The influence of various lipids on the activity of bovine milk galactosyltransferase

Purified bovine milk galactosyltransferase was combined with liposomes of different lipid composition. The activity was markedly affected by the nature of the lipid used. Thus phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol stimulated the activity, while phosphatidic acid and phosphatidylserine inhibited the activity of the transferase. Phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid had identical fatty acid compositions, yet phosphatidylcholine and phosphatidylglycerol stimulated the activity while phosphatidic acid inhibited the activity. The effect on the enzyme was probably related to the nature of the head group since the inhibition by phosphatidic acid could be converted to stimulation by methylating the phosphatidic acid. The properties of several of the head groups is discussed. The physical state of the lipid was shown to affect the activity markedly. When the enzyme was combined with dimyristylphosphatidylcholine the activity was markedly stimulated when the lipid was in the liquid-crystalline state i.e., above the phase transition.     

Stimulation of bovine milk galactosyltransferase activity by bovine colostrum N-acetylglucosaminyltransferase I

Purified bovine milk galactosyltransferase was stimulated by purified bovine colostrum N-acetylglucosaminyltransferase I by more than 10-fold. Only slight stimulation of the N-acetylglucosaminyltransferase I by galactosyltransferase was observed. Heat inactivation destroyed the ability of the N-acetylglucosaminyltransferase I to stimulate the galactosyltransferase. The stimulation of galactosyltransferase was accompanied by a decrease in Km of this enzyme from 9.7 to 3.3. mM and an increase in Vmax from 1.87 to 3.71 nmol galactose transferred/min per mg galactosyltransferase when GlcNAc was the substrate. When the Km for UDPgalactose was determined, it increased from 0.19 to 0.42 mM in the presence of N-acetylglucosaminyltransferase I and the Vmax increased from 0.66 to 2.76 nmol galactose transferred/min per mg galactosyltransferase. In phosphatidylcholine vesicles, no effect on Km values with GlcNAc as substrate was noted, while an increase in the Km of UDPgalactose was observed. The Vmax values were generally higher in the lipid vesicles. Complex formation between galactosyltransferase and N-acetylglucosaminyltransferase I was demonstrated both by glycerol density gradient centrifugation and Bio-Gel P-100 column chromatography. An approximate molecular weight for the complex was obtained on a calibrated Sephadex G-200 column and found to be about 75 000, consistent with a 1:1 complex. The stimulation of galactosyltransferase involved the N-acetyllactosamine synthetase activity of this enzyme and not the lactose synthetase activity, since the latter activity was only slightly affected. Since N-acetylglucosaminyltransferase I is not involved in the lactose synthetase reaction, the stimulation is consistent with the known biosynthetic role of N-acetylglucosaminyltransferase I in the biosynthesis of asparagine-linked oligosaccharides.     

The effect of acidic lipids on the activity of bovine milk galactosyltransferase in vesicles of different phosphatidylethanolamines

Bovine milk galactosyltransferase was incorporated into vesicles prepared from different phosphatidylethanolamines which varied widely in both their gel-liquid crystalline and their lamellar-hexagonal phase transition temperatures. Although all phosphatidylethanolamines stimulated the activity of the enzyme the extent of stimulation varied. Acidic lipids phosphatidylserine and phosphatidic acid inhibited the activity of the enzyme incorporated into all of the phosphatidylethanolamines except when the enzyme was in soya PE in which the acidic lipids had no effect.     

The effect of commercial bovine serum albumin and other proteins on the activity of bovine milk galactosyltransferase

The widespread use of bovine serum albumin preparations for the stabilization of purified glycosyltransferases has prompted us to study the effects of different preparations of albumins on the galactosyltransferase activity of bovine milk. For comparison, several other proteins were tested as well. The albumins caused a large stimulation of transferase activity (400-700%) which varied depending on the source of the albumin and the treatment to which it had been subjected. Several other unrelated proteins were tested for their effects on transferase activity. Some proteins stimulated, while others had little effect. Lysozyme stimulated the activity by 178% and poly-L-lysine had little effect. Other proteins stimulated to variable extents. The stimulations obtained with albumin and myelin basic protein were noteworthy. The stimulation was considerably less marked when the enzyme was incorporated into lipid vesicles. These results emphasize the need for caution when adding proteins such as bovine serum albumin to purified enzymes for the purpose of stabilizing the activity of the enzyme.     

The interaction of bovine milk galactosyltransferase with lipid and alpha-lactalbumin

The activation of galactosyltransferase (UDPgalactose: N-acetyl-D-glucosaminyl-glycopeptide 4-beta-D-galactosyltransferase, EC 2.4.1.38) by alpha-lactalbumin has been studied at low concentrations of alpha-lactalbumin where the relationship is sigmoidal. The sigmoidal shape of the activation curve was eliminated by neutral lipids such as phosphatidylcholine and phosphatidylethanolamine, detergents such as Triton X-100 or by an aggregated form of alpha-lactalbumin generated by crosslinking alpha-lactalbumin with dithiobissuccinimidylpropionate. It is proposed that these different reagents present a hydrophobic surface to the enzyme which is necessary for lactose synthase activity. In competition experiments, large amounts of alpha-lactalbumin were able to displace lipid from the enzyme as suggested by the loss of the lipid-activating effect in the presence of an excess of alpha-lactalbumin. Optimal lactose synthase activity was obtained when the ratio of lipid/alpha-lactalbumin/enzyme was 60:6:1. The mechanism by which the lipid effect was obtained probably involved a phase transition in the enzyme which was detected as a sharp break in the Arrhenius curve. The presence of phosphatidylcholine abolished the break demonstrating that full activity of the enzyme required both alpha-lactalbumin and lipid.     

Antimicrobial activity of lipids added to human milk,infant formula,and bovine milk

Lipids previously shown to have antiviral and antibacterial activity in buffers were added to human milk, bovine milk, and infant formulas to determine whether increased protection from infection could be provided to infants as part of their diet. Fatty acids and monoglycerides with chain lengths varying from 8 to 12 carbons were found to be more strongly antiviral and antibacterial when added to milk and formula than long chain monoglycerides. Lipids added to milk and formula inactivated a number of pathogens including respiratory syncytial virus (RSV), herpes simplex virus type 1 (HSV-1), Haemophilus influenzae, and Group B streptococcus. The results presented in this study suggest that increased protection from infection may be provided to infants at mucosal surfaces, prior to the digestion of milk and formula triglycerides, by the addition of antimicrobial medium chain monoglycerides to an infant's diet.     

Plasmin and the conversion of the molecular forms of bovine milk galactosyltransferase

Human plasmin catalyzed the conversion of the high molecular weight form (58 000) of galactosyltransferase to the low molecular weight form (44 000) in a manner similar to the conversion catalyzed by the contaminating proteolytic activity found in highly purified galactosyltransferase preparations. A protease partially purified from bovine milk was inhibited by plasmin inhibitors as was the contaminating proteolytic activity.     

Characterization of a soluble glycolipid galactosyltransferase which occurs in bovine milk.

Bovine milk was found to contain, in soluble form, an enzyme which transfers galactose from UDPgalactose to glucosylceramide. This enzyme was partially purified by the same procedure used to isolate the galactosyltransferase of lactose synthetase. The partially purified enzyme required detergents for activity, had a pH optimum of 7.2--7.3 and required Mn2+. The apparent Km calculated for glucosylceramide was 1.33 . 10(-4) M. With glucosylceramide as acceptor the product of the reaction was identified as lactosylceramide by autoradiography on thin-layer chromatograms. Lactosylceramide was also an effective acceptor for the transferase reaction but neutral glycosphingolipids or gangliosides with terminal galactose of N-acetylgalactosamine residues were ineffective or poorly effective as acceptors. Addition of alpha-lactalbumin inhibited the transferase reaction.     

Modulation of human 5-lipoxygenase activity by membrane lipids

Mammalian 5-lipoxygenase (5-LO) catalyzes the conversion of arachidonic acid (AA) to leukotrienes, potent inflammatory mediators. 5-LO is activated by a Ca(2+)-mediated translocation to membranes, and demonstrates the characteristic features of interfacially activated enzymes, yet the mechanism of membrane binding of 5-LO is not well understood. In an attempt to understand the mechanism of lipid-mediated activation of 5-LO, we have studied the effects of a large set of lipids on human recombinant 5-LO activity, as well as mutual structural effects of 5-LO and membranes. In the presence of 0.35 mM phosphatidylcholine (PC) and 0.2 mM Ca(2+), there was substrate inhibition at >100 microM AA. Data analysis at low AA concentrations yielded the following: K(m) approximately 103 microM and k(cat) approximately 56 s(-1). 5-LO activity was supported by PC more than by any other lipid tested except for a cationic lipid, which was more stimulatory than PC. Binding of 5-LO to zwitterionic and acidic membranes was relatively weak; the extent of binding increased 4-8 times in the presence of Ca(2+), whereas binding to cationic membranes was stronger and essentially Ca(2+)-independent. Polarized attenuated total reflection infrared experiments implied that 5-LO binds to membranes at a defined orientation with the symmetry axis of the putative N-terminal beta-barrel tilted approximately 45 degrees from the membrane normal. Furthermore, membrane binding of 5-LO resulted in dehydration of the membrane surface and was paralleled with stabilization of the structures of both 5-LO and the membrane. Our results provide insight into the understanding of the effects of membrane surface properties on 5-LO-membrane interactions and the interfacial activation of 5-LO.     

Modulation of bovine serum amine oxidase activity by hydrogen peroxide

Bovine serum amine oxidase (BSAO), reduced by excess amine under limited turnover conditions, was over 80% inactivated by H(2)O(2) upon oxygen exhaustion. The UV-Vis spectrum and the reduced reactivity with carbonyl reagents showed that the cofactor topaquinone (TPQ) was stabilized in reduced form. The protein large M(r) (170 kDa) prevented the identification of modified residues by amino acid analyses. Minor changes of the Cu(2+) EPR signal and the formation of a radical at g = 2.001, with intensity a few percent of that of the Cu(2+) signal, unaffected by a temperature increase, suggest that Cu(2+)-bound histidines were not oxidized and the radical was not the Cu(+)-semiquinolamine in equilibrium with Cu(2+)-aminoquinol. It may derive from the modification of a conserved residue in proximity of the active site, possibly the tyrosine at hydrogen-bonding distance of TPQ C-4 ionized hydroxyl. The inactivation reaction appears to be a general feature of copper-containing amine oxidases. It may be part of an autoregulatory process in vivo, possibly relevant to cell adhesion and redox signaling.     

Posttranslational ruling of xanthine oxidase activity in bovine milk by its substrates

The aims of this study were to test the hypothesis that the substrates of xanthine oxidase (XO), xanthine and hypoxanthine, are consumed while the milk is stored in the gland between milkings, and to explore how XO activity responds to bacteria commonly associated with subclinical infections in the mammary gland. Freshly secreted milk was obtained following complete evacuation of the gland and induction of milk ejection with oxytocin. In bacteria-free fresh milk xanthine and hypoxanthine were converted to uric acid within 30 min (T1/2 approximately 10 min), which in turn provides electrons for formation of hydrogen peroxide and endows the alveolar lumen with passive protection against invading bacteria. On the other hand, the longer residence time of milk in the cistern compartment was not associated with oxidative stress as a result of XO idleness caused by exhaustion of its physiological fuels. The specific response of XO to bacteria species and the resulting bacteria-dependent nitrosative stress further demonstrates that it is part of the gland immune system.     

Modulation of the in vitro activity of lysosomal phospholipase A1 by membrane lipids

Lysosomal phospholipases play a critical role for degradation of cellular membranes after their lysosomal segregation. We investigated the regulation of lysosomal phospholipase A1 by cholesterol, phosphatidylethanolamine, and negatively-charged lipids in correlation with changes of biophysical properties of the membranes induced by these lipids. Lysosomal phospholipase A1 activity was determined towards phosphatidylcholine included in liposomes of variable composition using a whole-soluble lysosomal fraction of rat liver as enzymatic source. Phospholipase A1 activity was then related to membrane fluidity, lipid phase organization and membrane potential as determined by fluorescence depolarization of DPH, 31P NMR and capillary electrophoresis. Phospholipase A1 activity was markedly enhanced when the amount of negatively-charged lipids included in the vesicles was increased from 10 to around 30% of total phospholipids and the intensity of this effect depended on the nature of the acidic lipids used (ganglioside GM1相似文献   

Enhancement of UDP-galactose:mucin galactosyltransferase activity by spermine

A microsomal preparation prepared from the mucosal lining of canine trachea catalyzed the transfer of galactose from its uridine diphosphate derivative to sialidase-treated ovine submaxillary mucin. Maximal incorporation occurred at 30 mm mn²⁺. When the concentration of mn²⁺ in the reaction mixture was reduced to 2.5 mm, approximately two-thirds of the enzymatic activity was lost, but full activity could be restored by the addition of 1 mm spermine. Under these conditions spermine did not affect the K_m for UDP-galactose, but lowered the K_m for sialidase-treated ovine submaxillary mucin and Mn²⁺ by a factor of 10. The effect of spermine was abolished with increasing concentrations of Mn²⁺, and in the absence of the metal, enzymatic activity was lost and could not be restored by the addition of spermine. Spermidine also stimulated activity at low levels of Mn²⁺, but to a lesser degree than spermine. A slight stimulatory effect was consistently derived from putrescine as well, while cadaverine, putreanine, and monoamines were ineffectual. Spermine had a similar effect on the enzymatic transfer of GalNAc to a protein core acceptor but had little or no effect on the enzymatic transfer of sialic acid to sialidase-treated ovine submaxillary mucin, galactose to N-acetylglucosamine, or fucose to sialidase-galactosidase-treated fetuin. Similar results were obtained with enzyme preparations prepared from canine submaxillary glands. Other polycationic compounds such as protamine, histone, and polylysine also stimulated enzymatic activity at suboptimal concentrations of mn²⁺.