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Multiple mutants of Escherichia coli defective in penicillin-bindingproteins (PBPs) were constructed, and into these strains CoEl plasmids carrying the genes for PBP-la, -lb, or -3 were introduced. From these plasmid-carrying strains, PBP-la and -lb were purified by ampicillin-Sepharose affinity chromatography and PBP-3 by cephalexin-Sepharose affinity chromatography. Improved purification was achieved by differential elution with NH20H. Purified PBP-lb synthesized murein when added to the membrane fraction of a PBP-lbdefective mutant, which by itself failed to support murein synthesis in vitro. The PBP-lb preparation was able to synthesize murein from the lipid intermediate extracted with chloroform/methanol but was unable to utilize UDP4inked precursors for murein synthesis. Murein synthesis was inhibited by van-comysin, ristocetin, moenomycin, and enduracidin, but not by f-lactam antibiotics. The synthesized murein was shown to contain crosslinked muropeptides. Their crosslinking was abolished by action of ,-lactam antibiotics. The PBP-la and -3 preparations showed substantially no activity for murein synthesis in the same reaction system. None of the three PBPs showed D-alanine carboxypeptidase activity with UDP-Nacetylmuramoyl-ntapeptide as substrate or endopeptidase activity with bis(dsaccharidepeptide) as substrate.

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Human plasma glutathione peroxidase (GSHPx) has been shown to be a selenium-containing enzyme immunologically distinct from cellular GSHPx. Oligonucleotide probes, based on the partial amino acid sequence of plasma GSHPx, were synthesized and used to screen a human placenta cDNA library. Nucleotide sequence analysis of the obtained clones revealed that GSHPx consisted of a 678-base pair open reading frame coding for a 226-amino acid polypeptide with a Mr of 25,389. About 50% of the deduced amino acid sequence was confirmed by partial amino acid sequencing of the peptides in a lysine endopeptidase-digest of the purified enzyme. The amino acid sequence exhibited only 44% homology with that of human cellular GSHPx. Northern blot analysis revealed a single transcript of 2.2 kilobases in the poly(A)+ RNA fractions of human placenta and HepG2 (a human hepatic cell line), but not that of human liver and endothelial cells.