Search

XOR (xanthine oxidoreductase) purified from human milk was shown to contain 0.04 atom of Mo and 0.09 molecule of molybdopterin/subunit. On the basis of UV/visible and CD spectra, the human enzyme was approx. 30% deficient in iron-sulphur centres. Mo(V) EPR showed the presence of a weak rapid signal corresponding to the enzyme of low xanthine oxidase activity and a slow signal indicating a significant content of desulpho-form. Resulphuration experiments, together with calculations based on enzymic activity and Mo content, led to an estimate of 50-60% desulpho-form. Fe/S EPR showed, in addition to the well-known Fe/S I and Fe/S II species, the presence of a third Fe/S signal, named Fe/S III, which appears to replace partially Fe/S I. Comparison is made with similarly prepared bovine milk XOR, which has approx. 15-fold higher enzymic activity and Mo content. Taken along with evidence of low Mo content in the milk of other mammals, these findings add further support to the idea that XOR protein plays a physiological role in milk (e.g. in secretion) equal in importance to its catalytic function as an enzyme.

Caseins and many other secretory proteins are phosphorylated during their transport through the secretory pathway by a protein kinase present within Golgi compartments. Molecular analysis of the Golgi casein kinase (GCK) has not been possible since it has not been purified to homogeneity or been cloned. Previous attempts have been made to purify GCK activity from mammary gland Golgi fractions, but these have not resulted in extensive purification of the enzyme. In the present study, we have demonstrated that substantial amounts of GCK activity, assayed using a specific peptide substrate, can be detected as a soluble form in bovine milk, and we have used milk as a source for purification. A purification protocol was established that allowed>80000-fold purification to a specific activity of GCK (approx. 700 nmoles/min per mg of protein) far higher than previously achieved. These findings cast doubts on previous claims for purification of GCK activity. In addition, ion-exchange chromatography resolved two closely eluting peaks of activity, suggesting the existence of two related, but distinct, GCK activities.

Much of the enzymic machinery required for the assembly of cell surface carbohydrates is located in the endoplasmic reticulum (ER) of eukaryotic cells. Structural information on these proteins is limited and the identity of the active polypeptide(s) is generally unknown. This paper describes the synthesis and characteristics of a photoaffinity reagent that can be used to identify and analyse members of the ER glycan assembly apparatus, specifically those glycosyltransferases, nucleotide phosphatases and nucleotide-sugar transporters that recognize uridine nucleotides or UDP-sugars. The photoaffinity reagent, P3-(4-azidoanilido)uridine 5'-triphosphate (AAUTP), was synthesized easily from commercially available precursors. AAUTP inhibited the activity of ER glycosyltransferases that utilize UDP-GlcNAc and UDP-Glc, indicating that it is recognized by UDP-sugar-binding proteins. In preliminary tests AAUTP[alpha-32P] labelled bovine milk galactosyltransferase, a model UDP-sugar-utilizing enzyme, in a UV-light-dependent, competitive and saturable manner. When incubated with rat liver ER vesicles, AAUTP[alpha-32P] labelled a discrete subset of ER proteins; labelling was light-dependent and metal ion-specific. Photolabelling of intact ER vesicles with AAUTP[alpha-32P] caused selective incorporation of radioactivity into proteins with cytoplasmically disposed binding sites; UDP-Glc:glycoprotein glucosyltransferase, a lumenal protein, was labelled only when the vesicle membrane was disrupted. These data indicate that AAUTP is a membrane topological probe of catalytic sites in target proteins. Strategies for using AAUTP to identify and study novel ER proteins involved in glycan assembly are discussed.

Lipoprotein lipase (LPL) purified from bovine milk showed variable abilities to stimulate the binding of low density lipoprotein (LDL) to J774 macrophages. The presence of a 37 kDa protein in the LPL sample seemed to be of importance for its stimulatory capacity. In order to investigate this, we isolated LPL from bovine milk via heparin Sepharose chromatography using a continuous salt gradient. Fractions containing the 37 kDa protein (as shown by SDS/PAGE under reducing conditions) eluted first from the column, followed by the 56 kDa LPL protein. The LPL enzymatic activity co-eluted with the 56 kDa protein, whereas the amount of 37 kDa protein fully paralleled the stimulatory effect on the binding of LDL to J774 cells. Samples not containing the 37 kDa protein were far less effective in stimulating the binding. Western blotting using a monoclonal antibody 5D2 against amino acids 396-405 in the carboxy-terminal domain of LPL, showed that the 37 kDa protein may be the C-terminal domain of LPL, presumably generated by proteolytic degradation of the mature LPL protein by milk proteases during its isolation. Furthermore, the functional mass of LPL for stimulation of the binding of LDL, as determined by radiation inactivation, was shown to be 30.9+/-1.8 kDa. We therefore suggest that cleavage of LPL at protease-sensitive sites causes a conformational change, generating an LPL protein which is more effective in mediating the binding and uptake of lipoproteins by cells.

Lipoprotein lipase (LPL) has been proposed to play a role in the uptake of chylomicron remnants by hepatocytes by mediating the binding of these lipoproteins to cell-surface glycosaminoglycans and to the low-density-lipoprotein receptor-related protein (LRP). This proposal is based on studies that examined the binding of chylomicrons to HepG2 cells, fibroblasts and Chinese hamster ovary cells in culture, in the presence of large amounts of LPL [Beisiegel (1995) Curr. Opin. Lipidol. 6, 117-122]. We have investigated whether LPL attached to the surface of chylomicrons enhances the binding and uptake of these lipoproteins to isolated hepatocytes maintained in culture. Bovine milk LPL was bound to mouse chylomicrons, double-labelled in vivo with [3H]retinol (in retinyl esters) and with [14C]palmitic acid (in triacylglycerols), collected from the mesenteric lymph of normal mice and from mice lacking the apoprotein E (apo E) gene. Normal chylomicrons (containing apo E) and apo E-free chylomicrons, with or without bound LPL, were incubated with cultured hepatocytes isolated from mice lacking the apo E gene. At 0 degree C LPL did not enhance the binding of the normal or apo E-free chylomicrons by the hepatocytes. When incubations were performed at 37 degrees C the triacylglycerols of normal and apo E-free chylomicrons were hydrolysed by LPL and there was a significant uptake of [14C]fatty acids and [3H]retinol by the hepatocytes. The addition of heparin or lactoferrin, a known inhibitor of hepatic uptake of chylomicron remnants, to the incubation medium inhibited the uptake of [3H]retinol, present in the lipoprotein core, but not the uptake of the [14C]fatty acids. We conclude that: (1) LPL attached to chylomicrons in amounts sufficient to effectively hydrolyse their core triacylglycerols does not enhance the binding of these lipoproteins to the surface of isolated hepatocytes; (2) the recognition and uptake of chylomicrons by hepatocytes requires that these lipoproteins be first hydrolysed by LPL; and (3) the uptake of lipolysed chylomicrons (remnants) by hepatocytes does not require the mediation of apo E.

We examined the effects of reproductive stage and fasting on lipoprotein lipase (LPL) activity and mRNA in the mouse mammary gland. Heparin-releasable and cell-associated LPL activity rose immediately after birth, followed 1-2 days later by an increase in LPL mRNA. Fasting decreased LPL activity in the mammary gland at all reproductive stages. During lactation, both milk and heparin-releasable LPL were substantially decreased by an overnight fast, whereas cell-associated LPL was less affected and LPL mRNA did not change. These studies indicate that the extracellular, heparin-releasable, fraction of mammary LPL activity responds most rapidly to alterations in physiological state, usually accompanied by smaller changes in cellular enzyme activity. Changes in the level of LPL mRNA were seen only during the transition from pregnancy to lactation, and these tended to follow, rather than precede, changes in enzyme activity. We conclude that in the mammary gland as in adipose tissue, LPL is regulated primarily at the translational and post-translational level.

The course of the reaction sequence hypoxanthine----xanthine----uric acid catalysed by xanthine:oxygen oxidoreductase from milk was investigated on the basis of u.v. spectra taken during the course of hypoxanthine and xanthine oxidations. It was found that xanthine accumulated in the reaction mixture when hypoxanthine was used as a substrate. The time course of the concentrations of hypoxanthine, xanthine intermediate and uric acid product was simulated numerically. The mathematical model takes into account the competition of substrate, intermediate and product and the accumulation of the intermediate at the enzyme. This type of analysis permits the kinetic parameters of the enzyme for hypoxanthine and xanthine to be obtained.

1. The catalytic properties of xanthine oxidase in bovine milk (EC 1.2.3.2) are dependent on the state of the enzyme, i.e. whether free or bound to the fat-globule membrane. Oxidase activity of the membrane-bound enzyme towards NADH is enhanced relative to that towards xanthine. This reflects a change in the relative K(m) values and enables the ratio of xanthine to NADH oxidase activities (X/N) to be used as a parameter for the relative amounts of free and membrane-bound xanthine oxidase in milk fractions. 2. Chromatography of buttermilk on Sepharose 2B yielded an excluded fraction, BM(1), with xanthine oxidase activity. The remaining xanthine oxidase activity was eluted as a single broad peak. This was further resolved on Sephadex G-200 into an excluded fraction, BM(2), and free xanthine oxidase. Fractions BM(1) and BM(2) had X/N values in the range 45-65, which is characteristic of membrane-bound xanthine oxidase. Purified xanthine oxidase has a mean X/N value of 110.3. Addition of fraction BM(1), heated to remove associated enzyme activities, to purified xanthine oxidase progressively enhanced its NADH oxidase activity to a value where its X/N value was characteristic of membrane-bound xanthine oxidase. This was shown to be due to binding of free enzyme to heated fraction BM(1). The binding constant and stoicheiometry were determined. 4. Proteolytic digestion of fraction BM(1) liberated free xanthine oxidase from the fat-globule membrane with a corresponding alteration in X/N value.

A unique ribonuclease, active only in the presence of Ca2+, was present in lactating mammary gland and milk of the rat. This enzyme was absent from virgin-rat mammary gland and non-mammary tissues of lactating rats. The presence of moderate activity in differentiated mammary tumours, together with an increase in activity in normal tissue parelleling development of mammary function, identify this enzyme as a marker of mammary differentiation in the rat.

1. Sulphydryl oxidase from bovine milk was purified by covalent affinity chromatography on cysteinylsuccinamidopropyl-glass. Selective immobilization of the oxidase occurs through formation of a mixed disulphide between the enzyme and the substrate cysteinyl-glass matrix. Reductive elution of the bound protein can be accomplished with small thiols such as reduced glutathione (GSH), dithiothreitol or cysteine. This method leads to approx. 4000-fold purification of the enzyme from whey. Furthermore, complete resolution of sulphydryl oxidase from gamma-glutamyltransferase was achieved with this procedure. 2. Antibodies prepared against this purified enzyme quantitatively precipitated 95% of the GSH-oxidative activity from detergent-solubilized skim-milk membranes, whereas 100% of the transferase activity remained in the supernatant fraction; these findings confirmed the distinction between these two enzymes. 3. Reverse-phase high-pressure-liquid-chromatographic analyses of assay mixtures containing both enzymes revealed an array of GSH derivatives generated by a combination of the oxidative and hydrolytic activities. However, purified sulphydryl oxidase yielded only GSSG with concomitant stoichiometric loss of GSH. 4. The chromatographic method described is simple and reproducible, and may be applicable to isolation of sulphydryl oxidase from other tissues.

1. Lipoprotein lipase (EC 3.1.1.34), which was previously shown to bind to immobilized heparin, was now found to bind also to heparan sulphate and dermatan sulphate and to some extent to chondroitin sulphate. 2. The relative binding affinities were compared by determining (a) the concentration of NaCl required to release the enzyme from polysaccharide-substituted Sepharose; (b) the concentration of free polysaccharides required to displace the enzyme from immobilized polysaccharides; and (c) the total amounts of enzyme bound after saturation of immobilized polysaccharides. By each of these criteria heparin bound the enzyme most efficiently, followed by heparan sulphate and dermatan sulphate, which were more efficient than chondroitin sulphate. 3. Heparin fractions with high and low affinity for antithrombin, respectively, did not differ with regard to affinity for lipoprotein lipase. 4. Partially N-desulphated heparin (40-50% of N-unsubstituted glucosamine residues) was unable to displace lipoprotein lipase from immobilized heparin. This ability was restored by re-N-sulphation or by N-acetylation; the N-acetylated product was essentially devoid of anticoagulant activity. 5. Partial depolymerization of heparin led to a decrease in ability to displace lipoprotein lipase from heparin-Sepharose; however, even fragments of less than decasaccharide size showed definite enzyme-releasing activity. 6. Studies with hepatic lipase (purified from rat post-heparin plasma) gave results similar to those obtained with milk lipoprotein lipase. However, the interaction between the hepatic lipase and the glycosaminoglycans was weaker and was abolished at lower concentrations of NaCl. 7. The ability of the polysaccharides to release lipoprotein lipase to the circulating blood after intravenous injection into rats essentially conformed to their affinity for the enzyme as evaluated by the experiments in vitro.

Enzymically inactive acetyl-CoA carboxylase [acetyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1.2] was found as a component of bovine milk-fat-globule membrane (MFGM). Acetyl-CoA carboxylase was present in MFGM at a higher concentration than in cytosolic or mitochondrial fractions of bovine mammary tissue, which makes it unlikely that its presence was due to simple contamination by these subcellular constituents.

E.p.r- (electron-paramagnetic-resonance) spectroscopy was used to compare chemical environment and reactivity of molybdenum, flavin and iron-sulphur centres in the enzyme xanthine dehydrogenase from Veillonella alcalescens (Micrococcus lactilyticus) with those of the corresponding centres in milk xanthine oxidase. The dehydrogenase is frequently contaminated with small but variable amounts of a species resistant to oxidation and giving a new molybdenum (V) e.p.r. signal, "Resting I". There is also a "desulpho" form of the enzyme giving a Slow Mo(V) signal, indistinguishable from that of the milk enzyme. Molybdenum of the active enzyme behaves in a manner analogous to that of the milk enzyme, giving a Rapid Mo(V) signal on partial reduction with substrates or dithionite. Detailed comparison shows that molybdenum in each enzyme must have the same ligand atoms arranged in the same manner. As with the milk enzyme, complex-formation between reduced dehydrogenase and purine substrate molecules, presumably interacting at the normal substrate-binding site, modifies the Rapid signal, confirming that such substrates interact near molybdenum. The dehydrogenase-flavin semiquinone signal is identical with that of the oxidase but, in contrast, there is only one iron-sulphur signal. The latter gives an e.p.r. spectrum similar to that of aldehyde oxidase.

Sulphydryl oxidase is known to catalyse the synthesis de novo of disulphide bonds in a variety of thiol-containing compounds. Reduced glutathione is the best thiol substrate; however, D- and L-cysteine, cysteamine and N-acetyl-L-cysteine, as well as cysteine-containing peptides and proteins, are also effectively oxidized. In contrast, oxidation of the thiol groups of mercaptoethanol, mercaptopyridine, dithiothreitol, dithioerythritol, mercaptoacetate, mercaptopropionate or lipoic acid is not detectably catalysed. In bovine milk, sulphydryl oxidase is closely associated with another glutathione-metabolizing enzyme, gamma-glutamyltransferase. Covalent chromatography of crude preparations on cysteinylsuccinamidopropyl-glass resolves the oxidase from the transferase, thus permitting the kinetic characterization of glutathione oxidation. Initial-rate data imply a Ter Bi substituted-enzyme mechanism, and the observed substrate inhibition by thiols suggest that O2 binds first. Independent, non-kinetic, data, namely the immobilization of sulphydryl oxidase on cysteinyl-matrices, support formation of a mixed-disulphide intermediate between the thiol and enzyme, as predicted by the proposed mechanism. The enzyme-catalysed reaction appears not to be mediated via a superoxide intermediate, since O2 consumption is not affected by the presence of Nitro Blue Tetrazolium. FAD, NAD+, NADP+ and Nitro Blue Tetrazolium are all inactive as electron acceptors for sulphydryl oxidase catalysis.

1. The mid-point reduction potentials of the various groups in xanthine oxidase from bovine milk were determined by potentiometric titration with dithionite in the presence of dye mediators, removing samples for quantification of the reduced species by e.p.r. (electron-paramagnetic-resonance) spectroscopy. The values obtained for the functional enzyme in pyrophosphate buffer, pH8.2, are: Fe/S centre I, -343 +/- 15mV; Fe/S II, -303 +/- 15mV; FAD/FADH-; -351 +/- 20mV; FADH/FADH2, -236 +/-mV; Mo(VI)/Mo(V) (Rapid), -355 +/- 20mV; Mo(V) (Rapid)/Mo(IV), -355 +/- 20mV. 2. Behaviour of the functional enzyme is essentially ideal in Tris but less so in pyrophosphate. In Tris, the potential for Mo(VI)/Mo(V) (Rapid) is lowered relative to that in pyrophosphate, but the potential for Fe/S II is raised. The influence of buffer on the potentials was investigated by partial-reduction experiments with six other buffers. 3. Conversion of the enzyme with cyanide into the non-functional form, which gives the Slow molybdenum signal, or alkylation of FAD, has little effect on the mid-point potentials of the other centres. The potentials associated with the Slow signal are: Mo(VI)/Mo(V) (Slow), -440 +/- 25mV; Mo(V) (Slow)/Mo(IV), -480 +/- 25 mV. This signal exhibits very sluggish equilibration with the mediator system. 4. The deviations from ideal behaviour are discussed in terms of possible binding of buffer ions or anti-co-operative interactions amongst the redox centres.

1. Xanthine oxidase (EC 1.2.3.2) was found to represent more than 8% of the intrinsic protein of the bovine milk-fat-globule membranes. 2. Less than 25% of the xanthine oxidase activity of the fat-globule membrane was solubilized with 0.1 M-sodium pyrophosphate buffer or 2M-NaCl. Of the particulate activity remaining 56% was solubilized with Triton X-100. 3. The xanthine oxidase activity solubilized with buffer, 2M-NaCl or Triton X-100 was not liberated as the free enzyme. Only tryptic digestion was found to release the free enzyme from the fat-globule membrane. Tryptic digestion also liberated free xanthine oxidase from those fractions solubilized by buffer or NaCl, but not from those fractions solubilized with Triton X-100 or by sonication. 4. The effect of membrane association on the catalytic properties of the enzyme could be mimicked by low pH or by the presence in the assay mixture of certain concentrations of 2-methyl-propan-2-ol, but not 1,4-dioxan, suggesting that hydrogen-bonding rather than low dielectric constant may be involved. 5. The origin of the milk-fat-globule membrane is discussed with reference to the intrinsic nature of the associated xanthine oxidase activity.

Lipoprotein lipase and salt-resistant lipase were isolated from human post-heparin plasma. The proteins of human post-plasma lipoprotein lipase and salt-resistant lipase were identified and demonstrated to be immunologically different. Significant differences between the two enzymes in their relative amino acid composition were demonstrated, which indicates that the two enzymes are different proteins. When analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the enzymes seemed to have monomer molecular weights similar to that of lipoprotein lipase purified from bovine milk.

It was deduced many years ago from indirect evidence that demolybdo xanthine oxidase is present in normal bovine milk. This has now been confirmed by isolation of this enzyme form by a method based on the folate-gel affinity-chromatography procedure described Nishino & Tsushima [(1986) J. Biol. Chem. 261, 11242-11246]. Enzymic and spectroscopic properties of demolybdo xanthine oxidase, which retains flavin and iron-sulphur centres, are generally in accordance with expectations. Like the normal enzyme, it yields on denaturation material fluorescing at 460 nm. Molybdenum cofactor activity measured by the Neurospora crassa nit-1 assay in the presence of added molybdate was 33% of that of the normal enzyme. The absorption spectrum in the near-u.v. region differs slightly, but significantly, from that of the active and desulpho forms of the enzyme. It is concluded that the molybdenum cofactor site contains a pterin-like material not identical with that in the normal enzyme. The significance of the occurrence of demolybdo xanthine oxidase in milk is discussed, and evidence in the literature for demolybdo forms of other molybdoenzymes is briefly reviewed. Additional studies on the use of the affinity procedure for large-scale preparation of high-activity xanthine oxidase are described. In agreement with our ability to isolate the demolybdo enzyme, the procedure appears less effective in eliminating the demolybdo than the desulpho enzyme.

In an incubation system in vitro with fully activated Intralipid as substrate, rat high-density lipoprotein inhibits the hydrolysis of triacylglycerol by lipoprotein lipase from rat adipose tissue, but does not inhibit hydrolysis by the enzyme from bovine milk. The pattern of inhibition suggests that substrate and high-density lipoprotein may compete for association with rat adipose-tissue lipoprotein lipase.

Lipoprotein lipases from a variety of sources have been shown previously to bind to heparin and some related polysaccharides. For the present studies lipoprotein lipase purified from bovine milk was used. 1. In batch experiments binding of the enzyme activity to heparin-Sepharose occurred relatively slowly, so that 30min was required for the system to come to near-equilibrium. In contrast, release of the enzyme activity from heparin-Sepharose by addition of salt to the liquid phase occurred rapidly. 2. Some binding was observed also with unsubstituted Sepharose, but this binding had a low capacity compared with that observed with heparin-Sepharose. High salt concentrations, heparin or deoxycholate decreased the binding to unsubstituted Sepharose. These factors also increase the solubility of the enzyme, which is low. 3. Addition of heparin to the liquid phase caused a concentration-dependent release of enzyme activity from the gel. These results suggested that the binding of the enzyme to heparin-Sepharose was mainly through interaction with heparin. 4. The enzyme activity was also quantitatively displaced to the liquid phase at increased concentrations of salt. Among the positive ions tested the following order of effectiveness was noted: Cs(+) approximately K(+)>Na(+)>Li(+); and among the negative the following: SCN(-)>I(-)> NO(3) (-)>Br(-) approximately Cl(-). The differences were quite large. Thus addition of 0.16m-KSCN (in addition to the 0.32m-NaCl originally present) displaced one-half of the enzyme activity to the supernatant, whereas 0.8m-LiCl only displaced one-quarter. 5. The distribution of heparin in the gel also profoundly influenced the binding. Two series of gels were studied. One series was made by mixing heparin-Sepharose with unsubstituted Sepharose. Results obtained with these gels were those expected from a series of decreasing volumes of heparin-Sepharose. In contrast, a series of heparin-Sepharoses made with different degrees of substitution gave quite different results. With these gels the amount of enzyme activity bound per amount of heparin increased markedly, whereas the salt concentration needed to displace the enzyme activity from the gel decreased markedly with decreased concentration of heparin in the gel. 6. On stepwise elution of small columns of heparin-Sepharose the enzyme activity was eluted over a remarkably wide range of salt concentrations. When enzyme eluted at one salt concentration was re-applied, it gave the same elution profile as enzyme previously eluted at other salt concentrations or the entire enzyme preparation. These and other results suggested that, whereas the enzyme preparation was rather homogeneous in its binding to heparin, the heparin preparation was polydisperse in binding of lipoprotein lipase.

Magnetic interaction between molybdenum and one of the iron-sulphur centres in milk xanthine oxidase [Lowe, Lynden-Bell & Bray (1972) Biochem. J. 130, 239-249] was studied further, with particular reference to the newly discovered Mo(V) e.p.r.(electron-paramagnetic-resonance) signal, Resting II [Lowe, Barber, Pawlik & Bray (1976) Biochem. J. 155, 81-85]. E.p.r. measurements at 35GHz near to 4.2K showed that the interaction has the same sign at all molybdenum orientations and is ferromagnetic. The predicted splitting of the e.p.r. signal from the reduced iron-sulphur centre, Fe/S I, was observed, Providing positive identification of this as the other interacting species. Chemical modification of the molybdenum environment in xanthine oxidase can change the size of the interaction severalfold, but interaction always remains approximately isotropic. The interaction in turkey liver xanthine dehydrogenase is indistinguishable from that in the oxidase. However, a bacterial xanthine dehydrogenase with different iron-sulphur centres shows rather larger interaction. Guanidinium chloride disturbs the iron-sulphur centres of the oxidase, and when this occurs there is a parallel and relatively small change in the interaction. Removal of flavin from the molecule, or raising the pH to 12.0, changes the interaction slightly without affecting the chromophores themselves. It is concluded that the Fe/S I centre and the Mo are at least 1.0nm and probably nearer 2.5nm apart, and that the conformation of the protein between them is relatively stable up to pH 12.

Molybdenum(V) e.p.r. signals from reduced functional milk xanthine oxidase molecules (the Rapid signals), obtained in the presence of purine substrates and products, were further investigated [cf. Bray & Vänngård, (1969) Biochem. J. 114, 725-734; Pick & Bray (1969) Biochem. J. 114, 735-742]. Xanthine forms two complexes with the enzyme that are believed to correspond to different orientations of the substrate molecule in the active site. Only one complex appears to undergo the catalytic reaction. Non-productive complexes, analogous to theone with xanthine, are not formed by 1-methylxanthine or purine. Uric acid forms more than one e.p.r.-detectable complex, one of which is analogous to the non-productive xanthine complex. The computer program used for handing the e.p.r. data is described briefly.

Milk samples were taken from rats feeding ten pups and from both the suckled and non-suckled glands of rats feeding two pups. The lipid, protein and lactose concentrations were similar in the milks from the secreting glands, but the fluid from the non-suckled glands contained less lactose and lipid but significantly higher total protein and transferrin concentrations. The fatty acid compositions of the milk from the three sources were very similar. The mammary tissue from the rats feeding ten pups had a higher DNA content/g wet wt. than either the suckled or non-suckled mammary tissue of the rats feeding two pups. The specific activities of several lipogenic enzymes were significantly lower in the non-suckled mammary tissue.

A procedure is described for isolation of the pterin molybdenum cofactor, in the active molybdenum-containing state, starting from purified milk xanthine oxidase. The method depends on the use of anaerobic-glove-cabinet techniques and on working in aqueous solution, in the presence of 1 mM-Na2S2O4. SDS was used to denature the protein, followed by ion-exchange chromatography and gel filtration. The cofactor, obtained at concentrations up to 0.5-1.0 mM, was fully active in the nit-1 assay [Hawkes & Bray (1984) Biochem. J. 214, 481-493], with a specific activity of 22 nmol of NO2-/min per pg-atom of Mo (with 15% molybdate-dependence). The Mr, determined by gel filtration, was about 610, consistent with the structure proposed by Kramer, Johnson, Ribeiro, Millington & Rajagopalan [(1987) J. Biol. Chem. 262, 16357-16363]. At pH 5.9, under anaerobic conditions, the cofactor was stable for at least 300 h at 20-25 degrees C.

Studies by e.p.r. (electron-paramagnetic-resonance) spectroscopy and by stopped-flow spectrophotometry on turkey liver xanthine dehydrogenase revealed strong similarities to as well as important differences from the Veillonella alcalescens xanthine dehydrogenase and milk xanthine oxidase. The turkey enzyme is contaminated by up to three non-functional forms, giving molybdenum e.p.r. signals designated Resting I, Resting II and Slow. Slow and to a lesser extent Resting I signals are like those from the Veillonella enzyme, whereas Resting II is very like a resting signal described by K. V. Rajagopolan, P. Handler, G. Palmer & H. Beinert (1968) (J. Biol. Chem. 243, 3784-3796) for aldehyde oxidase. Another non-functional form that gives the Inhibited signal is produced on treatment of the enzyme with formaldehyde. Stopped-flow measurements at 450 nm show that, as for the milk enzyme, reduction by xanthine is rate-limiting in enzyme turnover. The active enzyme gives rise to Very Rapid and Rapid molybdenum(V) e.p.r. signals, as well as to an FADH signal. That these signals are almost indistinguishable from those of the milk enzyme, confirms the similarities between the active sites. There are two types of iron-sulphur centres that give signals like those in the milk enzyme, though with slightly different parameters. Quantitative reduction titration of the functional enzyme with xanthine revealed two important differences between the turkey and the milk enzymes. First, the turkey enzyme FADH/FADH2 system has a redox potential sufficiently low that xanthine is incapable of reducing the flavin completely. This finding presumably explains the very low oxidase activity. Secondly, whereas the Fe/S II chromophore in the milk enzyme has a relatively high redox potential, for the turkey enzyme the value of this potential is lower and similar to that of its Fe/S I chromophore.

Immunochemical evidence is presented for the presence of blood-group-related carbohydrate structures on human milk galactosyltransferase and for the occurrence of the corresponding specificities among rabbit antibodies to this enzyme. Although these carbohydrate specificities constitute minor populations among antisera and affinity-purified antibodies to galactosyltransferase, their presence is important in the immunohistochemical approach to enzyme localization, since they give rise to strong reactivities with epithelial cells of the gastrointestinal tract.

A new non-functional modified form of milk xanthine oxidase is described. This contains molybdenum in a quinquivalent state, which is resistant to both oxidation and reduction. The new species is derived from the native enzyme in a two-step process. The first step is the conversion into the desulpho form, via loss of the 'persulphide' sulphur, and the second involves reaction with ethylene glycol or other reagents. The species gives a characteristic Mo(V) electron-paramagnetic-resonance signal, without proton splittings, designated Resting II. This is virtually identical with signals reported previously from resting turkey liver xanthine dehydrogenase and rabbit liver aldehyde oxidase. The possibility is discussed that species Resting II, prepared with ethylene glycol, contains a -COCH2OH residue bound to a nitrogen ligand of molybdenum.

1. The presence of xanthine was required for the inhibition of bovine milk xanthine oxidase by o-iodosobenzoate, iodoacetamide, hydrogen peroxide or p-chloromercuribenzoate. 2. Inactivation by p-chloromercuribenzoate was very rapid, was reversed by cysteine and was less in the presence of FAD. Lineweaver-Burk plots showed that the inactivation by p-chloromercuribenzoate was competitive with substrate. 3. Inactivation by o-iodosobenzoate, iodoacetamide or hydrogen peroxide could not be reversed by cysteine or xanthine. However, the presence of xanthine during the incubation with inhibitor protected the enzyme against o-iodosobenzoate but not against iodoacetamide or hydrogen peroxide. 4. p-Chloromercuribenzoate protected the enzyme against inactivation by hydrogen peroxide.

The composition of milk xanthine oxidase has been reinvestigated. When the enzyme is prepared by methods that include a selective denaturation step in the presence of sodium salicylate the product is obtained very conveniently and in high yield, and is homogeneous in the ultracentrifuge and in recycling gel filtration. It has specific activity higher than previously reported preparations of the enzyme and its composition approximates closely to 2mol of FAD, 2g-atoms of Mo and 8g-atoms of Fe/mol of protein (molecular weight about 275000). In contrast, when purely conventional preparative methods are used the product is also homogeneous by the above criteria but has a lower specific activity and is generally comparable to the crystallized enzyme described previously. Such samples also contain 2mol of FAD/mol of protein but they have lower contents of Mo (e.g. 1.2g-atom/mol). Amino acid compositions for the two types of preparation are indistinguishable. These results confirm the previous conclusion that conventional methods give mixtures of xanthine oxidase with an inactive modification of the enzyme now termed ;de-molybdo-xanthine oxidase', and show that salicylate can selectively denature the latter. The origin of de-molybdo-xanthine oxidase was investigated. FAD/Mo ratios show that it is present not only in enzyme purified by conventional methods but also in ;milk microsomes' (Bailie & Morton, 1958) and in enzyme samples prepared without proteolytic digestion. We conclude that it is secreted by cows together with the active enzyme and we discuss its occurrence in the preparations of other workers. Studies on the milks of individual cows show that nutritional rather than genetic factors determine the relative amounts of xanthine oxidase and de-molybdo-xanthine oxidase. A second inactive modification of the enzyme, now termed ;inactivated xanthine oxidase', causes variability in activity relative to E(450) or to Mo content and formation of it decreases these ratios during storage of enzyme samples including samples free from demolybdo-xanthine oxidase. We conclude that even the best purified xanthine oxidase samples described here and by other workers are contaminated by significant amounts of the inactivated form. This may complicate the interpretation of changes in the enzyme taking place during the slow phase of reduction by substrates. Attempts to remove iron from the enzyme by published methods were not successful.

Skim milks containing sodium chloride (0.75m) were centrifuged at 80000g for 2hr. and portions of the supernatants were submitted to gel filtration on columns of Sephadex G-200. Enzymes in the effluent fractions were assayed titrimetrically for their hydrolytic activities towards tributyrin, triolein and milk-fat emulsions, and triacetin solution. Summation of the measurements gave ratios of activities towards the various substrates similar to those of the original skim milks. Although only partial separation was obtained, five enzymes appeared to be present. They showed some differences in substrate specificity, but all appeared to be lipases in that they hydrolysed the emulsified substrates more rapidly than the dissolved triacetin.

1. The phosphorylation of milk alkaline phosphatase was studied under various conditions: maximum incorporation occurred at pH5.0 and 50% incorporation at pH6.6-7.0. 2. The phosphorylation was shown to be specific and the results suggest that the active centre of the enzyme is involved in the process. 3. Phosphoryl-enzyme is rapidly hydrolysed at alkaline pH. at pH7.0 the results suggest that a phosphoryl-enzyme could occur as a transient intermediate in the hydrolysis of phosphate esters by the phosphatase. 4. The catalytic-centre activity of the enzyme was found to be 2700sec.(-1) at pH10.0 and 25 degrees with p-nitrophenyl phosphate as substrate.

1. The xanthine oxidase of cow's milk, crude or purified, appears as an oxidase (type O), and can be converted almost completely into a NAD(+)-dependent dehydrogenase (type D) by treatment with dithioerythritol or dihydrolipoic acid, but only to a small extent by other thiols. 2. The D form of the enzyme is inhibited by NADH, which competes with NAD(+). 3. The kinetic constants of the two forms of the enzyme are similar to those of the corresponding forms of rat liver xanthine oxidase. 4. Milk xanthine oxidase is converted into an irreversible O form by pretreatment with chymotrypsin, papain or subtilisin, but only partially with trypsin. 5. The enzyme as purified shows a major faster band and a minor slower band on gel electrophoresis. The slower band is greatly reinforced after xanthine oxidase is converted into the irreversible O form by chymotrypsin.