Yoshihiro Masui

Synthetic substrates for vertebrate collagenase.

Abstract Seven kinds of 2,4-dinitrophenyl derivatives of hexa- to octapeptides were prepared in order to find a synthetic substrate for vertebrate collagenase. Analysis of the degree of hydrolysis of the peptides by tadpole collagenase and human serum peptidase(s) demonstrated that 2,4 - dinitrophenyl- l -prolyl- l -glutaminyl-glycyl- l -isoleucyl- l -alanyl-glycyl- l -glutaminyl - d - arginine was the best synthetic substrate for collagenase among the peptides tested. The peptide was successfully applied to the assay of collagenase in synovial fluids from patients with rheumatoid arthritis.

Synthesis of the mating factor of Saccharomyces cerevisiae and its truncated peptides : the structure-activity relationship.

Abstract The mating factor of Saccharomyces cerevisiae, Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr, has been synthesized to confirm the structure of the natural mating factor. The tridecapeptide has the same biological activity as the natural mating factor. From the studies on the biological activity of its truncated peptide synthesized the minimum sequence of the peptide require for the mating factor was deduced to be as His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln.

Conformations of yeast α‐mating factor and analog peptides as bound to phospholipid bilayer

The transferred nuclear Overhauser effects of yeast alpha-mating factor [(1-13)peptide] in the presence of various spin-labeled phosphatidylcholines in small unilamellar vesicles of perdeuterated phosphatidylcholine have been analyzed. From the analysis of the quenching effect by spin-labels, the depth of amino acid side chains of the mating factor in phospholipid bilayer has been elucidated. The Leu4 and Leu6 residues are buried deeply in the apolar region of the phospholipid bilayer while the hydrophilic residues such as Gln5 and Lys7 are in the shallow region of the bilayer. The interaction of the side chains of Trp1 and Trp3 residues of alpha-mating factor with the hydrophobic interior of the bilayer contributes to the binding of this peptide with the phosphatidylcholine bilayer. The conformation of des-Trp1-alpha-mating-factor [(2-13)peptide] in the membrane-bound state has been found to be similar to that of (1-13)peptide from the analysis of transferred nuclear Overhauser effects in the presence of mixed vesicles of perdeuterated phosphatidylcholine and perdeuterated phosphatidylserine. The incorporation of this acidic phospholipid in the vesicle remarkably enhances the binding of (1-13)peptide and analog peptides. However, such modifications that weaken the interaction with phospholipid bilayer (deletion of Trp1 and substitution of Trp3 by Gly or Ala) appreciably lower the physiological activity. Transferred nuclear Overhauser effect analyses have also been made of [DHis2]peptide, [DLeu6]peptide and [DLys7]peptide in the presence of the vesicles of perdeuterated phosphatidylcholine. The main-chain conformations of these three analogs in the membrane-bound state have been found to be similar to that of (1-13)peptide, although the side-chain conformations of the D-amino acid residues are naturally different from those of the L-amino acid ones. Thus, the physiological activities of the (1-13)peptide and a variety of analog peptides are found to correlate with the affinities to the phosphatidylcholine/phosphatidylserine membrane and with the molecular conformations in the membrane-bound state.

Physiological activities of peptides are correlated with the conformations of membrane-bound molecules: α-Mating factor from Saccharomyces cerevisiae and analog peptides

α‐Mating factor from Saccharomyces cerevisiae and analog peptides are found, by fluorescence analyses, to interact with phospholipid membrane. The circular dichroism analyses indicate that the physiological activities of α‐mating factor and analog peptide are correlated with the conformations of the membrane‐bound molecules rather than with the molecular conformations in aqueous solution. This suggests that the conformation change of peptide hormone upon binding with phospholipid membrane is an essential step for the recognition by the receptor.

Substrate specificity of vertebrate collagenase

Substrate specificity of purified tadpole collagenase (EC 3.4.24.3) has been studied using eleven synthetic peptides. A pentapeptide, t-butyloxycarbonylprolylalanylglycylisoleucylalanine amide, was susceptible to the action of the enzyme and an octapeptide, acetylprolylglutaminylglycylisoleucylalanylglycylglutaminylarginine ethyl ester, was proposed to be the best substrate for vertebrate collagenase among the peptides tested.

Amino acid substitution of mating factor of Saccharomyces cerevisiae: Structure-activity relationship

Analogs of the mating factor of Saccharomyces cerevisiae, Trp1- Trp3-Leu-Gln-Leu6-Lys7-Pro8-Gly-Gln-Pro11-Met12-Tyr13, from which amino acids were eliminated or substituted for other amino acid, were synthesized. These analogs showed lower biological activity than the natural mating factor if assayed after 6 hours incubation with a-mating type cells of S. cerevisiae. However, if assayed after 24 or 48 hours incubation, the situation changed, i.e. the analogs in which Leu6 or Lys7 were replaced by the corresponding D-isomer, showed higher mating factor activity than the unsubstituted mating factor. The same result was obtained with the analogs in which Met was replaced by norleucine.