Yuko Yamada

Kyoto University, Kioto, Kyōto, Japan

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Publications (20)47.57 Total impact

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    ABSTRACT: Recently, we found that dipeptide Arg-Phe (RF) had cholecystokinin (CCK)-dependent vasorelaxing activity. The RF sequence is often observed in the primary structure of natural food proteins. In the current study, we investigated enzymatic conditions for the release of RF-related peptides from rice glutelin, a major storage protein, using gastrointestinal proteases. RF-related peptides were then characterized. It was found that RF and Ile-His-Arg-Phe (IHRF) were released in the chymotrypsin digest of the partial structure of rice glutelin. We then focused on previously unidentified IHRF, corresponding to rice glutelin(155-158). IHRF had vasorelaxing activity in the mesenteric artery of spontaneous hypertensive rats (SHRs). Orally administered IHRF lowered systolic blood pressure in SHRs. The antihypertensive activity of IHRF was more potent and long-lasting than that of RF. IHRF-induced vasorelaxing activity was not blocked by inhibitors of nitric oxide synthase and cyclooxygenase, but by an antagonist for CCK1 receptor. IHRF also had CCK-like suppressive activities in food intake and gastrointestinal transit. IHRF increased intracellular Ca(2+) flux and CCK release in the enteroendocrine cell STC-1. IHRF, a novel CCK-dependent vasorelaxing peptide, decreases both blood pressure and food intake in rodents.
    Molecular Nutrition & Food Research 09/2013; · 4.31 Impact Factor
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    ABSTRACT: Novokinin (RPLKPW) was designed based on ovokinin (FRADHPFL), a vasorelaxing peptide derived from ovalbumin. Novokinin relaxed a mesenteric artery isolated from the spontaneously hypertensive rat (SHR) at 10-5 M, and reduced SHR blood pressure at a dose of 0.1 mg/kg (po.) emulsified in 30% egg yolk. Novokinin exhibited an affinity for the AT2 receptor (Ki = 7 x 10-6M), and its antihypertensive and vasorelaxing activities were blocked by PD123319, an AT2 receptor antagonist. The hypotensive effect of novokinin in normotensive mice was not observed in the AT2 receptor-knockout mice. Its antihypertensive and vasorelaxing activities in SHR were also blocked by CAY-10441, an antagonist of the IP receptor for prostaglandin I2 (PGI2) suggesting that these activities are mediated by the AT2 receptor, followed by the prostaglandin I2-IP receptor pathway. Novokinin suppressed food intake after icv. or po. administration in mice. The anorexigenic activity was not observed in the AT2 receptor-knockout mice, but was observed in the AT1 receptor-knockout mice. The anorexigenic activities of novokinin and angiotensin II were blocked by PD123319, and ONO-AE3-208, an antagonist of the EP4 receptor suggesting that the anorexigenic activities of the AT2 agonists are mediated by the PGE2-EP4 receptor pathway downstream of the AT2 receptor. Novokinin given icv in mice antagonized the antinociceptive effect of morphine. The antiopiod activites of novokinin and angiotensin II were blocked by PD123319, and by ONO-AE3-240, an antagonist of the EP3 receptor suggesting that the antiopioid activity of AT2 agonists are mediated by the PGE2-EP3 receptor downstream of the AT2 receptor.
    Current pharmaceutical design 11/2012; · 4.41 Impact Factor
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    ABSTRACT: We found that a dipeptide, Arg-Phe (RF), had vasorelaxing activity in mesenteric artery isolated from spontaneously hypertensive rats (SHRs) (EC(50) = 580 nM). We then investigated its mechanism of action, and elucidated its physiological functions. Vasorelaxing activities of RF-related peptides were tested. The retro-sequence dipeptide FR was inactive, suggesting that the RF sequence is important for a potent vasorelaxing effect. RA and AF were also inactive. RF-nh(2) had vasorelaxing activity, implying that the C-terminal amidation of RF is tolerated. Nitric oxide (NO) and prostaglandins (PGs) are known to be vasorelaxing factors; however, the vasorelaxing activity of RF was inhibited by neither N(G) -nitro-l-arginine methyl ester (l-NAME), an NO synthase inhibitor, nor indomethacin, a COX inhibitor. Interestingly, the activity was blocked by lorglumide, an antagonist of the cholecystokinin (CCK)(1) receptor; however, RF had no affinity for CCK receptors, suggesting that RF stimulates CCK release. Orally administered RF decreased blood pressure in SHRs, and this antihypertensive activity was also blocked by a CCK(1) antagonist. RF had CCK-like suppressive effects on food intake and gastrointestinal transit. RF increased intracellular Ca(2+) flux and CCK release in enteroendocrine STC-1 cells. A novel CCK-dependent vasorelaxing RF decreases both blood pressure and food intake.
    Molecular Nutrition & Food Research 09/2012; 56(9):1456-63. · 4.31 Impact Factor
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    ABSTRACT: Soymorphin-5 (YPFVV) derived from soybean β-conglycinin β-subunit is a μ-opioid agonist peptide having anxiolytic-like activity. Here, we show that soymorphin-5 improves glucose and lipid metabolism after long-term oral administration to KKAy mice, a type 2 diabetes model animal. Soymorphin-5 inhibited hyperglycemia without an increase in plasma insulin levels in KKAy mice. Soymorphin-5 also decreased plasma and liver triglyceride (TG) levels and liver weight, suggesting that soymorphin-5 improved lipid metabolism. Soymorphin-5 increased plasma adiponectin concentration and liver mRNA expression of AdipoR2, a subtype of adiponectin receptor that is involved in stimulating the peroxisome proliferator-activated receptor (PPAR)α pathway and fatty acid β-oxidation. The expressions of the mRNA of PPARα and its target genes acyl-CoA oxidase, carnitine palmitoyltransferase 1 A, and uncoupling protein-2, in the liver were also increased after oral administration of soymorphin-5. Furthermore, des-Tyr-soymorphin-5 (PFVV) without μ-opioid and anxiolytic-like activities did not decrease blood glucose levels in KKAy mice. These results suggest that μ-opioid peptide soymorphin-5 improves glucose and lipid metabolism via activation of the adiponectin and PPARα system and subsequent increases of β-oxidation and energy expenditure in KKAy mice.
    AJP Endocrinology and Metabolism 11/2011; 302(4):E433-40. · 4.51 Impact Factor
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    ABSTRACT: Activation of peroxisome proliferator-activated receptor (PPAR)-α which regulates lipid metabolism in peripheral tissues such as the liver and skeletal muscle, decreases circulating lipid levels, thus improving hyperlipidemia under fasting conditions. Recently, postprandial serum lipid levels have been found to correlate more closely to cardiovascular diseases than fasting levels, although fasting hyperlipidemia is considered an important risk of cardiovascular diseases. However, the effect of PPARα activation on postprandial lipidemia has not been clarified. In this study, we examined the effects of PPARα activation in enterocytes on lipid secretion and postprandial lipidemia. In Caco-2 enterocytes, bezafibrate, a potent PPARα agonist, increased mRNA expression levels of fatty acid oxidation-related genes, such as acyl-CoA oxidase, carnitine palmitoyl transferase, and acyl-CoA synthase, and oxygen consumption rate (OCR) and suppressed secretion levels of both triglycerides and apolipoprotein B into the basolateral side. In vivo experiments revealed that feeding high-fat-diet containing bezafibrate increased mRNA expression levels of fatty acid oxidation-related genes and production of CO(2) and acid soluble metabolites in enterocytes. Moreover, bezafibrate treatment suppressed postprandial lipidemia after oral administration of olive oil to the mice. These findings indicate that PPARα activation suppresses postprandial lipidemia through enhancement of fatty acid oxidation in enterocytes, suggesting that intestinal lipid metabolism regulated by PPARα activity is a novel target of PPARα agonist for decreasing circulating levels of lipids under postprandial conditions.
    Biochemical and Biophysical Research Communications 06/2011; 410(1):1-6. · 2.28 Impact Factor
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    ABSTRACT: Rapakinin, Arg-Ile-Tyr, is a vasorelaxing, anti-hypertensive and anorexigenic peptide derived from rapeseed napin. In this study, we found that rapakinin intracerebroventricularly administered to mice inhibited the analgesic effect of morphine, evaluated by the tail-pinch test. The anti-opioid activity of rapakinin was blocked by LY225910, an antagonist of the cholecystokinin (CCK) CCK(2) receptor, but not by lorglumide, an antagonist of the CCK(1) receptor. The anti-opioid activity of rapakinin was also blocked by CAY10441, an antagonist of the prostaglandin (PG) IP receptor. These results suggest that the anti-opioid activity of rapakinin is mediated by the CCK(2) and IP receptors. The anti-opioid activity induced by ciprostene, an IP receptor agonist, was blocked by LY225910, while that of CCK-8 was not blocked by CAY10441. Thus, it is demonstrated that the CCK-CCK(2) system was activated downstream of the PGI(2)-IP receptor system. Taken together, rapakinin shows anti-opioid activity via the activation of the PGI(2)-IP receptor system followed by the CCK-CCK(2) receptor system.
    Peptides 02/2011; 32(2):281-5. · 2.52 Impact Factor
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    ABSTRACT: Novokinin (Arg-Pro-Leu-Lys-Pro-Trp, RPLKPW) is a new potent antihypertensive peptide based on the sequence of ovokinin (2-7) derived from ovalbumin. We previously generated transgenic rice seeds in which eight novokinin were fused to storage protein glutelins (GluA2 and GluC) for expression. Oral administration of these seeds to spontaneously hypertensive rats (SHRs) reduced systolic blood pressures at a dose of 1 g seed/kg of SHR. Here, 10- or 18-tandem repeats of novokinin with an endoplasmic reticulum (ER) retention signal (Lys-Asp-Glu-Leu, KDEL) at the C terminus were directly expressed in rice under the control of the glutelin promoter containing its signal peptide. Only small amounts of the 18-repeat novokinin accumulated, and it was unexpectedly deposited in the nucleolus. This abnormal intracellular localization was explained by an endogenous signal for nuclear localization. The GFP reporter protein fused to this sequence targeted to nuclei by a transient assay using onion epidermal cells. Transgenic seed expressing the 18-repeat novokinin exhibited significantly higher antihypertensive activity after a single oral dose to SHR even at one-quarter the amount (0.25 g/kg) of the transgenic rice seed expressing the fusion construct; though, its novokinin content was much lower (1/5). Furthermore, in a long-term administration for 5 weeks, even a smaller dose (0.0625 g/kg) of transgenic seeds could confer antihypertensive activity. This high antihypertensive activity may be attributed to differences in digestibility of expressed products by gastrointestinal enzymes and the unique intracellular localization. These results indicate that accumulation of novokinin as a tandemly repeated structure in transgenic rice is more effective than as a fusion-type structure.
    Plant Biotechnology Journal 11/2010; 9(7):729-35. · 6.28 Impact Factor
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    ABSTRACT: The anti-hypertensive peptide Arg-Ile-Tyr, which was isolated based on its inhibitory activity (IC(50)=28microM) for angiotensin I-converting enzyme (ACE) from the subtilisin digest of rapeseed protein, exhibited vasorelaxing activity (EC(50)=5.1microM) in an endothelium-dependent manner in the mesenteric artery of spontaneously hypertensive rats (SHRs). We named the peptide rapakinin. ACE inhibitors are reported to induce nitric oxide (NO)-dependent vasorelaxation by elevating the endogenous bradykinin level; however, the vasorelaxation induced by 10microM of rapakinin was blocked only insignificantly by HOE140 or N(G)-nitro-l-arginine methyl ester (l-NAME), antagonists of bradykinin B(2) receptor and an inhibitor of NO synthase, respectively. On the other hand, the vasorelaxation induced by 10microM rapakinin was significantly blocked by indomethacin and CAY10441, a cyclooxygenase (COX) inhibitor and an antagonist of the IP receptor, respectively. The vasorelaxing activity of rapakinin was also blocked by lorglumide, an antagonist of the cholecystokinin (CCK) CCK(1) receptor, although rapakinin has no affinity for the IP and CCK(1) receptors. The vasorelaxation induced by 10microM iloprost, an IP receptor agonist, was also blocked by lorglumide, suggesting that CCK-CCK(1) receptor system is activated downstream of the PGI(2)-IP receptor system. The anti-hypertensive activity of rapakinin after oral administration in SHRs was also blocked by CAY10441 and lorglumide. These results suggest that the anti-hypertensive activity of rapakinin might be mediated mainly by the PGI(2)-IP receptor, followed by CCK-CCK(1) receptor-dependent vasorelaxation.
    Peptides 02/2010; 31(5):909-14. · 2.52 Impact Factor
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    ABSTRACT: Novokinin (RPLKPW), LPYPR, and rubiscolin (YPLDLF) are bioactive peptides with respective hypotensive, hypocholesterolemic, and memory-enhancing activities. We generated transgenic soybean lines that expressed modified forms of the alpha' subunit of seed storage protein beta-conglycinin containing tandem repeats of these bioactive peptides. The modified alpha' subunits constituted up to 0.2% of extracted proteins from the transgenic seeds.
    Bioscience Biotechnology and Biochemistry 01/2009; 72(12):3301-5. · 1.27 Impact Factor
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    ABSTRACT: Novokinin (Arg-Pro-Leu-Lys-Pro-Trp) is a vasorelaxing and hypotensive peptide acting through the angiotensin AT(2) receptor. Centrally administrated novokinin (30nmol/mouse) inhibited the antinociceptive effect of micro agonist morphine in mice, as evaluated by the tail-pinch test. The anti-opioid effect of novokinin was blocked by PD123319, an antagonist of the AT(2) receptor. Angiotensin II (0.01nmol/mouse, i.c.v.) and [p-aminophenylalanine(6)]-angiotensin II [p-NH(2)Phe(6)]-Ang II (0.1nmol/mouse, i.c.v.), a highly selective AT(2) receptor agonist, also inhibited the antinociceptive effect of morphine, and the effects were also blocked by PD123319. Angiotensin II did not suppress the antinociceptive effect induced by kappa or delta agonists. Novokinin, angiotensin II and [p-NH(2)Phe(6)]-Ang did not have affinity for the micro receptor. The anti-opioid effects induced by these peptides were blocked by ONO-AE3-240, an antagonist of the EP(3) receptor. These results suggest that the anti-opioid effects of AT(2) agonists are mediated by the PGE(2)-EP(3) receptor system downstream of the AT(2) receptor.
    Peptides 12/2008; 30(4):735-9. · 2.52 Impact Factor
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    ABSTRACT: Enterostatin (APGPR) found in the gastrointestinal tract and brain is an anorectic pentapeptide. We found that APGPR inhibited morphine-induced analgesia after intracerebroventricular administration in mice at a dose of 10nmol/mouse. The anti-analgesic effect of APGPR was inhibited by pretreatment with lorglumide and LY225910, antagonists for cholecystokinin 1 (CCK1) and cholecystokinin 2 (CCK2) receptors, respectively. The anti-analgesic effect of APGPR may be mediated by CCK release, since APGPR does not have affinity for CCK receptors.
    Peptides 05/2008; 29(4):559-63. · 2.52 Impact Factor
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    ABSTRACT: Novokinin (Arg-Pro-Leu-Lys-Pro-Trp) is a potent hypotensive peptide previously designed based on the structure of ovokinin(2-7) (Arg-Ala-Asp-His-Pro-Phe), a vasorelaxing and hypotensive peptide derived from ovalbumin. Novokinin exhibited an affinity for the angiotensin AT(2) receptor (Ki=7.35 microM). Novokinin significantly lowered systolic blood pressure at a dose of 0.03 and 0.1 mg/kg after intravenous and oral administration, respectively, in spontaneously hypertensive rats (SHRs), and the hypotensive activity was blocked by PD123319, an antagonist of the AT(2) receptor. Novokinin lowered blood pressure in C57BL/6J mice after oral administration at a dose of 50 mg/kg. However, in AT(2) receptor-deficient mice, novokinin did not reduce blood pressure. These results demonstrate that the hypotensive activity of novokinin is mediated by the AT(2) receptor. The hypotensive activity of novokinin in SHRs was completely blocked by indomethacin and CAY10441, an inhibitor of cyclooxygenase and an antagonist of the prostaglandin IP receptor, respectively. These suggest that the hypotensive activity is mediated by prostacyclin and the IP receptor downstream of the AT(2) receptor.
    Peptides 04/2008; 29(3):412-8. · 2.52 Impact Factor
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    ABSTRACT: Novokinin (Arg-Pro-Leu-Lys-Pro-Trp), which has been designed based on the structure of ovokinin (2-7), significantly reduces the systolic blood pressure at a dose of 100 microg/kg after oral administration in spontaneously hypertensive rats (SHRs). In this study, we generated a transgenic soybean which accumulates novokinin. A vector encoding a modified beta-conglycinin alpha' subunit (4novokinin-alpha') in which four novokinin sequences have been incorporated by site-directed mutagenesis was introduced into somatic embryos by whisker-mediated gene transformation to produce a transgenic soybean. The 4novokinin-alpha' occupied 0.5% of total soluble protein and 5% of the beta-conglycinin alpha' subunit in the transgenic soybean seeds. Protein extracted from the transgenic soybean reduced systolic blood pressure after single oral administration in SHRs at a dose of 0.15 g/kg. Defatted flour from the transgenic soybean also reduced the systolic blood pressure at a dose of 0.25 g/kg. Thus, the 4novokinin-alpha' produced in soybean exhibited an anti-hypertensive activity in SHRs after oral administration.
    Peptides 04/2008; 29(3):331-7. · 2.52 Impact Factor
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    ABSTRACT: In this study, we found that novokinin (Arg-Pro-Leu-Lys-Pro-Trp), a potent hypotensive peptide acting through the AT(2) receptor, has vasorelaxing activity in the mesenteric artery isolated from spontaneously hypertensive rats. The vasorelaxing activity was significantly blocked by PD123319, indomethacin, and CAY10441, which are an AT(2) receptor antagonist, a cyclooxygenase inhibitor, and an IP receptor antagonist, respectively. N(G)-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase, did not block the vasorelaxing activity. These results suggest that the vasorelaxing activity of novokinin, which contributes to the hypotensive effect, is mainly mediated by prostaglandin I(2) (prostacyclin) and the IP receptor downstream of the AT(2) receptor.
    Bioscience Biotechnology and Biochemistry 02/2008; 72(1):257-9. · 1.27 Impact Factor
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    ABSTRACT: Previously, we introduced the RPLKPW sequence, a highly potent hypotensive peptide designed based on ovokinin (2-7), into three homologous sites in the soybean beta-conglycinin alpha' subunit by site-directed mutagenesis. The modified protein expressed in Escherichia coli reduced blood pressure of spontaneously hypertensive rats (SHRs) after oral administration at a dose of 10 mg/kg, which suggested about 30% of the introduced peptide was released in vivo. In this study amino acid residues around the RPLKPW sequence were optimized with a use of synthetic peptides to facilitate release of RPLKPW by gastrointestinal proteases. Then, fourth RPLKPW was also introduced into the extension domain of the protein. The newly modified protein, which was produced in E. coli, significantly lowered blood pressure in SHRs at a dose of 2.5 mg/kg 4 h after oral administration. Furthermore, we produced an extension domain that corresponds to residues 1-143 of the modified alpha' subunit containing four RPLKPW sequences by introducing a termination codon. The minimum effective dose of the modified extension domain was 1.0 mg/kg, which is 1/2000 that of ovalbumin.
    Peptides 02/2004; 25(1):37-43. · 2.52 Impact Factor
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    ABSTRACT: Food proteins can be a source of various bioactive peptides including such possessing anti-hypertensive activity. While most orally active anti-hypertensive peptides derived from food proteins inhibit the angiotensin I-converting enzyme (ACE), ovokinin (2-7) (RADHPF), a peptide isolated from a chymotryptic digest of ovalbumin, has been shown to induce nitric oxide-dependent vasorelaxation in an isolated mesenteric artery as well as anti-hypertensive effect after oral administration in spontaneously hypertensive rats (SHRs). Rational amino acid replacement lead to the ovokinin (2-7) analog, RPLKPW, which had the highest anti-hypertensive activity among the tested peptides. Furthermore, oral administration (0.1 mg/kg) of the peptide lowered the blood pressure of SHR but not of normotensive Wistar-Kyoto (WKY) rats. In order to develop a novel use of this potent anti-hypertensive peptide for prevention of hypertension, RPLKPW has been genetically introduced into the homologous sequences in soybean beta-conglycinin alpha' subunit by site-directed mutagenesis. The recombinant RPLKPW-incorporated alpha' subunit expressed in E. coil has been shown to exert anti-hypertensive activity after oral administration in SHR. Thus, RPLKPW-incorporated alpha' subunit is the first example of a genetically modified food protein possessing physiological activity based on a bioactive peptide.
    Current Medicinal Chemistry - Cardiovascular & Hematological Agents 07/2003; 1(2):197-202.
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    ABSTRACT: Ovokinin(2-7) (RADHPF), an orally active antihypertensive peptide derived from ovalbumin, lowers blood pressure in SHRs at a dose of 10 mg/kg. Attempts were made to potentiate its anti-hypertensive activity by replacing the amino acid residues in [Pro2, Phe3]-ovokinin(2-7), which was previously reported to have 33-fold stronger activity than ovokinin(2-7). The anti-hypertensive activity of [Pro2, Phe3]-ovokinin(2-7) was improved by replacement of the C-terminal Phe residue with Trp. Then, the best amino acid residues at other positions for the anti-hypertensive effect were selected. RPLKPW, the most potent derivative obtained, showed significant anti-hypertensive activities at a dose of 0.1 mg/kg after oral administration in spontaneously hypertensive rats (SHRs). Thus, RPLKPW showed 100-fold more potent anti-hypertensive activity than ovokinin(2-7).
    Bioscience Biotechnology and Biochemistry 07/2002; 66(6):1213-7. · 1.27 Impact Factor
  • Bioscience Biotechnology and Biochemistry - BIOSCI BIOTECHNOL BIOCHEM. 01/2002; 66(6):1213-1217.
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    ABSTRACT: RPLKPW is a highly potent anti-hypertensive peptide obtained by replacing four amino acid residues in ovokinin (2-7) (RADHPF) that is a vasorelaxing peptide isolated from a chymotryptic digest of ovalbumin. In previous study, RPLKPW sequence was introduced into three homologous sites in soybean β-conglycinin α' subunit by site- directed mutagenesis and the mutatedα' subunit expressed in Escherichia coli exerted an anti-hypertensive effect in spontaneously hypertensive rats at a dose of 10 mg/kg (per os). In this study, we found that RPLKPW was released from only one site among three RPLKPW-sites by in vitro digestion experiment with model peptides around RPLKPW-sites of modifiedα' subunit. In order to release RPLKPW efficiently, we optimized amino residues around individual RPLKPW-sites. The yields of RPLKPW from 3 RPLKPW-sites were increased markedly. Furthermore, we introduced fourth RPLKPW sequence to the α' subunit. The minimum effective dose of α' subunit containing four RPLKPW was 2.5 mg/kg, about one-fourth of that of modified α' subunit that we previously reported. The anti-hypertensive activity of α' subunit containing four RPLKPW was larger than ovalbumin by about eight hundred times. Soy Protein Research, Japan5, 26-30, 2002.
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  • Bioscience Biotechnology and Biochemistry - BIOSCI BIOTECHNOL BIOCHEM. 01/2001; 65(3):736-739.