Expression of recombinant diaminopimelate epimerase in Escherichia coli. Isolation and inhibition with an irreversible inhibitor.

Recombinant diaminopimelate epimerase is overproduced to give 1% of soluble protein when grown under the appropriate conditions in Escherichia coli. This compares with 0.02% of the constitutive level of wild-type enzyme. A new purification procedure now yields milligram quantities of homogeneous enzyme of high specific activity (192 U/mg). This has enabled sufficient amounts of enzyme both to compare with wild-type enzyme and to enable active site modification studies to be performed. Incubation of the enzyme with 2-(4-amino-4-carboxybutyl)-2-aziridine-carboxylic acid (AZIDAP), results in time-dependent irreversible inhibition. Tryptic digestion of the inactivated enzyme and peptide-mapping show that AZIDAP is specifically and covalently bound to the enzyme at a unique peptide. Determination of the amino acid sequence of this peptide and comparison with the sequence deduced from the DNA sequence of the dapF gene shows that Cys73 is labelled. Finally based on limited sequence similarities around this cysteine and active-site cysteines of proline racemase and 1-hydroxyproline 2-epimerase, together with mechanistic considerations, we propose that all three non-pyridoxal-phosphate-containing racemases/epimerases derive from a common evolutionary origin.

High-sensitivity amino acid analysis by derivatization with O-phthalaldehyde and 9-fluorenylmethyl chloroformate using fluorescence detection: applications in protein structure determination.

An amino acid analysis method using a commercially available analyzer that accurately quantitates protein-derived amino acids in the 10-100 pmol range is described. The method utilizes the robotic capability of the analyzer's autosampler to perform precolumn derivatization of both primary and secondary amino acids with o-phthalaldehyde and 9-fluorenylmethyl chloroformate, respectively. The derivatized amino acids are then separated on a C-18 reverse-phase amino acid column and quantitated in a single run by fluorescence detection. The characterization of beta-lactoglobulin and two tryptic peptides from the bacterial enzyme diaminopimelic acid epimerase is used to demonstrate the sensitivity and utility of this method.

Isolation and characterization of four heparin-binding cyanogen bromide peptides of human plasma apolipoprotein B.

Apolipoprotein B-100 (apoB-100) is the major protein constituent of human plasma low-density lipoproteins (LDL). On the basis of its amino acid sequence [Chen, S.-H., Yang, C.-Y., Chen, P.-F., Setzer, D., Tanimura, M., Li, W.-H., Gotto, A. M., Jr., & Chan, L. (1986) J. Biol. Chem. 261, 12918-12921], apo B-100 is one of the largest monomeric proteins known with a calculated molecular weight of 512937. Heparin binds to the LDL surface by interacting with positively charged amino acid residues of apoB-100, forming soluble complexes in the absence of divalent metals and insoluble complexes in their presence. The purpose of this study was to isolate and characterize the heparin-binding domain(s) of apoB-100. Human plasma LDL were fragmented with cyanogen bromide (CNBr). After delipidation and reduction-carboxymethylation, the CNBr peptides were fractionated by sequential chromatography on DEAE-Sephacel, Mono S, and high reactive heparin (HRH) AffiGel-10; HRH was purified by chromatography of crude bovine lung heparin on LDL AffiGel-10. Heparin-binding peptides were further purified by reverse-phase high-performance liquid chromatography. Heparin-binding activity was monitored by a dot-blot assay with 125I-HRH. The amino-terminal sequences of four CNBr heparin-binding peptides (CNBr-I-IV) were determined. CNBr-I-IV correspond to residues 2016-2151, 3109-3240, 3308-3394, and 3570-3719, respectively, of the amino acid sequence of apoB-100. Each CNBr peptide contains a domain(s) of basic amino acid residues which we suggest accounts for their heparin-binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural organization of apolipoprotein B-100 of human plasma low density lipoproteins. Comparison to B-48 of chylomicrons and very low density lipoproteins.

The present study characterizes the substructural organization of apolipoprotein B-100 (B-100) of human plasma low density lipoproteins (LDL) and its relation to B-74 and B-26 of LDL and B-48 of chylomicrons and very low density lipoproteins. LDL were digested with human kallikrein and thrombin to yield two major fragments: K1 (Mr 410,000) and K2 (Mr 145,000) and T1 (Mr 385,000) and T2 (Mr 170,000), respectively. The antigenic sequences, Mr, and amino acid compositions of K1 and K2 were identical to those of plasma B-74 and B-26; B-26 and K2 had identical NH2-terminal sequences and correspond to the NH2-terminal region of B-100. K1 was further degraded by kallikrein to give K3 (Mr 235,000) and K4 (Mr 170,000); these peptides correspond immunochemically to two newly discovered plasma LDL peptides B-44 and B-30 and are assigned as complementary fragments of B-74. The thrombin cleavage fragments, T1 and T2, did not correspond to B-74 and B-26. Neither kallikrein nor thrombin generated a fragment from B-100 corresponding to B-48 in chylomicrons. However, B-48 showed antigenic homology with B-26 and to the of B-74 adjoining B-26, indicating that its structure is represented in the NH2-terminal half of B-100. The structural studies further clarify the relatedness among the B-100, B-74, B-26, and B-48 polypeptides and should now make possible the delineation of the functional domains mediating the interactions of apolipoprotein B in the circulation and arterial wall.