsin was observed suggesting that a conformational change in the protein is responsible for the observed protection.

A study on the effect of lysolecithin and phospholipase A on membrane-bound galactosyltransferase.

Addition of lysolecithin caused very marked activation of UDP-galactose:glycoprotein galactosyltransferase in rat liver microsomes and in Golgi-rich membranes. Lysolecithin activated galactosyltransferase when the enzyme was assayed both with endogenous acceptor and with exogenous proteins or monosaccharides as acceptors. Lactose synthetase activity in presence of α-lactalbumin was also stimulated by lysolecithin. Lecithin, lysophosphatidylethanolamine, lysophosphatidic acid, and glycerophosphorylcholine did not activate the enzyme, suggesting that both fatty acyl and phosphorylcholine groups of the lysolecithin molecule are required for the observed activation. The degree of activation was about the same when myristoyl-, palmitoyl-, oleoyl-, or stearoyllysolecithin were tested. The activation by lysolecithin was observed well within the physiological concentration of the lipid in the liver cell. Saturating amounts of Triton masked the effect of lysolecithin.Brief preincubation with phospholipase A activated the enzyme and generated lysolecithin in the membranes. Triton and lysolecithin activated the enzyme without any lag time, whereas phospholipase A activation was dependent on preincubation and also on an alkaline pH favorable for the hydrolysis of phospholipid. EDTA blocked the activation effect of phospholipase A but had no effect on activation by lysolecithin. Albumin and cholesterol opposed the effects of lysolecithin and phospholipase A on the enzyme. Two successive incubations of the microsomes with lysolecithin caused considerable release of the enzyme into the soluble fraction. The role of lysolecithin in the activation of the enzyme is probably related to the solubilization of the membrane and consequent enhanced interaction of the enzyme with substrate. Lysolecithin also activated N-acetylglucosaminyl- and sialyltransferase activities in microsomes. A possible role of lysolecithin is indicated in the regulation of glycosylation reactions in mammalian system.

The correlation between the apparent molecular weight and the enzymic activity of lactose synthetase

FEBS LettersVolume 47, Issue 1 p. 94-97 Full-length articleFree Access The correlation between the apparent molecular weight and the enzymic activity of lactose synthetase G.S. Challand, G.S. Challand Department of Biochemistry, University College, London, Gower Street, London WC1 e 6BT, England Department of Pathological Biochemistry, Royal Infirmary, Castle Street, Glasgow, Scotland Search for more papers by this authorM.A. Rosemeyer, M.A. Rosemeyer Department of Biochemistry, University College, London, Gower Street, London WC1 e 6BT, EnglandSearch for more papers by this author G.S. Challand, G.S. Challand Department of Biochemistry, University College, London, Gower Street, London WC1 e 6BT, England Department of Pathological Biochemistry, Royal Infirmary, Castle Street, Glasgow, Scotland Search for more papers by this authorM.A. Rosemeyer, M.A. Rosemeyer Department of Biochemistry, University College, London, Gower Street, London WC1 e 6BT, EnglandSearch for more papers by this author First published: October 01, 1974 https://doi.org/10.1016/0014-5793(74)80433-3Citations: 4 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 K. Brew, T.C. Vanaman, R.L. Hill, Proc. Natl. Acad. Sci. U.S., 59, (1968), 491– 497. 2 U. Brodbeck, K.E. Ebner, J. Biol. Chem., 241, (1966), 762– 764. 3 U. Brodbeck, W.L. Denton, N. Tanahashi, K.E. Ebner, J. Biol. Chem., 242, (1967), 1391– 1397. 4 K.O. Pedersen, Biochem. J., 30, (1936), 948– 960. 5 P. Andrews, FEBS Letters, 9, (1970), 297– 300. 6 I.P. Trayer, R.L. Hill, J. Biol. Chem., 246, (1971), 6666– 6675. 7 G.S. Challand, Ph. D. Thesis (1970), University of London 8 R.J. Ivatt, M.A. Rosemeyer, FEBS Letters, 28, (1972), 195– 197. 9 P. Andrews, Biochem. J., 91, (1964), 222– 233. 10 W.A. Klee, C.B. Klee, J. Biol. Chem., 247, (1972), 2336– 2344. 11 K. Brew, T.C. Vanaman, R.L. Hill, J. Biol. Chem., 242, (1967), 3747– 3749. Citing Literature Volume47, Issue1October 01, 1974Pages 94-97 ReferencesRelatedInformation

Simple flow-cell for the fluorimetric study of conformational changes of proteins on agarose matrix

The construction and utilization of a new cylindrical cell for fluorescence measurements on protein-Sepharose 4B conjugates is described. This experimental device proved very convenient for fluorimetry of proteins covalently bound to agarose gels, for measurements on proteins in solution, and finally for monitoring the adsorption of proteins in the course of affinity chromatography. With the aid of this cell, the fluorescence spectra of human α-lactalbumin in solution and in an insoluble state were compared. The α-lactalbumin-Sepharose 4B complex gives a spectrum which closely resembles that of the native protein. Fluorescence spectra were recorded with as little as 50 μliters gel in the cell, which corresponds to approximately 0.015–23 nmoles of chemically bound protein. The fluorescence intensity was within experimental error proportional to protein concentration from 0.03 to 0.20 nmole bound protein/mg dry resin. The application of this fluorimetric method to conformational studies on membrane bound enzymes such as the proteins of the lactose synthetase function is discussed.

Studies on the thiol group of lactose synthetase A protein from human milk and on the binding of uridine diphosphate galactose to the enzyme.

The lactose synthetase activity of A protein from human milk was much decreased but not abolished by reaction with thiol-group reagents. Protection experiments indicated that a free thiol group on the enzyme is situated near the UDP-galactose binding site and inactivation of the enzyme with p-hydroxymercuribenzoate was probably due to prevention of UDP-galactose binding. Affinity chromatography showed that the mercuribenzoate substituent also decreased the affinity of A protein for N-acetylglucosamine but complex-formation between A protein-N-acetylglucosamine and alpha-lactalbumin was relatively unaffected. UDP-galactose appears to be bound to the enzyme mainly through its pyrophosphate group with Mn(2+) ion and through the cis hydroxyls of ribose, whereas its hexose moiety has little if any affinity for the enzyme. Lactose synthetase activity remaining after the reaction with thiol-group reagents indicates that a free thiol group is not an essential part of the A protein active site.

Metabolic Adaptations in Fatty Acid and Lactose Biosynthesis by Sheep Mammary Tissue During Cessation of Lactation

The effect of involution of fatty acid synthesis, lactose synthesis, and carbon dioxide production by sheep mammary tissue was examined. Mammary tissue from lactating or nonlactating (9 days after weaning) sheep was used for measurements of biosynthetic capacities (in vitro incubations) and enzymatic activities. Termination of lactation decreased biosynthetic rates of fatty acid and lactose synthesis 99 and 97%. Similarly, rates of acetate and glucose oxidation to carbon dioxide by mammary tissue from nonlactating sheep relative to lactating sheep decreased 84 and 98%. Of enzymes in fatty acid synthesis, acetyl-CoA carboxylase decreased the most dramatically as a result of involution (99%). Other major decrease were for acetyl-CoA sythetase (79%) and fatty acid synthetase (85%) while enzymes involved in reducing equivalent generation (glucose-76-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and NADP-isocitrate dehydrogenase) decreased less dramatically. for enzymes in lactose synthesis, involution decreased UDP-glucose 4-epimerase (76%), UDP-glucose pyrophosphorylase (87%), and lactose synthetase (93%) while activity of phosphoglucomutase remained unchanged. With the termination of lactation, the magnitude of alterations in rates of fatty acid sythesis and lactose synthesis compared most closely to decreases in acetyl-CoA carboxylase activity and lactose synthetase activity, respectively.

Some kinetic properties of human-milk galactosyl transferase.

Reactions catalyzed by human milk N-acetyl lactosamine synthetase in the presence and absence of ä-lactalbumins have been investigated by steady-state kinetics. N-Acetyl lactosamine synthesis and lactose synthesis in the absence of α-lactalbumin appear to proceed by an ordered sequential reaction, with substrates attaching in the order: Mn2+, UDP-galactose and monosaccharide. Under the conditions used (pH 7.4, 37 °C) the attachment of Mn2+ is not at thermo-dynamic equilibrium and it appears that the enzyme can accept either free UDP-galactose or its Mn2+ complex as substrate. Evidence is presented which suggests that the Mn2+ complex of UDP may be the final product released from the enzyme. Reactions in the presence of α-lactalbumin proceed by a similar ordered mechanism. Kinetic effects observed in the presence of human α-lactalbumin with three different monosaccharide acceptors, and in the presence of bovine α-lactalbumin with glucose, can be reasonably explained only by assuming that α-lactalbumin attaches to the enzyme immediately before monosaccharides, contrary to suggestions by other workers. It is proposed that α-lactalbumin attaches to an enzyme Mn2+ UDP-galactose complex at thermodynamic equilibrium, producing a new enzyme form with increased affinity for monosaccharides. The inhibitory effects of ä-lactalbumin on N-acetyl lactosamine synthesis are attributed to inhibition resulting from attachment of the protein to a central complex in an alternative pathway in the reaction scheme. The kinetic effects of four α-lactalbumins with human galactosyl transferase are characterized and intrinsic differences shown to be independent of the source of galactosyl transferase. The function deduced for α-lactalbumin in the lactose synthetase system is discussed in relation to its structure and a procedure is indicated for quantitatively measuring activity differences in α-lactalbumins.

The occurrence and properties of soluble UDP-galactose:glycoprotein galactosyltransferase in human amniotic fluid.

Human amniotic fluid obtained by amniocentesis during the third trimester of pregnancy has been found to contain the enzyme for transferring galactose from UDP-galactose into various exogenous acceptors. The enzyme galactosyltransferase is present in soluble form and does not require Triton for activity. The optimum pH (pH 6.8), divalent cation requirement (Mn2+, 12.5 mM), optimum temperature (37 °C), and linearity with time and enzyme concentrations have been established. The amniotic fluid enzyme catalyzed the transfer of 60 nmol of galactose to fetuin acceptor per milliliter of amniotic fluid per hour and its apparent Km for UDP-galactose is 9.1 × 10−5 M. The enzyme is stable to storage at −20 °C for at least 2 months. Acid hydrolysis and β-galactosidase treatment of the labelled reaction product released all of the radioactivity as 14C-galactose. Uridine nucleotides inhibited the soluble enzyme whereas the adenine and cytidine nucleotides had no appreciable effect. The addition of α-lactalbumin in the assay caused the appearance of lactose synthetase activity and inhibition of N-acetyllactosamine synthetase activity. The specific activity of the enzyme in the amniotic fluid in human and rat is 30- to 40-fold higher than in their serum. Placenta, fetal liver, and fetal lungs of rat also showed considerable enzyme activity.Enzymes that transfer N-acetylglucosamine, sialic acid, and N-acetyl-4-galactosamine from their respective nucleotide sugars to exogenously added acceptors are also present in human amniotic fluid.

The regulatory effect of α-lactalbumin in the lactose synthetase system

The regulatory effect of α-lactalbumin in the lactose synthetase system

Selective Sulfenylation of Tryptophan Residues in α-Lactalbumin of Bovine Milk

Abstract Tryptophan residues of bovine α-lactalbumin, one of the two protein components of lactose synthetase, were selectively sulfenylated with 2-nitrophenylsulfenyl chloride. When the reaction was performed in 0.1 m acetic acid for 1 hour, a modified protein containing 1.0 nitrophenylsulfenyl tryptophan residue per α-lactalbumin molecule was formed and isolated in 42% yield. Upon reduction, carboxymethylation, and trypsin digestion, this sulfenylated α-lactalbumin (NPS1-αLA) yielded two yellow peptides. Amino acid composition of these peptides indicated that in NPS1-αLA, tryptophan residues 60 and 118 were equally sulfenylated to the extent of 0.5 nitrophenylsulfenyl tryptophan per α-lactalbumin. NPS1-αLA cross-reacts with anti-α-lactalbumin antibodies, but, unlike the native protein, it has no biological activity in the lactose synthesis reaction, and its electrophoretic mobility on polyacrylamide gels is different from that of native α-lactalbumin.

Progesterone and the metabolic control of the lactose biosynthetic pathway during lactogenesis in the rat

1. Lactogenesis was initiated in pregnant rats by ovariectomy, thereby causing progesterone withdrawal, after which the mammary tissue was analysed for contents of enzymes and metabolites concerned with the biosynthesis of lactose. 2. Lactose synthesis increased about 126-fold with little or no accompanying change in the contents of most metabolic intermediates or in the adenine nucleotide energy charge. 3. Comparison of mass-action ratios with equilibrium constants showed that phosphoglucomutase (EC 2.7.5.1), UDP-glucose pyrophosphorylase (EC 2.7.7.9) and UDP-glucose epimerase (EC 5.1.3.2.) catalysed reactions close to equilibrium. Nucleoside diphosphokinase (EC 2.7.4.6.) activity was very high and probably equilibrates the UTP-UDP and ATP-ADP couples. Lactose synthetase and hexokinase (EC 2.7.1.1) appeared to catalyse rate-limiting reactions. 4. Large increases were seen of UDP-glucose pyrophosphorylase (5-fold), lactose synthetase A protein (3.8-fold) and alpha-lactalbumin (28-fold), but not of hexokinase, phosphoglucomutase, UDP-glucose epimerase, nucleoside diphosphokinase or glucose 6-phosphate dehydrogenase (EC 1.1.1.49) activities. 5. It appeared that the increased lactose synthesis was largely accounted for by the increased lactose synthetase A protein activity and alpha-lactalbumin.

Use of affinity chromatography for the quantitative study of acceptor–ligand interactions: the lactose synthetase system (Short Communication)

From the effects of N-acetyl-d-glucosamine and d-glucose on the elution of the A protein of human lactose synthetase from a column of Sepharose-alpha-lactalbumin, values of 200m(-1) and 0.57m(-1) are deduced for the association constants describing the interaction between the enzyme and the respective monosaccharides.

Metabolic adaptations during lactogenesis. Fatty acid and lactose synthesis in cow mammary tissue.

1. Mammary-tissue biopsies were obtained from multiparous cows at 30 and 7 days pre partum and 7 and 40 days post partum. Investigations of the effect of lactogenesis on fatty acid and lactose synthesis involved measurements of biosynthetic capacity (tissue-slice incubations in vitro) and activities of relevant enzymes. 2. Fatty acid synthesis from acetate increased over 20-fold from 30 days pre partum to 40 days post partum. Changes in the lipogenic capacity of mammary-tissue slices more closely paralleled increases in the activities of acetyl-CoA carboxylase (EC 6.4.1.2) and acetyl-CoA synthetase (EC 6.2.1.1) than of other enzymes involved in acetate incorporation into fatty acids or in NADPH generation. 3. Lactose biosynthesis by mammary-tissue slices, lactose synthetase activity (EC 2.4.1.22) and alpha-lactalbumin concentration were all negligible at 30 days pre partum but increased 2.5-4-fold between 7 days pre partum and 40 days post partum. Phosphoglucomutase (EC 2.7.5.1), UDP-glucose pyrophosphorylase (EC 2.7.7.9) and UDP-glucose 4-epimerase (EC 5.1.3.2) had substantial activities at 30 days pre partum and increased less dramatically during lactogenesis. 4. Results are consistent with acetyl-CoA carboxylase and perhaps acetyl-CoA synthetase representing the regulatory enzyme(s) in fatty acid synthesis, with lactose synthetase (alpha-lactalbumin) serving a similar function in lactose biosynthesis.

Lactogenesis in the diabetic rat.

The transition of the mammary gland from the quiescent state of pregnancy to the secretory state characteristic of lactation requires the presence of several hormones. Thus, when lactogenesis in rats or mice is monitored by increases of mammary lactose or casein, or of biosynthetic enzymes such as lactose synthetase, it shows a minimum requirement for prolactin and corticosteroid. Cultured mammary explants from pregnant mice undergo similar changes when given a minimum hormonal supplement of insulin, prolactin and a corticosteroid (for review see Denamur, 1971). The insulin is required in vitro at high concentrations (about 50 mu./ml) and apparently stimulates a wave of cell division from which the daughter cells undergo 'functional differentiation' in the presence of the other two hormones (for review see Topper, 1968, 1970). Lactogenesis in vivo is accompanied or preceded by active mitosis according to some authors (Jeffers, 1935; Baldwin & Milligan, 1966) but not according to

Further Evidence of Plasma Membrane Material in Skim Milk

Membrane fragments and microvillus like sacs were concentrated from bovine skim milk by differential centrifugation into a fraction resting on the casein pellet. The ratios of the free cholesterol, lipid phosphorus, and triglyceride contents of the fraction were similar to those for plasma membrane of the lactating cell. Assays for marker enzymes of Golgi apparatus (lactose synthetase), rough endoplasmic reticulum (nicotinamide adenine dinucleotide phosphate-cytochrome c reductase), and plasma membrane (glycosyl hydrolase) demonstrated the presence of only the latter. Suspension and recentrifugation of the membrane fraction produced a supernatant and a second (purified) membrane fraction resting as fluff on a second small casein pellet. Lipid analyses revealed that triglyceride with traces of free cholesterol and phospholipid were released from the original membrane fraction to the second supernantant. The relative content of these three lipid classes in conjunction with the enzyme assays provided further evidence for the co-identity of skim milk membrane material and plasma membrane of the lactating mammary cell.

Maleyl-α-lactalbumin as lactose synthetase specifier protein

We have studied the effects on the function and conformation of bovine α-lactalbumin after reaction with the protein-dissociating reagent maleic anhydride. The 13 amino groups of α-lactalbumin were accessible to the reagent and when all of these were maleylated a highly acidic and more expanded protein resulted. Despite these differences in physical properties, maleyl-α-lactalbumin was as effective as native α-lactalbumin in the lactose synthetase reaction. On the other hand, when α-lactalbumin was treated with trinitrobenzenesulphonic acid, inactivation occurred.

Incorporation of Galactose into Blood‐Groups (ABH) Precursor Substance by Lactose Synthetase from Human Milk

It has been shown that an enzyme preparation from human milk having lactose synthetase activity is capable of incorporating galactose residues into water‐soluble blood‐group substance. A suitable acceptor substance for investigating the enzymatic galactose transfer was obtained from blood‐group H‐substance by sequential removal of fucose and terminal galactose residues. When galactose was incorporated into this acceptor substance with the help of a preparation of galactosyl transferase from human milk in the presence of UDP‐galactose, a remarkable increase in cross‐reactivity with anti‐type‐14 pneumococcus sera could be observed. Since a glycoprotein with “type‐14 specificity”, having galactose as the serologically determinant group, is the precursor of the water‐soluble blood‐group A, B, H and Le substances, this enzymatic galactose transfer would appear to be an essential step in the biosynthesis of the blood‐group‐active compounds. Evidence for the identity of the enzyme responsible for the synthesis of type‐14‐specific sites with lactose synthetase of human milk was obtained from competition experiments.

An Early Effect of Prolactin on the Formation of α-Lactalbumin by Mouse Mammary Epithelial Cells

Abstract Epithelial cells from freshly isolated mammary glands of mature virgin mice make minute amounts of casein and exhibit low levels of lactose synthetase A-protein (a UDP-galactosyltransferase) activity. They have barely detectable lactose synthetase B-protein (α-lactalbumin) activity. When cultured in the presence of insulin, hydrocortisone, and prolactin, mammary explants from mature virgin mice develop the capacity to synthesize these milk proteins asynchronously. Casein synthesis and A-protein activity reach their maxima after 72 hours of exposure to the hormones and 24 hours before the emergence of B-protein activity. Estradiol-17β, or prolactin, priming of mature virgin mice reduces the time required for the development of B-protein activity in the mammary explants, while the time course of casein synthesis and appearance of A-protein activity is not appreciably affected. As a result, the secretory proteins, casein and α-lactalbumin, develop synchronously. This represents a heretofore undiscovered manifestation of the influence of prolactin in this system.

Apparent heterogeneity of bovine α-lactalbumin A and B in DEAE-Sephadex chromatography

The major component of bovine α-lactalbumin B exhibits a bimodal pattern on elution from columns of DEAE-Sephadex A-50 or DEAE-cellulose using a linear salt gradient in buffers at pH 6.3–7.9. This bimodality is not observed on zone electrophoresis, sedimentation and Sephadex gel filtration under the conditions studied. It originates in an interacting system and not true heterogeneity, since a bimodal pattern is observed on re-chromatography of protein from either peak. The relative areas of the peaks depend on the previous treatment of the protein: application of samples eluted from DEAE-Sephadex and Sephadex G-75, with or without concentration by ultrafiltration, give predominantly the first peak, but concentration by (NH 4) 2SO 4 precipitation favours formation of the second peak. Exhaustive dialysis against column buffers and the presence of Mg 2−, glucose, lactose, “A protein” do not affect the relative size of the peaks. The A variant behaves similarly to B, but is eluted at higher salt concentration. No difference in lactose synthetase activity or optical rotation between 195 nm and 400 nm can be detected between protein samples from each peak. Possible causes of the interaction are discussed.