PubMed · 1211626
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K C Chen, R M Krause. 1975. A peptide mapping technique--a three map system.. https://doi.org/10.1016/0003-2697(75)90579-5
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Class A high-molecular-weight penicillin-binding protein 1a (PBP1a) and PBP1b of Escherichia coli have both transglycosylase (TG) and transpeptidase (TP) activity. These enzymes are difficult to assay, since their substrates are difficult to prepare. We show the activity of PBP1a or PBP1b can be measured in membranes by cloning the PBP into an E. coli ponB::Spcr strain. Using this assay, we show that PBP1a is approximately 10-fold more sensitive to penicillin than PBP1b and that the 50% inhibitory concentration (IC50) of moenomycin, a TG inhibitor, is approximately 10-fold higher in the PBP transformants than in wild-type membranes; this increase in IC50 in transformants can be used to test the specificity of test compounds for inhibition of the TG. Alternatively, the coupled TG-TP activity of PBP1b can be directly measured in a two-step microplate assay. In the first step, radiolabeled lipid II, the TG substrate, was made in membranes of the E. coli ponB::Spcr strain by incubation with the peptidoglycan sugar precursors. In the second step, the TG-TP activity was assayed by adding a source of PBP1b to the membranes. The coupled TG-TP activity converts lipid II to cross-linked peptidoglycan, which was specifically captured by wheat germ agglutinin-coated scintillation proximity beads in the presence of 0.2% Sarkosyl (B. Chandrakala et al., Antimicrob. Agents Chemother. 48:30-40, 2004). The TG-TP assay was inhibited by penicillin and moenomycin as expected. Surprisingly, tunicamycin and nisin also inhibited the assay, and paper chromatography analysis revealed that both inhibited the transglycosylase. The assay can be used to screen for novel antibacterial agents.
The crude polysaccharide was extracted from Leave in Hippohae rhamnoides L. with hot water, and precipitated by ethanol. The crude polysaccharide has been fractionated by acidic ethanol. Three fractions (SJ1, SJ2, SJ3) were got respectively. SJ2 deproteinizationed by the combined methods of enzyme and Seveage, purified by DEAE-Sephadex A-25 gel filtration. PC analysis indicated that SJ22 is composed of Xyl,Ara,Glc,Gal,GaL A. The identification of purify by Sepharose CL-4B, paper chromatography and cellulous acetate electrophoresis showed it was homogeneous. Typical absorption of polysaccharides was shown in its IR spectrum. It contained a-glucosidic bonds by IR analysis. It had typical absorption of protein by UV scaning. SJ22 is first isolated from Leave in Hippohae rhamnoides L Scavenging free radical experiment showed that SJ22 was effective in scavenging superoxideradical and hydroxylradical, but only a little effective in scavenging lipid radical.
A cytoplasmic peptide: N-glycanase (PNGase) has been implicated in the proteasomal degradation of aberrant glycoproteins synthesized in the endoplasmic reticulum. The reaction is believed to be important for subsequent proteolysis by the proteasome since bulky N-glycan chains on misfolded glycoproteins may impair their efficient entry into the interior of the cylinder-shaped 20S proteasome, where its active site resides. This cytoplasmic enzyme was first detected in 1993 by a simple, sensitive assay method using 14C-labeled glycopeptide as a substrate. The deglycosylation reaction by PNGase brings about two major changes on substrate the peptide; one is removal of the N-glycan chain and the other is the introduction of a negative charge into the core peptide by converting the glycosylated asparagine residue(s) into an aspartic acid residue(s). The assay method we developed monitors these major changes in the core peptide, and the respective changes were detected by distinct analytical methods: i.e., paper chromatography and paper electrophoresis. This chapter will describe the simple, sensitive in vitro assay method for PNGase.