Characteristics of purified cows' milk xanthine oxidase and its submolecular characteristics.

Xanthine oxidase (EC 1.2.3.2) was purified from fresh cows' milk by differential centrifugation and hydroxylapatite chromatography in the absence of reducing agents and proteases. The purified isolate possessed an absorbance at 280 nm:absorbance at 450 nm ratio of 4.84; an absorbance (1 cm at 280 nm 1%) of 11.9; an activity:absorbance at 450 nm of 141, a specific activity of 3.59 units/mg; and detectable dehydrogenase activity. The enzyme preparation was obtained in a reversible oxidase form that could be partially converted to xanthine dehydrogenase in the presence of 10mM dithiothreitol or 1% mercaptoethanol. Amino acid analyses revealed that the enzyme was hydrophobic in nature and that lysine constituted its N-terminal residue. The protein contained 22 disulfide and 38 sulfhydryl groups, four of which were detectable in the undenatured protein complex. Discontinuous PAGE in the presence of selected dissociation agents did not result in further resolution. Sodium dodecyl sulfate-PAGE of the purified enzyme revealed a sharp zone with a molecular weight of 151,000 +/- 4000 (i.e., monomer). The purified enzyme exhibited oxidase activity in the presence of 6 M urea and following limited proteolysis by trypsin, chymotrypsin, plasmin, pancreatin, pepsin, and papain. Proteolyzed xanthine oxidase migrated as a single zone in polyacrylamide gels in the presence and absence of dissociating agents such as 1% mercaptoethanol and 6 M urea. Restricted digestion of xanthine oxidase by proteases was indicated by the presence of three major zones with molecular weights ranging from 85,000 to 100,000, 30,000 to 35,000, and 18,000 to 20,000 commonly observed in SDS gels. Amino acid profiles of the principal peptidyl fragments of trypsin-cleaved xanthine oxidase indicated their hydrophobic nature and lysine as the N-terminal residue for all fragments.

Blue-green color and composition of Stejneger's beaked whale (Mesoplodon stejnegeri) milk.

Two hundred ml of milk were obtained from a lactating Stejneger's beaked whale stranded at Ninilchik, Alaska on 21 Oct, 1980. Total solids (41%) were similar to values reported for sperm and belukha whales, while fat (17%) was half as great and crude protein (17%) was 2-4 times greater than in milk of these species. Lactose was not detected. Calcium (0.22%) was greater than reported for pigmy sperm whales but less than for blue whales. Phosphorus (0.07%) was less than for any of the above species. Sodium and potassium concentrations were 0.13% and 0.11%, respectively. Values (microgram/g) for other elements analyzed (magnesium, 42; iron, 35; copper, 2.6; zinc, 1.5; manganese, 0.3; selenium, 0.36) have not been reported for whale milk. Based on SDS-gel electropherograms, this whale milk did not contain a whey protein corresponding to cattle milk alpha-lactalbumin. A blue-green pigment in the milk was identified as biliverdin.

Compositional homology of membrane-protein systems and membrane-associated proteins: comparison with milk fat globule membrane and "membrane"-derived xanthine oxidase.

Amino acid compositions of the milk fat globule membrane protein and plasma membrane protein from other sources, as well as the compositions of milk fat globule membrane-derived xanthine oxidase and selected plasma membranes-associated proteins were compared by statistical difference index. Additionally, the average hydrophobicity of xanthine oxidase and selected membrane proteins were compared. These comparisons indicate high orders of apparent compositional homology between the various plasma membranes and membrane-associated proteins. Because the biological functions of membrane proteins are widely diverse, it is speculated that their "relatedness" may reflect on evolutionary convergence to similar amino acid compositions, necessitated by their in situ environment--the lipoidal bilayer. However, compositional relatedness should not imply sequential homology.

Homogenized milk: is it really the culprit in dietary-induced atherosclerosis?

Xanthine oxidase activity was assayed in commercial samples of homogenized milk subjected to pH ranging from 6.7 to 2.0 and held at room temperature for 5 min. Activity decreased sharply between pH 5.5 and 3.2. Below pH 3.2 no activity was detected. Also, rabbit anti-bovine xanthine oxidase failed to crossreact immunologically with xanthine oxidase of mouse milk. These results cast doubt on the hypothesis that dietary xanthine oxidase participates in the formation of atherosclerotic plaques.

Nomemclature of the proteins of cow's milk: fourth revision.

This report reviews the nomenclature of the milk proteins of cow's milk in light of more recent advances in our knowledge. With the establishment of the primary structures of a number of these proteins, we now have a definite identification of alphas1-, kappa-, beta-, and the gamma-caseins as well as beta-lactoglobulin and alpha-lactalbumin. On the basis of new information on their primary structures and relationship to beta-casein polymorphs, changes in nomenclature have been recommended for proteins of the gamma-casein fraction. Although the primary structure serves as the unambiguous definition of proteins for which it is known, a more practical identification is necessary. We recommend that their behavior in gel electrophoresis under suitable conditions be employed for this purpose for all of the "major" milk proteins of raw skim milk except the immunoglobulins where, because of their heterogeneity and molecular genetics, physical parameters are less useful and their identification must be based upon antigenic determinants and their homology with their human counterparts. More work is needed and, with the accumulation of more information, additional changes in nomenclature can be expected for such proteins as the minor components of alphas- and kappa-caseins, alpha-lactalbumin, and the proteose-peptone fraction as well as further confirmation of the presence of immunoglobulins IgE and additional IgG subclasses. Additional components and genetic variants also can be expected.

Molecular weight profile of fat globule membrane proteins.

Fat globule membrane material, isolated by churning cream that had been washed four times, was extracted sequentially with .6 M potassium chloride and centrifuged to yield pellet and supernatant fractions. Compositional data indicated that lipid components were removed preferentially into the supernatant fractions. Electropherograms of the pellet and supernatant fraction in 10% and 5% sodium dodecyl sulfate-containing polyacrylamide gels revealed that the protein species originally present in the membrane associated into higher molecular weight species concomitant with the removal of lipid moieties. Thus, a contribution of the lipid moiety to membrane integrity is suggested. Approximately 17 protein-stained zones, ranging in molecular weight from 13,500 to 208,000 daltons, were observed in gels of the membrane material.