Yasuo Ariyoshi

Angiotensin-converting enzyme inhibitors derived from food proteins

Abstract Angiotensin-converting enzyme (ACE) acts in blood pressure regulation, converting angiotensin I to the potent vasoconstrictor angiotensin II and inactivating the vasodilator bradykinin to raise blood pressure. Various synthetic ACE inhibitors are currently in use as antihypertensive agents. Recently, ACE-inhibitory peptides have been isolated from enzymatic digests of food proteins. The possible roles of these food-derived ACE inhibitors are discussed.

Complete Amino Acid Sequence of the Sweet Protein Monellin

The sweet protein monellin consists of two noncovalently associated polypeptide chains, the A chain of 44 amino acid residues and the B chain of 50 residues. Two different primary structures have been reported for each of these chains. The complete amino acid sequence of monellin was determined by a combination of FAB- and ESI-mass spectrometry, and by automatic Edman degradation.

SYNTHESIS AND CHARACTERIZATION OF THE SWEET PROTEIN BRAZZEIN

The sweet protein brazzein isolated from the fruit of the African plant, Pentadiplandra brazzeana Baillon is 2000-500 times sweeter than sucrose and consists of 54 amino acid residues with four intramolecular disulfide bonds. Brazzein was prepared by the fluoren-9-yl-methoxycarbonyl solid-phase method, and was identical to natural brazzein by high performance liquid chromatography, mass spectroscopy, peptide mapping, and taste evaluation. The D enantiomer of brazzein was also synthesized, and was shown to be the mirror image of brazzein. The D enantiomer (ent-brazzein) was devoid of any sweetness and was essentially tasteless.

Structure of an Enantiomeric Protein, D-Monellin at 1.8 A Resolution.

The D-enantiomer of a potently sweet protein, monellin, has been crystallized and analyzed by X-ray crystallography at 1.8 A resolut ion. Two crystal forms (I and II) appeared under crystallization conditions similar, but not identical, to the crystallization conditions of natural L-monellin. There are four molecules per asymmetric unit in crystal form I and one in crystal form II. Crystal form I is not reproducible and is equivalent to that of monoclinic L-monellin. Intermonomer contacts in crystal form II are very different from those found in natural L-monellin crystals. The backbone trace of D-monellin resembles very closely the mirror image of that of L-monellin, but the N- and C-terminus backbones as well as several side-chain conformations of D-monellin are different from those of natural L-monellin. Most of these apparent differences may be attributable to the crystal packing differences.

Inhibition of Prolyl Endopeptidase by Synthetic Peptide Fragments of Human β-Casein

It has been suggested that peptide inhibitors of prolyl endopeptidase (PEP) may act as anti-amnestic agents. In the hope of finding PEP inhibitors in milk proteins, we synthesized a total of 37 human beta-casein peptide fragments containing proline residues. It was found that the peptides with PEP inhibition activity in vitro were located in the region of amino acid residues 49-59 of human beta-casein. The most potent inhibitor was Ile-Tyr-Pro-Phe-Val-Glu-Pro-Ile (IC50 = 8 microM).

Effects of synthetic peptides on giant neurons identified in the ganglia of an African giant snail (Achatina fulica Férussac)—II

Thirteen synthetic biologically-active peptides, which were classified into the peptides proposed as neurotransmitters in mammals and invertebrates and neural venom peptides, were investigated for their effects on the following six identifiable giant neurons of an African giant snail (Achatina fulica Férussac): RAPN (right anterior pallial neuron), INN (intestinal nerve neuron), RPeNLN (right pedal nerve large neuron), LPeNLN (left pedal nerve large neuron), d-LPeLN (dorsal-left pedal large neuron) and d-LPeCN (dorsal-left pedal constantly firing neuron). Oxytocin and proctolin at 10(-4)M excited the RAPN membrane potential, whereas FMRFamide at the same concentration inhibited the same neuron. FMRFamide at 10(-4)M markedly inhibited the d-LPeLN membrane potential, sometimes produced inhibition of RPeNLN and LPeNLN, showed varied effects (excitatory or inhibitory) on INN, and had no effect on d-LPeCN. The other peptides examined had almost no effect on any of the neurons tested.

ASSIGNMENT OF THE DISULFIDE BONDS IN THE SWEET PROTEIN BRAZZEIN

The thermostable sweet protein brazzein consists of 54 amino acid residues and has four intramolecular disulfide bonds, the location of which is unknown. We found that brazzein resists enzymatic hydrolysis at enzyme/substrate ratios (w/w) of 1:100‐1:10 at 35–40°C for 24–48 h. Brazzein was hydrolyzed using thermolysin at an enzyme/substrate ratio of 1:1 (w/w) in water, pH 5.5. for 6 h and at 50°C. The disulfide bonds were determined, by a combination of mass spectrometric analysis and amino acid sequencing of cystine‐containing peptides, to be between Cys4‐Cys52, Cys16‐Cys37, Cys22‐Cys47, and Cys26‐Cys49. These disulfide bonds contribute to its thermostability.

Enzymatic ligation for synthesis of single-chain analogue of monellin by transglutaminase* †

Monellin, a sweet protein, consists of two noncovalently associated polypeptide chains: an A chain of 44 amino acid residues and a B chain of 50 residues. Microbial transglutaminase (MTGase) was used for ligation of the monellin subunits without any protecting groups, and without activation of the C alpha-carboxyl group at the C-terminus. Since a peptide fragment LLQG is a good substrate for MTGase to form an amide bond between the gamma-amide group of the Gln residue and the epsilon-amino group of Lys, a monellin B chain analogue in which LLQG was elongated at the C-terminus (B-LLQG) was synthesized by solid-phase synthesis. The monellin A chain analogue in which KGK was elongated at the N-terminus (KGK-A) was synthesized by the same method as that of the B chain analogue. The KGK-A chain and the B-LLQG chain were coupled by MTGase to give single-chain analogue of monellin. The single-chain analogue of monellin was characterized by analytical reverse phase high performance liquid chromatography, electrospray ionization, and amino acid analyses. All analyses gave satisfactory results. The single-chain analogue of monellin was more heat stable than natural monellin.

Chemical synthesis and characterization of the sweet protein mabinlin II

The sweet protein mabinlin II isolated from the seeds of Capparis masaikai consists of the A chain with 33 amino acid residues and the B chain composed of 72 residues. The B chain contains two intramolecular disulfide bonds and is connected to the A chain through two intermolecular disulfide bridges. The A chain was synthesized by the stepwise fluoren-9-ylmethoxycarbonyl (Fmoc) solid-phase method in a yield of 5.9%, while the B chain was synthesized by a combination of the stepwise Fmoc solid-phase method and fragment condensation in a yield of 6.0%. Disulfide formation and combination of the A and B chains followed by purification by ion-exchange high-performance liquid chromatography (HPLC) gave mabinlin II in a yield of 47.4%. The characterization of the synthetic mabinlin II by HPLC, electrospray ionization mass spectrometry, amino acid analysis, and disulfide bond determination fully supported the expected structure. A 0.1% solution of the synthetic mabinlin II had an astringent-sweet taste.

Structure and dynamic studies by NMR of the potent sweet protein monellin and a non-sweet analog: Evidence on the importance of residue AspB7 for sweet taste

Monellin, an intensely sweet protein and a non‐sweet analog in which the AspB7 in monellin has been replaced with AbuB7 were studied by NMR. The results of our investigations show that the 3‐dimensional structure of these two proteins are very similar indicating that the lack of the β‐carboxyl group in the AbuB7 analog is responsible for the loss of sweet potency. Selectively labeled monellin was prepared by solid‐phase peptide synthesis by incorporating 15N‐labeled amino acids into 10 key positions including AspB7. The internal mobility of these 10 key residues in monellin was estimated by the method of model‐free analyses and our NMR studies show that AspB7 is the most flexible of these 10 residues. The flexibility of the AspB7 side chain may be important for receptor binding.