Arginase in lactating bovine mammary glands: implications in proline synthesis.

The occurrence and subcellular distribution of arginase have been studied in mammary glands from lactating dairy cattle. The enzyme appears to be localized in the mitochondrial fraction, although a significant amount has been found to be associated with the cytosolic fraction. Both mitochondrial and cytosolic arginase are activated by heating with Mn2+. The Michaelis constants for the two fractions, however, are different: 49.5 and 18.5 mM for the mitochondrial fraction and cytosolic fraction, respectively. Overall the total enzyme concentration in the gland suggests that these enzymes contribute to the conversion of arginine to ornithine. Ornithine, in turn, may be converted by ornithine aminotransferase into an intermediate, L-delta 1-pyrroline-5-carboxylate; concurrently. alpha-keto-glutarate is transformed into glutamic acid. Finally, pyrroline-5-carboxylate reductase yields proline, an important amino acid that is needed for casein synthesis. Because pyrroline-5-carboxylate reductase requires NADPH, and because ornithine aminotransferase uses alpha-ketoglutarate, this new pathway is linked to the Krebs cycle through the cytosolic isocitrate dehydrogenase, which is the source of both of these intermediates.

Pyrroline-5-carboxylate reductase in lactating bovine mammary glands.

The occurrence and subcellular distribution of pyrroline-5-carboxylate reductase have been studied in lactating bovine mammary glands. The enzyme appears to have only a cursory association with the mitochondrial fraction, because significant amounts of the enzyme are found in other membrane-containing fractions and in the cytosol. Polyamines stimulate the enzyme in vitro, supporting the mediation of cursory attachment to membrane fractions by these compounds. The enzyme is selective for NADPH but can utilize NADH as well. Long-chain coenzyme A derivatives, which are generated during lipid metabolism, almost completely inhibit this enzyme, which is responsible for the synthesis of a portion of the proline needed for casein production. Overall, the enzyme concentration in the gland correlates well with a role in the conversion of an intermediate, L-delta 1-pyrroline-5-carboxylate, into proline, an important amino acid for the mammary secretory process, especially casein synthesis.

Ornithine-delta-aminotransferase in lactating bovine mammary glands.

The occurrence and subcellular distribution of ornithine-delta-aminotransferase have been studied in lactating bovine mammary glands. The enzyme is localized in the mitochondria and has a unique thermal reaction profile that distinguishes it from putative liver and kidney isozymes. The enzyme concentration in the gland correlates well with a role in the conversion of ornithine into the proline precursor, L-delta 1-pyrroline-5-carboxylate. However, an unusually high Michaelis constant for the mitochondrial enzyme (8.4 mM) raises the question of enzyme efficiency in vivo such that this pathway needs to be considered in estimating barriers to protein secretion into milk.

Chromatographic and electrophoretic methods used for analysis of milk proteins.

Current knowledge of milk proteins and their behavior in dairy foods is based on early applications of chromatography and electrophoresis. Electrophoretic identification of the number and genetic variety of milk proteins inaugurated a research effort in which chromatographic techniques were successfully applied to the isolation of each milk protein, thus facilitating the characterization and further study of milk and dairy products. This review focuses on recent applications of chromatography for separations and analysis and on analytical applications of electrophoresis.

Distribution of adenosine-5'-diphosphatase activity in the lactating bovine mammary gland.

A Ca2+- and Mg(2+)-stimulated adenosine-5'-diphosphatase has been found in lactating bovine mammary glands. The enzyme is associated with membranes of mitochondrial, microsomal, and Golgi apparatus fractions. The pH activity curves for the Golgi apparatus and microsomal fractions display two distinct maxima, one at pH 5.8 and the other between pH 7.4 and 8.4. Studies with activators and inhibitors indicate the enzyme is similar to adenosine-5'-diphosphatase found in other tissues. Its occurrence in the Golgi apparatus fraction indicates a possible role for this enzyme in the milk secretory process, particularly in ATP cycling in vesicles. Its occurrence in the microsomal fraction suggests a role in plasma membrane functioning.

Differential release of proteins from bovine fat globule membrane.

Alkaline phosphatase and 5'-nucleotidase are covalently linked to phosphatidylinositol in bovine fat globule membrane, as demonstrated by their release following treatment with phospholipase C specific for phosphatidylinositol. The failure of this treatment to liberate phosphodiesterase I may indicate that it has a variant linkage resistant to release. In a test of exposure at the membrane surface, alkaline phosphatase and phosphodiesterase I, but not 5'-nucleotidase, were released from fat globule membrane by treatment with proteinase K. These apparent differences in accessibilities of membrane surface proteins suggest that attachment to phosphatidylinositol does not necessarily impart greater exposure to proteins with which it is linked.

Identification of the milk fat globule membrane proteins. II. Isolation of major proteins from electrophoretic gels and comparison of their amino acid compositions.

The fat globule membranes of milk are derived from the apical plasma membrane of the mammary secretory cells. The nature of the membrane proteins, as isolated from cows' milk, has been studied by the use of discontinuous and continuous SDS-gel electrophoresis. Six methods of preparation of milk fat globule membrane suggested by various authors were tested; gel electrophoresis showed that five major bands were present, independent of the method of preparation. The apparent molecular masses of these proteins as determined on SDS-gels (15% T) were 167, 142, 64, 49 and 46 kDa, respectively. The 167 kDa band stained only with periodic acid-Schiff reagent, while the 142 kDa band stained only with Coomassie blue; the last three bands stained with both. Delipidated membranes were extracted stepwise with water, 0.02 M NaCl and 0.6 M NaCl. The 64 kDa band appears to be nearly insoluble, while the bands of 142, 49 and 46 kDa are fractionated by this procedure. The resolution of all of these proteins by electrophoresis was superior to that achieved by molecular sieve chromatography, and so electrophoretic extraction was used to isolate the major proteins. Dansyl chloride derived proteins were used as markers. Amino acid compositions of the recovered proteins were obtained and are compared.

Charge separation of proteins complexed with sodium dodecyl sulfate by acid gel electrophoresis in the presence of cetyltrimethylammonium bromide.

Globular proteins, casein, and membrane proteins which were reacted with sodium dodecyl sulfate were studied by acid urea gel electrophoresis. The sodium dodecyl sulfate bound tightly to the proteins, producing a more acidic charge which prevented migration into the gel. When cetyltrimethylammonium bromide was added to the sodium dodecyl sulfate-protein complexes, the sodium dodecyl sulfate apparently reacted with cetyltrimethylammonium bromide and dissociated so that the proteins migrated in acid gel in a normal manner as compared to the proteins without any added detergent. The sodium dodecyl sulfate-cetyltrimethylammonium bromide complex could be removed from the proteins by centrifugation. Thus, cetyltrimethylammonium bromide used in conjunction with acid gel electrophoresis allows direct comparison by charge of proteins fractionated in the presence of sodium dodecyl sulfate with the starting mixture of proteins not exposed to detergent. The reaction of cetyltrimethylammonium bromide with sodium dodecyl sulfate in acidic urea also provides a simple convenient method of removal of sodium dodecyl sulfate from proteins.

Identification of the milk fat globule membrane proteins. I. Isolation and partial characterization of glycoprotein B.

The salt soluble proteins from the fat globule membrane of cow's milk were resolved into three fractions by Sephadex column chromatography in sodium dodecyl sulfate. One of the fractions, termed glycoprotein B, was purified by rechromatography to essentially one band on sodium dodecyl sulfate gel electrophoresis. It was found to contain 14% carbohydrate including sialic acid, mannose, galactose, glucose, glucosamine and galactosamine. The amino acid composition of glycoprotein B was determined; it has amino terminal serine and carboxyl terminal leucine. The molecular weight of this glycoprotein as estimated by sodium dodecyl sulfate gel electrophoresis is 49 500.