Francis J. Morgan

Complete amino acid sequence of Embden goose (Anser anser) egg-white lysozyme☆

Abstract The complete amino acid sequence of Embden goose ( Anser anser ) egg-white lysozyme (EC 3.2.1.17) has been proposed. The structure was established by comparing the citraconylated tryptic peptides of Embden goose lysozyme with the corresponding peptides from the black swan goose-type lysozyme. Those peptides which differed in amino acid composition were subjected to sequence analysis; the alignment of the tryptic peptides was achieved by analogy with other goose-type lysozymes. Embden goose lysozyme is a single polypeptide chain of 185 amino acids. It closely resembles the goose-type lysozymes from the black swan and the ostrich, differing in 6 and 30 residues, respectively.

Isolation of protein S6 from rat liver ribosomes by reversed-phase high-performance liquid chromatography

A rapid procedure for the isolation of ribosomal protein S6 from rat liver ribosomes has been developed in which proteins were separated by reversed-phase HPLC using wide-pore n-butyl-, n-octyl-, or diphenyl-bonded silica phases. Rapid processing of whole ribosomal material was achieved by the extraction of proteins in 6 M guanidinium hydrochloride and subsequent precipitation of RNA by acidification. Highly purified S6 was obtained in two chromatographic steps as shown by sodium dodecyl sulfate-gel electrophoresis, amino acid analysis, and automated microsequencing. The purification of S6 was monitored using 32P-labeled S6 as a marker which cochromatographed with unphosphorylated S6 under the low-pH elution conditions employed. Other ribosomal proteins were also purified using these reversed-phase supports, although in the case of more hydrophobic proteins such as S4 and S10 further optimization of the gradient conditions was required.

A crossreactive antipeptide monoclonal antibody with specificity for lysyl-lysine

Abstract Synthetic peptides meeting certain guidelines have been used as immunogens to generate antibodies with predefined specificity. We have raised and characterized using established methods a monoclonal antibody against a synthetic peptide corresponding to the 18-amino acid carboxyterminal sequence (A194-211) of the platelet-derived growth factor (PDGF) A chain expressed by the U343 human glioma cell line. This antibody was generated in order to carry out structure-function studies on this region of PDGF whose biological significance is not yet clear. Anti-PDGF-A194-211 was found to be a low titre, IgMκ molecule, with Krmd of 2.8 × 10−7M. When antibody reactivity was tested with parent PDGF-AAL (A chain homodimer containing a carboxyterminal extension) significant binding was observed. Suprisingly, 125I-PDGF-AAS, consisting of truncated A chains but lacking the extension was also bound. Moreover, poly- l -lysine, β-thromboglobulin, PDGF-A194-211, and myoglobin competed dose-dependently with 125I-PDGF-AAL for antibody, 125I-bovine serum albumin was also bound. Examination of the primary sequence of proteins and peptides bound by the antibody revealed only one shared structural motif: a lysyl-lysine moiety. Selected small synthetic peptides containing this and other sequences were used as potential competitors of 125I-PDGF-A194-211 in antibody binding. Lysyl-lysyl-glycyl-glutamine and lysyl-lysine competed, whereas lysyl-leucine did not. These results suggest that as few as two amino acid residues constitute a functional antigenic determinant and contrast with most previous estimates of the minimum number of residues required. Furthermore, we show that guidelines governing the design of synthetic peptides for their use as antigens to produce monoclonal antibodies of predetermined specificity may be unreliable.

A microprocessor-based controller and program for a protein sequenator☆

Abstract A microprocessor-based controller suitable for use with a commercial sequenator (Beckman Sequencer 890C) is described. The program allows flexible and very reliable performance of protein and peptide sequencing operations.

A crossreactive antipeptide monoclonal antibody with specificity for lysyl-lysine (J. Immunol. Methods 140 (1991) 249–258)

Synthetic peptides meeting certain guidelines have been used as immunogens to generate antibodies with predefined specificity. We have raised and characterized using established methods a monoclonal antibody against a synthetic peptide corresponding to the 18-amino acid carboxyterminal sequence (A194-211) of the platelet-derived growth factor (PDGF) A chain expressed by the U343 human glioma cell line. This antibody was generated in order to carry out structure-function studies on this region of PDGF whose biological significance is not yet clear. Anti-PDGF-A194-211 was found to be a low titre, IgM kappa molecule, with a Kd of 2.8 x 10(-7) M. When antibody reactivity was tested with parent PDGF-AAL (A chain homodimer containing a carboxyterminal extension) significant binding was observed. Surprisingly, 125I-PDGF-AAS, consisting of truncated A chains but lacking the extension was also bound. Moreover, poly-L-lysine, beta-thromboglobulin, PDGF-A194-211, and myoglobin competed dose-dependently with 125I-PDGF-AAL for antibody. 125I-bovine serum albumin was also bound. Examination of the primary sequence of proteins and peptides bound by the antibody revealed only one shared structural motif: a lysyl-lysine moiety. Selected small synthetic peptides containing this and other sequences were used as potential competitors of 125I-PDGF-A194-211 in antibody binding. Lysyl-lysyl-glycyl-glutamic acid [corrected] and lysyl-lysine competed, whereas lysyl-leucine did not. These results suggest that as few as two amino acid residues constitute a functional antigenic determinant and contrast with most previous estimates of the minimum number of residues required. Furthermore, we show that guidelines governing the design of synthetic peptides for their use as antigens to produce monoclonal antibodies of predetermined specificity may be unreliable.