Biomedical subjects

R Anderegg

Publications and source records attributed to R Anderegg.

4 recordsLinked to original sources

PubMed1992

Human interleukin-5 expressed in Escherichia coli has N-terminal modifications.

Recombinant human interleukin-5 exists as four major isoforms all possessing N-terminal methionine. Peptide mapping and subsequent analysis by fast-atom-bombardment mass spectrometry (f.a.b.-m.s.) have shown that N-terminal modifications are the cause of the charge heterogeneity. In order of decreasing abundance, these are unmodified methionine, retention of N-terminal formyl group, oxidation of N-terminal methionine to sulphoxide and carbamoylation of the N-terminus. These results were confirmed by analysis of the reduced and alkylated intact protein by electrospray-ionization mass spectrometry. The implications of these findings for the production and characterization of recombinant proteins are briefly discussed.

Chromatography, High Pressure Liquid↗

PubMed1992

Rapid optimization of enzyme substrates using defined substrate mixtures.

A strategy is described for the rapid optimization of kcat/Km for protease substrates. Selected positions of a given peptide substrate sequence are varied through synthesis with mixtures of amino acids. Incubation of the resulting peptide mixture with the enzyme of interest and analysis by high pressure liquid chromatography provides a direct measure of analogs with enhanced kcat/Km. High performance liquid chromatography/continuous flow fast atom bombardment mass spectrometry is used to assign structure to each peak in the chromatogram. As an example of the utility and efficiency of "substrate mapping" we describe optimization of the collagenase substrate Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 (where Dnp is dinitrophenyl) at the P'1 and P'2 positions. Six different mixtures were prepared for evaluation, representing the synthesis of 128 different synthetic substrates. "Substrate mapping" has led to Dnp-Pro-Leu-Gly-Cys(Me)-His-Ala-D-Arg-NH2, a substrate that possesses a 10-fold better kcat/Km than Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2.

Amino Acid Sequence↗

PubMed1992

Sequencing and characterization of the soybean leaf metalloproteinase : structural and functional similarity to the matrix metalloproteinase family.

A novel zinc endoproteinase has been sequenced and characterized from soybean leaves (Glycine max var Williams 82) and has been designated as Protein Identification Resource accession No. A41820 SMEP1 (soybean metalloendoproteinase 1). Comparison of the primary amino acid sequence with other zinc proteinases revealed the enzyme to be a new member of the matrix metalloproteinase (MMP) family of enzymes. SMEP was found to have MMP cleavage specificity toward peptide substrates and the enzyme is specifically inhibited by naturally occurring tissue inhibitors of MMPs through a high-affinity interaction (inhibitor concentration resulting in an approximate 50% decrease in enzyme activity = 23 x 10(-9) molar). Together, these results suggest that the origin of the MMP family of enzymes and their cognate inhibitors predates the divergence of plants and animals.

Journal Article↗

PubMed1978

Primary structure of the lambda repressor.

The complete covalent structure of the bacteriophage lambda repressor has been determined by sequential Edman degradation, gas chromatographic-mass spectrometric peptide sequencing, and DNA sequencing of the repressor gene cI. The repressor is a single-chain, acidic protein containing 236 amino acids. The amino terminal 40 residues are highly polar and basic. Lysines and arginines in the sequence tend to be clustered.

Amino Acid Sequence↗