Sialic acid-9-phosphatase

A number of nonspecific phosphatases can act on NAN-9-PO4 to dephosphorylate it. However, it appears that in rat liver there is a specific sialic acid-9-phosphatase (Warren and Felsenfeld, 1962). A similar enzyme has been purified some 800-fold from human erythrocyte lysates (Jourdian et al., 1964) and has been shown to be highly specific for sialic acid 9-phosphate (both A/ -acetyl and A/ -glycolyl). This enzyme completes the synthesis of sialic acid in animal tissues. In bacteria, however, another pathway does exist and may be widespread. Extracts of Neisseria meningitidis synthesize sialic acid utilizing free N-acetyl-... 

Jourdian, G. W., Swanson, A. L., Watson, D., and Roseman, S., 1964, Isolation of sialic acid 9-phosphatase from human erythrocytes, 7. Biol. Chem. 239 pc2714-pc2715. 

The frequent occurrence of sialylated enzymes, or even of multiple forms, which are sometimes tissue-dependent, with a varying number of sialyl residues as, for example, in y-glutamyltranspeptida.se (EC 2.3.2.2),456,457 is not yet fully understood. Although the activity of most of these enzymes is not influenced by removal of sialic acid,454 the activity of monoamine oxidase A (EC 1.4.3.4) of outer mitochondrial membranes of rat liver has been shown to be destroyed by treatment with sialidase438 the substrate specificity of acetylcholinesterase (EC 3.1.1.7) is altered,459 the kinetic properties of human acid and alkaline phosphatases (EC 3.1.3.1 and 3.1.3.2) are changed, and the stability of a-D-galactosidase (EC 3.2.1.22) is drastically lowered.415 In these cases, an influence of sialyl residues on the conformation of the enzyme is assumed, but awaits firm evidence. 

The relationship between the various tissue alkaline phosphatases has been under discussion for many years (24). Bodansky established that inhibition by bile acids could be used to distinguish between intestinal and bone or kidney isoenzymes (25). The organ-specific behavior of rat tissue phosphatases toward a variety of compounds was investigated by Fishman (26). Of particular importance was the observation that l-phenylalanine is a stereospecific inhibitor for the intestinal isoenzyme (27). Immunochemical (28, 29) and electrophoretic techniques (30, 31) have shown that there are also physical differences between the tissue phosphatases. It is not yet clear what the precise nature of these differences is (32), although in part it results from a variability in sialic acid content. 

Beckman et al. (28) have studied the electrophoretic separation of the acid phosphatase activity in tissue extracts on starch gel at pH 8. They described four electrophoretic bands A, B, C, and D. Table IV (28) shows the distribution of activity in different organ extracts. The ABD pattern predominated in kidney BD in liver, intestine, heart, and skeletal muscle B in skin and D in pancreas. The C component was present in a large number of placentae but not in other adult organs. All four electrophoretic components were inhibited by d-(- -)-tartrate A contained sialic acid, D had a lower pH optimum and was more heat resistant than A, B, and C. Components C and D showed parallel electrophoretic behavior. In human skin fibroblasts grown in tissue culture, the acid phosphatase was generally high and the most common pattern was BD. Almost every culture showed some activity. The BD... 

Alkaline phosphatase is an enzyme of the cellular membranes. Its isoforms can be found in liver, digestive tract, placenta, and some tumor tissues. Bone isoform, BALP, is a membrane enzyme of the osteoblasts. Bone, liver, and intestinal isoforms are the posttranslational modifications of the same isoenzyme exprimed by the same gene, and the difference among them lies in various ways of reaction with a saccharide component, sialic acid (M9). 

Many enzymes are glycoproteins, and variations in carbohydrate side chains are a common cause of nonhomogeneity of preparations of these enzymes. Some carbohydrate moieties, notably h/-acetylneuraminic acid (sialic acid), are strongly ionized and consequently have a profound effect on some properties of enzyme molecules. For example, removal of terminal sialic acid groups from human liver and/or bone alkaline phosphatase with neuraminidase greatly reduces the electrophoretic heterogeneity of the enzyme. 

Does the failure of a protein, following neuraminidase treatment, to undergo change in electrophoretic migration mean that it is not a sialoprotein Also, does the change in migration represent the release of one, two, or more sialic acid molecules per mole of enzyme If alkaline phosphatase is a substrate for neuraminidase, what are the pH and substrate requirements for optimal hydrolysis What is the evidence for the identity of the product of neuraminidase hydrolysis ... 

Fig. 28. Light absorption spectrum of the pigment in the sialic acid color reaction (Warren-thiobarbiturate technique) (A) pure sialic acid, and (B) the product of hydrolysis of placental alkaline phosphatase (G6a).

Fig. 29) for the liberation of sialic acid from purified placental alkaline phosphatase. 

It is reasonable to assume that all human alkaline phosphatases are sialoproteins, but the distinction in the case of intestine is that the sialic acid residues may not be terminal or that the terminal residues are buried in the three-dimensional structure of the enzyme. 

Of interest are recent observations from Harris laboratory demonstrating that graded neuraminidase concentrations in placental alkaline phosphatase digests yielded up to eight bands (R16). Participation of neuraminidase-sensitive sialic acid in the K -dependent nitrophenyl phosphatase in isolated rat liver plasma membranes (El) has been reported. 

Fig. 29. Optimum pH of hydrolysis of human placental alkaline phosphatase by neuraminidase. The liberated sialic acid was measured by the Warren-thiobarbiturate procedure, using iV-acetylneuraminic acid as the standard (G6a).

Mammals produce sialic acid by aldolic condensation of phosphoenolpyruvate and Ai-acetylmannosamine 6-phosphate (reaction 12.1). A kinase enzyme catalyses the phosphorylation of A -acetylmannosamine and a phosphatase catalyses the hydrolysis of the phosphate of sialic acid. These phosphorylation and dephosphorylation steps are irreversible, such that the synthesis can be total even with low concentrations of the substrate. A variation of reaction (12.1), observed with the bacterium Neisseria meningitidis, uses non-phosphated /-acetylmannosamine. However, these were not the enzymes used in the preparative synthesis, which used instead a microbial aldolase which catalyses equilibrium (12.2). This enzyme probably plays a catabolic role in these organisms, but it functions in the synthetic sense in the presence of an excess of pyruvate. 

In diabetic rats orally administered 2 g/kg aqueous extract of parsley leaf daily for 28 days, a reduction in blood glucose, serum alkaline phosphatase activity, sialic acid, uric acid, potassium, and sodium levels, and liver lipid peroxidation and non-enzymatic glycosylation levels was observed, along with an increase in GSH levels (Ozsoy-Sacan et al. 2006). A reduction in blood glucose levels was observed in diabetic rats orally administered 2 g/kg of an aqueous extract of parsley leaf daily for 28 days (Yanardag et al. 2003). 

A synthetase purified from liver and submaxillary gland produces N-acetylneuraminic add 9-phosphate and Pj by condensing acetylmannosamine-6-phos-phate and phosphoenolpyruvic acid. Sialic acid is formed by the action of phosphatase on N-acetylneuraminic acid-9-phosphate. The biosynthetic pathway of sialic acid is summarized in Fig. 3-32. 

---