Glycosylation labeling

Fischer projection of acyclic form, 56-57 glycosides, 132-135 C-glycosyl compounds, 139-140 N-glycosyl derivatives, 137-139 glycosyl halides, 136-137 glycosyl residues, 125 isotopic substitution and isotopic labelling, 91 me so forms, 59 optical rotation, 59 parent structure choice, 53... 

The type of intermediate that is formed in the slow inhibition with D-gly-cals was identified, with the aid of the ) -D-glucosidase A3 from Asp. wentii, as an ester of 2-deoxy-D-araA/ o-hexose with an aspartic acid side-chain. The same aspartoyl residue had already been shown, by labeling with con-duritol B epoxide (see Section 111,1), to be essential for -D-glucoside hydrolysis. In addition, this aspartate was found to form a glycosyl -enzyme... 

If k2 > kj, the glycosyl-enzyme intermediate will accumulate, and may be trapped by the rapid denaturation of the enzyme in the presence of (saturating) amounts of substrate. With -glucoside Aj from Asp. wentii and 4-nitrophenyl [ C]-2-deoxy-) -D-irra />jo-hexopyranoside, it was possible to identify the intermediate as a glycosyl ester (acylal) of 2-deoxy-D-arabino-hexose bound to the same aspartate residue that had previously been labeled with the active-site-directed inhibitor conduritol B epoxide and with D-glucal." This constituted an important proof that the carboxylate reacting with the epoxide is directly involved in catalysis. 

Upper panel The hydropathy profile of the entire 69 kD precursor protein is shown. The abscissa is amino acid residues and the ordinate, positive values indicate hydrophilic. The black and hatched rectangles at the bottom of the figure denote the calculated signal sequence and amino-terminal propeptide domains, respectively. The mature and carboxyl-terminal domains are labeled. N-linked core glycosylation consensus sites are depicted by branched structures. 

Fig. 3. (A) Disposition of afi unit in the membrane, based on sequence information [14,15], selective proteolytic digestion of the a subunit [5,6] and hydrophobic labelling (Table 1). The model for the (S subunit is based on sequencing of surface peptides and identification of S-S bridges [64,65]. T, T2 and C3 show location of proteolytic splits. CHO are glycosylated asparagines in the P subunit. (B) Peptide fragments remaining in the membrane after extensive tryptic digestion of membrane-bound Na,K-ATPase from outer medulla of pig kidney as described by Karlish et al. [7,58].

Add 106-108cells/ml in a PBS solution (lOmM sodium phosphate, 0.15M NaCl, pFI 7.4) containing ImM sodium periodate and incubate on ice for 30 minutes in the dark. This level of periodate addition will target the oxidation only to sialic acid residues (Chapter 1, Section 4.4). If additional sites of glycosylation also are to be labeled, increase the periodate concentration to 10 mM and do the reaction at room temperature in the dark. 

Dissolve the antibody to be labeled in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.5, at a concentration of at least lOmg/ml. The immunoglobulin must be glycosylated to work in this procedure. 

The following protocol describes the use of biotin-hydrazide to label glycosylated proteins at their carbohydrate residues. Control of the periodate oxidation level can result in specific labeling of sialic acid groups or general sugar residues (Chapter 1, Section 4.4). 

Scheme 10 Migration in 1-glycosyl radicals with a labeled benzoate group...

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