Mammary tissue enzymes

Ruminant milk fats are also rich in medium-chain fatty acids. These are synthesized in the mammary gland via the usual malonyl CoA pathway (section 3.5) and are released from the synthesizing enzyme complex by thioacylases presumably, the higher levels of medium chain acids in ruminant milk fats compared with those of monogastric animals reflect higher thioacylase activity in the mammary tissue of the former. 

Synthesis of fatty acids via the malonyl CoA pathway does not proceed beyond palmitic acid (C16 0) and mammary tissue contains an enzyme, thioacylase, capable of releasing the acyl fatty acid from the carrier protein at any stage between C4 and C16. Probable interspecies differences in the activity of thioacylase may account for some of the interspecies differences in milk fatty acid profiles. 

Isolation and characterization. Alkaline phosphatase is concentrated in the fat globule membrane and hence in cream. It is released into the buttermilk on phase inversion consequently, buttermilk is the starting material for most published methods for the purification of alkaline phosphatase. Later methods have used chromatography on various media to give a homogeneous preparation with 7440-fold purification and 28% yield. The characteristics of milk alkaline phosphatase are summarized in Table 8.2. The enzyme appears to be similar to the alkaline phosphatase of mammary tissue. 

In many in vitro studies the acylation of the sn-3 position appears to be the rate-limiting step in TG synthesis. It has been suggested that the intracellular concentration of medium chain fatty acids may limit the final acylation reaction in TG synthesis (Dimmena and Emery 1981). Another theory is that the concentration of phosphatidate phosphatase, the enzyme that hydrolyzes the phosphate bond in phospha-tidic acid, yielding DG, may be the limiting factor (Moore and Christie 1978). The DG acyltransferase responsible for the final acylation of milk TG has been studied in mammary tissue from lactating rats (Lin et al. 1976). It was observed to be specific for the sn-1,2 DG, with very little activity observed with the sn-1,3 or sn-2,3 DG. It exhibited a broad specificity for acyl donors. The acyl-CoA specificity was not affected by the type of 1,2 DG acceptor offered, which implies that the type of fatty acid introduced into the glycerol backbone was not influenced by the specificity of subsequent acylation steps. However, the concentration of acyl donors will affect the final acylation. It was ob-... 

Five transcripts of the gene for ACCa have been described (Kim, 1997). These occur by two independent promoters, PI and PII, and differential splicing of the primary transcripts. Transcripts derived from PI have been characterized in adipose tissue while those from PII are found in mammary tissue (Kim, 1997). A third promoter (PHI) has been characterized in ovine mammary glands it generates a transcript encoding an enzyme with an alternative N-terminus. Whereas PI was strongly expressed in bovine... 

Shirley, J.F., Emery, R.S., Convey, E.M., Oxender, W.D. 1973. Enzymic changes in bovine adipose and mammary tissue, serum and mammary tissue hormonal changes with initiation of lactation. J. Dairy Sci. 56, 569-574. 

Expression of recombinant proteins in the milk of transgenic animals is gaining popularity due to simple and cost effective production. Over the past several years, the feasibility of this approach has been demonstrated by the production, at mg mb1 levels, of pharmaceutically relevant monomeric proteins such as human al -antitrypsin, human tPA, human protein C and hexameric fibrinogen [166]. However there are limitations of mammary tissue in making the meaningful post-translational modifications, which has been overcome by co expression of key pro cessing enzymes [167]. 

Casein kinases exist as two distinct sets of enzymes [88]. The casein kinase in mammary tissue that normally provides in vivo phosphorylation of nascent proteins is distinct from the multisubstrate and ubiquitous casein kinase (CK-2) that is responsible for in vitro casein phosphorylation. The substrate specificity of the two enzymes is also different. For the casein kinase from mammary gland, the recognition sequence corresponds to the tripeptide Ser/Thr-X-Glu/Ser-P or Ser/ Thr-X-Glu/Ser in nonphosphorylated proteins, where X is any amino acid [89]. For CK-2, the recognition sites have been identified as Ser-Glu-Ala-Glu-Glu-Glu and Ser-Ala-Ala-Glu-Glu-Glu [88]. 

The mere presence of peroxidase, H2O2, and iodide are not necessarily sufficient to ensure formation of I2. Tissues must be capable of concentrating iodide at a sufficient threshold such that the iodide anion can itself compete for binding to the enzyme-bound hypoiodous acid intermediate to yield I2. Currently no evidence exists outside of thyroid and mammary tissue to support the formation of iodinated by-products, although both the salivary gland and ovaries concentrate iodide and express a peroxidase. 

C24 Chen, M.-H. and Larson, B. L. Pyrimidine synthesis pathway enzymes and orotic acid in bovine mammary tissue. J. Dairy Sci., 54, 842-846 (1971)... 

Mammalian multiple-enzyme complexes have been purified from rat liver (540,000 dalton), rat mammary tissue (478,000 dalton), and rabbit mammary gland (910,000 dalton). No active subunits have as yet been prepared. 

General Discussion. (See also Chapter XIV.) Enzymic syntheses of lactose from D-glucose have also been carried out (70). Mammary tissue and other tissues not only catalyze the condensation of two molecules of hexose but also the transformation of D-glucose to D-galactose. 

Fraser et al. (126), for example, found that the incorporation of amino acids into RNA by the pH 5 enzymes from guinea pig mammary tissue was a biphasic reaction. One, occurring at low concentrations of amino acids (about 0.2 mil/) used by most workers, followed a course characteristic of the reaction described above, and seemed to be a specific reaction. If, however, higher concentrations of amino acid (10-100 mAf, which is about the concentration of total amino acids in intracellular pools) were employed, extensive further labeling of RNA occurred, indicating the existence of sites which are not as reactive as the original ones, although... 

Ornithine aminotransferase (EC 2.6.1.13) Ubiquitous" Mitochondrial matrix Hepatic enzyme induced by dietary protein, glucagon kidney enzyme induced by estrogens. Increased in mammary tissue with lactogenesis... 

The recent identification of 9-cw-retinol dehydrogenase in the mouse embryo reveals a pathway for 9-cw-RAs synthesis in this species [60]. This membrane-bound enzyme is able to oxidize 9-c/5-retinol into 9-c/5-retinaldehyde which can be subsequently oxidized to 9-cis-RA. The expression of this enzyme is temporally and spatially controlled during embryogenesis in parts of the nervous system, sensory organs, somites and myotomes, and several tissues of endoder-mal origin. Mertz et al. have also identified a stereospecific human enzyme that catalyzes 9-cis-retinol oxidation and is likewise a member of the short chain alcohol dehydrogenase protein family [61]. The mRNA for the protein is most abundant in human mammary tissues. 

The SAT that catalyzes the synthesis of sialyllactose was first detected in rat mammary tissue by Roseman and his co-workers (Jourdian et al., 1963 Carlson et al., 1973a). In fact, this was the first SAT to be characterized in an animal system, being detected later in embryonic chicken brain (Kaufman and Basu, 1966). However, the SAT activities that catalyze the transfer of sialic acid to lactose and to lactosylceramide appear to be different from the latter by all kinetic parameters (S. Basu, 1966 Kaufman and Basu, 1966 M. Basu et al., 1987 S. C. Basu, 1991). In addition to CMP-NeuAc lactose a2-3 sialyltransferase, CMP-NeuAc lactosamine a2-6 sialyltransferase (ST6N) was first characterized by Roseman and his co-workers (Bartholomew et al., 1973) from colostrum. The ST6N enzyme activity is ubiquitous and was purified by Hill and his associates... 

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