Neuraminidase treatment

Fig. 4.—Mass Fragmentography (m/e 161) of Methylated Hexose Derivatives from the Disialosy] Ganglioside. [Top, before treatment with neuraminidase bottom, after treatment with Vibrio cholerae neuraminidase. The methylated hexose derivatives identified are (A) 2,4,6-tri-O-methylgalactose (B) 2,3,6-tri-O-methylglucose (C) 2,3,4,6-tetra-O-methylgalactose. The peaks eluting before C are unrelated signals that were also detected in a blank experiment employing the neuraminidase treatment. Conditions 3% of QF-1, at 190°. Reproduced, by permission, from Ref. 80.]...

Fig. 4.5.4 Identification of mutations in the transferrin protein by neuraminidase treatment. Unusual patterns in the IEF of serum transferrin might lead to pitfalls in CDG diagnostics. These varying patterns are often due to mutations of charged amino acids in the protein backbone of the transferrin molecule, which might lead, for example, to an accumulation of trisialo transferrin bands (lane 3, indicated by a question mark). Further investigations are carried out by cleaving off charged sialic acid monosaccharide moieties from transferrin-linked oligosaccharides by neuraminidase treatment, followed by IEF and transferrin antibody staining. In the case of protein mutations, additional bands below (lane 4) or above (not shown) the desialylated transferrin form appear...

To compare sera from patients suffering from unclear CDG types, sera of healthy persons and patients with already defined CDG are used as controls for neuraminidase treatment studies. 

Data analysis is carried out by comparing IEF patterns from patients suspected of having a CDG with healthy controls and already defined CDG types. Following neuraminidase treatment, controls and CDG patients will predominantly present with the asialo form of transferrin. In the case of mutations that affect the protein backbone of transferrin, additional bands appear (Fig. 4.5.4). 

Aggregation of Neuraminidase-Treated Red Cells by Dextrans. Following the depletion of RBC surface charge by neuraminidase treatment, the cells can be aggregated by Dx 20 with concentrations above... 

Agglutination of RBCs by polycations (e.g. polylysines) involves electrostatic attraction between the positively charged lysine groups of the polylysine molecule and the negatively charged N-acetylneuraminic acid on the RBC surface. Reduction of RBC surface charge by neuraminidase treatment markedly decreases the effectiveness of RBC agglutination by polylysine. 

Figure 2. Glycolipids of parental and WgaR CHO cells after neuraminidase treatment...

Mild acid and neuraminidase treatment and DEAE cellulose chromatography were used to further characterize the Thy-1 active compounds (Table I, previous page). Neuraminidase treatment and mild acid conditions, which result in the removal of sialic acid, destroyed the anti-Thy-1 PFC response to these antigens. Furthermore, both Thy-1.1 and Thy-1.2 glycolipids bound to DEAE cellulose, confirming their acidic nature. 

Thy-1.2 activity decreased rapidly after 1 hour of treatment and diminished to background levels after 24 hours. Thus we have found that the Thy-1 activity of purified glycolipid and glycoprotein was destroyed by neuraminidase treatment. 

The existence of specific receptors for transferrin on the reticulocyte membrane was implied by the work of Jandl and associates, who observed that trypsin virtually abolished the ability of reticulocytes to take up iron from transferrin without affecting other metabolic functions of the cells (8). Whether the effect of the enzyme was to cleave the receptor from the cell membrane or to degrade it proteolytically was not clear. Neuraminidase treatment also depressed iron uptake by reticulocytes, but to a much lesser degree than trypsin and only at much higher concentrations than needed to abolish the hemagglutinating effects of influenza virus. Subsequent work from Morgan s laboratory has confirmed these results and has shown further that binding of transferrin to the receptor protects it from proteolytic enzymes (70). 

According to Rolla, ionic bonds are important in the associations between bacterial polysaccharides and protein-coated tooth surfaces (21). This was based on in vitro experiments on the afiinity of dextran for hydroxyapatite powder coated with salivary glycoprotein specifically, adsorption of dextran was inhibited by 0.5M. Prior treatment of the coated hydroxyapatite with neuraminidase also reduced adsorption of dextran. Neuraminidase would be expected to reduce the negative charge of the protein coat by removing ionized sialic acid moieties. Of course, reduced adsorption of dextran could result from conformational changes induced in the pellicle protein by the neuraminidase treatment, as was apparently effected by 4M or 8M urea, in other experiments. 

Bourne (S3).] Moss and King (M36) separated different zones of human alkaline phosphatase by starch-gel electrophoresis and determined their Michaelis constants. They also found a high degree of overlap of the various bands on starch-gel electrophoresis of purified human tissue alkaline phosphatases. In another study, purified intestinal alkaline phosphatase (M34, M35) was found to travel to several positions including the typical slow zone. More recently Moss et al. (M38) reported the neuraminidase sensitivity of human liver alkaline phosphatase. Butter-worth and Moss (B45) showed that purified human renal alkaline phosphatase is also neuraminidase-sensitive, as the electrophoretic mobility of the enzyme in the starch gel was considerably reduced after neuraminidase treatment. 

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 ... 

The alkaline phosphatase of both human intestine and placenta are L-phenyl-alanine-sensitive and undergo uncompetitive inhibition to the same extent (nearly 80%) by 0.005 M L-phenylalanine. However, we have been able to find several distinguishing biochemical characteristics of the two enzymes (1) the anodic mobility of intestinal alkaline phosphatase remains unchanged after neuraminidase treatment, whereas the placental enzyme is sialidase-seusitive and hence the electrophoretic mobility on starch gel is considerably reduced by such treatment, (2) the Michaelis constant of placental alkaline phosphatase at a definite pH is appreciably higher than that of the intestinal enzyme (at pH 9.3 the Km values of placenta and intestine are 316 and 160 ixM, respectively), and (3) the pH optima (with 0.018 Af phenyl phosphate as substrate) of the two enzymes are different the values for intestinal and placental enzymes with 0.006 Af n-phenylalanine are 9.9 and 10.6, respectively, and the respective values in the presence of 0.005 Af L-phenylalanine are 10.2 and 11.1. Finally, contrary to the behavior of intestinal alkaline phosphatase, placental enzyme is completely heat stable (P19). 

In our opinion, a single set of this laboratory s partition values on serum is not diagnostic in itself of the relative degree of liver and bone involvement. Additional parameters need to be evaluated such as starch gel, neuraminidase treatment, clinical status (Yi), etc. 

In the present state of knowledge and in the absence of information of the patient s diagnosis, one cannot yet expect to correctly identify a serum as mostly bone or mostly liver in origin with respect to its alkaline phosphatase by heat inactivation or L-phenylalanine inhibition. With the inclusion of additional studies, such as starch-gel electrophoresis, neuraminidase treatment, and continued study over a period of time, one can increase the certainty of the interpretation of the origin of the serum alkaline phosphatase in patients. 

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