ABSTRACT
The influence of the P1 amino acid on the substrate selectivity, the catalytic parameters K(m) and k(cat), of carboxypeptidase M (CPM) (E.C. 3.4.17.12) was systematically studied using a series of benzoyl-Xaa-Arg substrates. CPM had the highest catalytic efficiency (k(cat)/K(m)) for substrates with Met, Ala and aromatic amino acids in the penultimate position and the lowest with amino acids with branched side-chains. Substrates with Pro in P1 were not cleaved in similar conditions. The P1 substrate preference of CPM differed from that of two other members of the carboxypeptidase family, CPN (CPN/CPE subfamily) and CPB (CPA/CPB subfamily). Aromatic P1 residues discriminated most between CPM and CPN. The type of P2 residue also influenced the k(cat) and K(m) of CPM. Extending the substrate up to P7 had little effect on the catalytic parameters. The substrates were modelled in the active site of CPM. The results indicate that P1-S1 interactions play a role in substrate binding and turn-over.
Subject(s)
Metalloendopeptidases/metabolism , Base Sequence , Binding Sites , Cell Line , Chromatography, High Pressure Liquid , DNA Primers , GPI-Linked Proteins , Kinetics , Metalloendopeptidases/chemistry , Models, Molecular , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
Genetically altered mice may exhibit highly variable phenotypes due to the variation in genetic background, which can only be circumvented by generation of inbred, isogenic gene-targeted and control mice. Here we report that an embryonic stem (ES) cell culture medium conditioned by a rabbit fibroblast cell line transduced with genomic rabbit leukemia inhibitory factor allows efficient derivation and maintenance of ES cell lines from all of 10 inbred mouse strains tested, including some that were presumed to be nonpermissive for ES cell derivation (129/SvEv, 129/SvJ, C57BL/6N, C57BL/6JOla, CBA/CaOla, DBA/2N, DBA/1Ola, C3H/HeN, BALB/c, and FVB/N). Germline transmission was established by blastocyst injection of established ES cell lines after 10 or more passages from all of seven strains tested (129/SvJ, C57BL/6N, C57BL/6JOla, DBA/2N, DBA/1Ola, BALB/c, and FVB/N), by diploid aggregation of ES cell lines from all of four strains tested (129/SvEv, C57BL/6N, CBA/ CaOla, and FVB/N), or by tetraploid aggregation of ES cell lines from all of three strains tested (129/SvEv, C57BL/6N, and CBA/CaOla). Thus, these inbred ES cell lines may constitute useful tools to derive gene-targeted mice and isogenic controls in selected genetic backgrounds.
