Biomedical subjects

R M Valerio

Publications and source records attributed to R M Valerio.

4 recordsLinked to original sources

PubMed1991

Synthesis of peptide analogues using the multipin peptide synthesis method.

Modification of the multipin peptide synthesis method which allows the simultaneous synthesis of large numbers of different peptide analogues is described. Peptides were assembled on polyethylene pins derivatized with a 4-(beta-alanyloxymethyl)benzoate (beta-Ala-HMB) handle. For comparative purposes, peptides were also assembled on the diketopiperazine-forming handle N epsilon-(beta-alanyl)lysylprolyloxylactate. In model studies it was demonstrated that beta-Ala-HMB-linked peptides were cleaved from polyethylene pins with dilute sodium hydroxide or 4% methylamine/water to yield analogues with beta-Ala-free acid (beta-Ala-CO2H) and beta-Ala-methylamide (beta-Ala-CONHCH3), respectively. To assess the suitability of this approach for T-cell determinant analysis, analogues of a known T-cell determinant were synthesized with the various C-terminal endings. Peptides were characterized by amino acid analysis and fast atom bombardment-mass spectrometry. HPLC of the crude cleaved peptides indicated that 22 of the 24 peptides were greater than 95% pure. These crude peptide solutions were nontoxic in sensitive cell culture assays without further purification. All three cleavage procedures gave comparable activities in T-cell proliferation assays. These results demonstrate the potential of the multipin peptide synthesis method for the production of large numbers of different peptide analogues.

Amino Acid Sequence↗

PubMed1990

Phosphorylation of src-phosphopeptides by casein kinases-1 and -2: favourable effect of phosphotyrosine.

The synthetic phosphotyrosyl tridecapeptide H-Arg-Leu-Ile-Glu-Asp-Asn-Glu-Tyr(P)-Thr-Ala-Arg-Gln-Gly-OH, reproducing a major phosphoacceptor site of protein tyrosine kinases of the src-family, can be phosphorylated at Thr-9 by both casein kinases -1 and -2. Its shorter derivative H-Asn-Glu-Tyr(P)-Thr-Ala-OH is not affected by casein kinase-1 while representing a substrate as good as the tridecapeptide for casein kinase-2. The unphosphorylated analogue H-Asn-Glu-Tyr-Thr-Ala-OH, however, is a much poorer substrate, and no significant phosphorylation could be observed of its O-methyl ether derivative H-Asn-Glu-Tyr(Me)-Thr-Ala-OMe. These data on one side corroborate the concept that casein kinase-1 recognizes residues located on the C-terminal edge of acidic stretches, providing, on the other, the evidence that phosphotyrosyl side chains can act as specificity determinants for casein kinase-2.

Amino Acid Sequence↗

PubMedDate not supplied

Systematic screening for bioactive peptides.

Using simultaneous multiple peptide synthesis by the multipin approach, the feasibility of systematic large-scale pharmacological screening of peptide ligands was investigated. The method involves the assembly of small quantities of peptides (ca. 50 nmol) on plastic pins derivatized with an ester linker based on glycolate and 4-(hydroxymethyl)benzoate. These esters are stable under peptide synthesis and side-chain deprotection conditions but cleave under relatively mild basic conditions to generate peptides having C-terminal acid, amide and methylamide. A two-step approach to side-chain deprotection and cleavage from the solid support allows potentially toxic reagents to be removed (washed) from the peptide prior to cleavage. Consequently, the resulting peptide solutions can be used in bioassays with minimal processing. A series of angiotensin II and substance P analogs were synthesized and evaluated in an in vivo rat model and in vitro radioreceptor assay, respectively. Structure-activity studies on analogs of these bioactive peptides are well documented. The data obtained were consistent with that reported in the literature.

Amino Acid Sequence↗

PubMedDate not supplied

Effects of end groups on the stimulatory capacity of minimal length T cell determinant peptides.

We studied the effect of end groups on the capacity of two closely related peptides to stimulate a human T cell clone. Using multipin peptide synthesis technology, we generated solution phase peptides with six combinations of end groups for each sequence. The end modifications examined were amino-terminal acetylation, carboxy-terminal methylamidation or the addition of a dipeptide containing a diketopiperazine ring. The response to the less stimulatory of the two peptide sequences was significantly increased by acetylation of the amino-terminus, a finding which was consistent at different peptide doses. Amino-terminal acetylation was found to be more significant in affecting responses than any of the carboxy-terminal modifications tested. The use of peptides with a diketopiperazine ring structure at the carboxy-terminus did not interfere with presentation and recognition of peptides and may enhance the effectiveness of peptides for T cell epitope scanning.

Amino Acid Sequence↗