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Antifungal peptides with a molecular mass of 9 kDa and an N‐terminal sequence demonstrating remarkable similarity to those of nonspecific lipid transfer proteins (nsLTPs) were isolated from seeds of the vegetable Brassica campestris and the mung bean. The purified peptides exerted an inhibitory action on mycelial growth in various fungal species. The antifungal activity of Brassica and mung bean nsLTPs were thermostable, pH‐stable, and stable after treatment with pepsin and trypsin. In contrast, the antifungal activity of mung bean chitinase was much less stable to changes in pH and temperature. Brassica LTP inhibited proliferation of hepatoma Hep G2 cells and breast cancer MCF 7 cells with an IC50 of 5.8 and 1.6 µm, respectively, and the activity of HIV‐1 reverse transcriptase with an IC50 of 4 µm. However, mung bean LTP and chitinase were devoid of antiproliferative and HIV‐1 reverse transcriptase inhibitory activities. In contrast to the mung bean LTP, which exhibited antibacterial activity, Brassica LTP was inactive. All three antifungal peptides lacked mitogenic activity toward splenocytes. These results indicate that the two LTPs have more desirable activities than the chitinase and that there is a dissociation between the antifungal and other activities of these antifungal proteins. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.

The binding ability of bovine milk caseins with mutagenic heterocyclic amines was investigated. Binding was determined with 2 mg of casein and 20 micrograms of heterocyclic amine in .40 ml of pH 7.4, 50 mM phosphate buffer, at 37 degrees C, in a shaker for 10 min. The unbound heterocyclic amine in protein-free ultrafiltrate was analyzed by HPLC. The binding ability of whole casein, alpha s-casein, beta-casein, and kappa-casein, respectively, was 90.08, 83.06, 90.92, and 96.70% with 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole; 85.48, 51.54, 63.62, and 82.71% with 3-amino-1-methyl-5H-pyrido[4,3-b]indole; and 87.03, 59.77, 97.04, and 88.30% with 2-amino-6-methyldipyrido[1,2-a:3',2'-d]-imidazole. Higher binding of 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole and 3-amino-1-methyl-5H-pyrido[4,3-b]indole to alpha s-casein, beta-casein, and kappa-casein was observed at pH above 7.4, and the binding was inhibited at pH below 6.5. The maximum binding of 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole to these caseins was at pH 6.6 and 7.4. The binding was inhibited at alkaline pH above 8.5 and acidic pH below 6.5.

The binding ability of bovine milk proteins with mutagenic heterocyclic amines was investigated. Binding was determined with 2 mg of beta-lactoglobulin and 20 micrograms of heterocyclic amine in .4 ml of pH 7.4, 50 mM phosphate buffer, at 37 degrees C, in a shaker for 10 min. The unbound heterocyclic amine in protein-free ultrafiltrate was analyzed by HPLC method. The binding of alpha-lactalbumin, beta-lactoglobulin A and beta-lactoglobulin B were 90.44, 81.38, and 89.18%, respectively, with 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole; 37.85, 34.04, and 43.90%, respectively, with 3-amino-1-methyl-5H-pyrido[4,3-b]indole; and 49.11, 43.25, and 57.44%, respectively, with 2-amino-6-methyldipyrido[1,2-a:3',2'-d] imidazole. Binding of beta-lactoglobulin and alpha-lactalbumin to 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole and 3-amino-1-methyl-5H-pyrido [4,3-b]-indole was higher at pH conditions above 7.4, and binding was lost at pH less than 5.5. Maximum binding of both proteins to 2-amino-6-methyl-dipyrido[1,2-a:3',2'-d]imidazole was at pH 7.4, and binding was inhibited at pH conditions above 8.5 and less than 6.5.

Lactoperoxidase and lactoferrin were isolated by carboxymethyl cation-exchange chromatography using .05 M phosphate buffer (pH 7.7) and a linear gradient of NaCl from 0 to .55 M . Carboxymethyl-Toyopearl adsorbed only lactoperoxidase and lactoferrin from the albumin fraction of bovine milk acid whey. Lactoperoxidase was released between .10 to .15 M NaCl with a recovery of 91.4%. Lactoferrin was released between .4 to .55 M NaCl and was separated into lactoferrin-a and lactoferrin-b. Yields were 41mg of lactoperoxidase, 21mg of lactoferrin-a, and 67mg of lactoferrin-b as protein from 1L of acid whey. Several minor peaks eluted between 0 to .55M NaCl, and a strong muddy peak was observed during the regenerating phase using .2 N NaOH. The latter peak was estimated as the Ig.

牛乳中の各種蛋白質、即ち whole casein, αs-casein, β-casein, κ-casein, β-lactoglobulin A, β-lactoglobulin B, α-lactalbumin, lactoferrin, lactoperoxidase を分離精製し、これらの蛋白質が発癌性ヘテロサイクリックアミンである 3-amino-1, 4-dimethyl-5Hpyrido[4, 3-b]indole (Trp-P-1) と給合するか否かについて実験を行った。さらに我々自身で分離調製した蛋白質と比較検討する為にSigma社製のα-casein, β-casein, κ-casein, β-lactoglobulin A, β-lactoglobulin B, α-lactalbumin, lactoferrin, lactoperoxidase ついても同様の実験を行った。凍結乾燥した蛋白質2mg, Trp-P1 20μgを0.4mlのpH7.4, 50mM燐酸バッファーに溶解し、37℃で10分間振盪し反応させる。つづいて、日本ミリポアリミテッド製のUFC3LGC00を用いて反応液中の非結合型 Trp-P-1 を限外濾過により分離し、この Trp-P-1 を HPLC 法により測定する。HPLC は日立 L-6000ポンプ・L-4000UV 検出機に GL-Science 製 Inertsil ODS-2(4.6×150mm)カラムを使用し、acetonitrile-H2O-triethylamine 50:50:0.05 で展開した。254nmに於ける吸収を SIC System Instruments 製Labchart 12で記録する。Whole casein:54.04%, αs-casein:40.02%, β-casein:56.24%, κ-casein:33.75% で反応液中の Trp-P-1 を比較的に高い割合で結合した。Sigma 社製の α-Casein:59.46%, β-casein:40.43%, κ-casein:23.75% で類似の傾向を示した。β-Lactoglobulin A:23.61%, β-lactoglobulin B:17.87%, β-lactoglobulin A・Sigma:15.78%, β-lactoglebulin B・sigma:3.65% であった。Lactoperoxidase:15.20%, lactoperoxidase・Sigma:6.41%, lactoferrin-a:22.53%, lacteferrin-b:6.92% であったが、α-lactalbumain, α-lactalbumin・Sigma, lacteferrin・Sigma は Trp-P-1 と結合しなかった。, The binding ability of bovine milk proteins with mutagenic heterocyclic amine was investigated. Binding was determined with 2 mg of protein and 20 g of 3-amino-1, 4-dimethyl-5H-pyrido[4, 3-b]indole (Trp-P-1) in 0.4 ml of pH 7.4, 50 mM phosphate buffer, at 37°C, in a shaker for 10 min. The unbound Trp-P-1 in protein-free ultrafiltrate was prepared using Ultrafree UFC3LGC00 and was analyzed by HPLC method. The binding of whole casein, αs-casein, β-casein and κ-casein were 54,03, 40.02, 56.24 and 33.75%, respectively. β-Lactoglobulin A, β-lactoglobulin B, lactoperoxidase, lactoferrin-a and lactoferrin-b were 23.61, 17.87, 15.20, 22.53 and 6.92% respectively. However, α-lactalbumin could not bind Trp-P-1 in this experiment. The binding of these milk proteins which were prepared by ourselves were compared with purchased proteins. They showed similar tendency to our proteins.