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Biomedical subjects

R B Merrifield

Publications and source records attributed to R B Merrifield.

At least 91 records · Page 5Linked to original sources
PubMed1976

Affinity purification of synthetic peptides.

A general strategy and a specific tactic for affinity purification of polypeptides synthesized on solid supports are desbribed and demonstrated. The desired peptide chains were distinguished from terminated peptide chains before removal from the support by attachment of an affinity reagnet (cysteinyl-methionine) bearing an affinity group (thiol) and a binding group (carboxylic acid). After cleavage from the synthetic support, the affinity-labeled peptides (Cys-Met-peptides) were bound to an affinity receptor (organomercurial-agarose) and thus separated from terminated peptides and all other peptides lacking the affinity group. The desired synthetic peptide was obtained by separation of the affinity reagent (loss of Cys-Met by cyanogen bromide cleavage). This general affinity purification strategy is independent of the length or amino acid sequence of the desired peptide. After assembly of ribonuclease-(111-124)-tetradecapeptide, using radiolabeled acetic anhudride for termination of uncoupled in termediates, essentially all (greater than 98.5%) of the acetylated delection peptides were removed by employing the organomercurial Cys-Met tactic. Similarly, the purity of crude synthetic histone H4-(1-37)-heptatriacontapeptide was increased six-fold by using this tactic to remove terminated peptides. A related dimeric Cys-Met tactic is outlined for affinity purification of peptides containing internal cysteine and methionine residues.

Amino Acids↗
PubMed1975

The role of serine-123 in the activity and specificity of ribonuclease. Reactivation of ribonuclease 1-118 by the synthetic COOH-terminal tetradecapeptide, ribonuclease 111-124, and its O-methylserine and alanine analogs.

The COOH-terminal tetradecapeptide of ribonuclease A, Glu-Gly-Asn-Pro-Tyr-Val-Pro-Val-His-Phe-Asp-Ala-Ser-Val, and two analogs, [Ser(Me)-123]-RNase 111-124 and [Ala-123]-RNase 111-124, were synthesized by the solid phase method and were purified to chromatographic and electrophoretic homogeneity. Methods are described for the hydrolysis and quantitative amino acid analysis of peptides containing O-methylserine. The peptides were combined noncovalently with RNase 1-118 and examined for ability to regenerate enzymatic activity in the presence of the substrates C greater than p, U greater than p, poly(C) poly(U), and poly(AF). The dissociation constants of the peptide-protein complexes, and the Michaelis constants for C greater than p and U greater than p with the reconstituted enzymes were determined. The data were used to test hypotheses, drawn from x-ray crystallographic and other studies, for the role of serine-123 in the binding of substrates by ribonuclease. It was found that Ser-123- and Ala-123-containing peptides were equally active for the hydrolysis step when measured with C greater than p as substrate and for the transphosphorylation step as measured in the assays with poly(C). The serine and alanine analogs were also equally active for the transphosphorylation step when poly AF was the substrate. With U greater than p as substrate the alanine analog was 4 times less active than the serine derivative and with poly U it was 2 times less active. The semisynthetic enzyme composed of RNase 1-118 and [Ala-123]-RNase 111-124, therefore, shows appreciable selectivity for substrates containing cytosine. It was concluded that a hydrogen bond between the hydroxyl of serine-123 and the C4 amino group of cytidine or the C-7 amino group of formycin is not important for substrate binding and catalytic activity. In contrast, the hydrogen bond between the hydroxyl of serine 123 and the C-4 carbonyl oxygen of uridine contributes significantly to substrate binding and catalytic activity. The data with serine-O-methyl ether at position 123 in the tetradecapeptide were less clear because it was difficult to separate steric effects from the contributions of hydrogen bonding. Substrate binding to ribonuclease was rationalized in terms of a binding energy equivalent to a total of two hydrogen bonds per pyrimidine.

Alanine↗
PubMed1975

Synthetic position 5 analogs of adrenocorticotropin fragments and their in vitro lipolytic activity.

Twenty-three analogs of the ACTH-(4-10)-heptapeptide sequence, which forms the "active core" of adrenocorticotropin (ACTH) and related hormones, have been synthesized by the solid-phase method. These analogs all contain structural modifications at or near the 5-glutamic acid residue of ACTH. The peptides were purified to electrophoretic and chromatographic homogeneity. The peptides were assayed for lipolytic activity in an isolated cell system derived from rabbit adipose tissue. In this system, it was determined that residue 5 plays a very important "spacer" role in the peptide, but that this spacer function is not very dependent on the nature of the side chain of the position 5 amino acid. It was found, however, that a number of analogs containing basic residues (arginine or lysine) in position 3 and/or position 5 of ACTH-(3-10) and ACTH-(4-10) fragments have 5 to 10 times the activity of the respective parent peptides. The presence of a latent anionic locus in the rabbit fat-cell receptor for ACTH is suggested by this study.

Adipose Tissue↗