Bovine submandibular gland

Neu7Ae5Gc in sialic acid mixtures from bovine, submandibular glands, based on analysis by g.l.c.-m.s.82... 

Histochemical demonstration of most of the O-acetylated sialic acids is possible because substituents on the side chain of Neu hinder periodate oxidation of this part of the molecule to an extent dependent on the number and position of the O-acetyl groups already mentioned. Correspondingly, removal of these ester groups by alkaline treatment (0.5% KOH in 70% ethanol91) may increase the staining reaction of a sialic acid. For example, the presence of O-acylated sialic acids has been demonstrated in colonic, epithelial mucin of man and various mammals (summarized in Ref. 91), in healthy and diseased, human small-intestine,188-190 in bovine submandibular gland,182 in mouse and rat erythrocyte-membranes,191 and in human lymphocytes.192... 

The group of 7- and 8-mono-O-acetylated, 7,9- or 8,9-di-O-acetylated, and 7,8,9-tri-O-acetylated sialic acids (the existence of which in bovine, submandibular glands is well established82) is specifically stained in the following way. Tissue slices are treated with 0.5 M periodate for 120 min at 25°, causing complete oxidation of the side chain to the C7 aldehyde of the unsubstitutecl and 9-mono-O-acetylated sialic acids. 

The mode, and subeellular site, of biosynthesis of Neu5Gc is similar in bovine, submandibular glands, although the activity is less pronounced.216 Neu5Ac-mono-oxygenase activity has also been determined in liver and serum of the pig.216 Because of the wide occurrence of Neu5Gc in Nature, the mono-oxygenase is, presumably, also widespread. 

N-Acylneuraminate-9(7)-0-acetyltransferase seems to be widespread in Nature, as sialic acids O-acetylated in the side chain are found in many animal species, including man (see Section II). However, enzyme activity has thus far been measured only in bovine, submandibular gland and in bovine serum. The 4-O-acetyltransferase appears... 

Lrhorfi LA, Srinivasan GV, Schauer R (2007) Properties and partial purification of sialate 0-acetyltransferase from bovine submandibular glands. Biol Chem 388 297-306... 

Neu5Ac or Neu5Gc with 0-acetyl groups at positions 7, 8, and 9 may be prepared from bovine submandibular gland mucin. Isolation of the crude mucous glycoprotein before hydrolysis is advisable to improve the resolution of sialic acids... 

One solution to this problem (Reid et al. 1978 a) was to extend the initial periods of oxidation in both the methods. Under these conditions the Cp class of sialic acids would be expected to oxidize slowly and therefore the red staining of the PBT/KOH/PAS would be expected to gradually diminish while staining with the PAT/KOH/PAS would become progressively bluer. These theoretical results were obtained in practice on sections of bovine submandibular gland, a known site... 

To investigate the latter possibility, sections of bovine submandibular gland and human colon were oxidized for periods of either 10 or 30 minutes and were then reduced with sodium borohydride. The sections were then reoxidized with periodate for various periods and then stained with Schiff reagent. The results obtained showed that when human colonic mucins were oxidized for an initial period of 10 minutes, some PAS reactivity could be regenerated by a second oxidation period of 60 minutes. When however, the initial oxidation period was extended to 30 minutes, no further PAS reactivity could be generated by a second period of oxidation. These results could be interpreted in two ways either they were the result of the incomplete oxidation of a relatively small number of 9-0-acyl sialic acids or the failure to oxidize other tissue diols. The probability of the latter was lessened by the observation that the oxidation of the crypt mucins of rabbit small intestine which do not contain 0-acylated sialic acids was apparently complete in 10 minutes. 

In contrast, in bovine submandibular gland mucin, neither of the initial periods of oxidation abolished subsequent PAS reactivity although the PAS reactivity was somewhat weaker in sections exposed to an initial period of 30 minutes. We interpret these results as evidence for the presence of 9-0-acyl sialic acids although not all such acids can be demonstrated by such a method since presumably some oxidation of the 9-0-acyl sialic acids will occur within the initial period of oxidation. 

An alternative procedure was adopted by Veh et al. (1979). They demonstrated that a mild (ImM) periodate oxidation PAS was specific for the sialic acid residues of bovine submandibular gland. Since these oxidation conditions were not expected to oxidize 9-0-acyl sialic acids, they considered that the procedure demonstrated the Q and Oj classes of sialic acids, whereas using such mild oxidation procedures, the PBT/KOH/PAS demonstrated the Cg and the Q classes of sialic acids. Oxidation with 0.5 M periodate for 120 minutes at room temperature, however, resulted in the oxidative cleavage of 9-0-acyl sialic acids between positions C7 and Cg. Subsequent steps of the PBT/KOH/PAS then demonstrated the Cg class of sialic acids. 

The anabolic route to NeuSAc proceeds via a N-acetyl-D-neuraminyl-9-phosphate (Neu5Ac9P) synthase which catalyzes Neu5Ac9P formation from ManNAc-6-P and PEP. The enzyme has been found in rat liver and bovine submandibular glands (Roseman et al. 1961, Warren and Felsenfeld 1961 a,b, 1962). 

Sialidases require a negatively charged carboxyl function on the a-linked sialic acid-containing substrate for catalytic activity. V. cholerae sialidase did not cleave the sialic acid methyl esters of bovine submandibular gland mucus glycoproteins (Gottschalk 1962) or IPNeu5AcLac (Kuhn / al 1966). In colominic acid, esters... 

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