X.Y. Ye

Sativin: A novel antifungal miraculin-like protein isolated from legumes of the sugar snap Pisum sativum var. macrocarpon

An antifungal protein designated sativin was isolated from the legumes of the sugar snap (also known as honey pea) Pisum sativum var. macrocarpon. The procedure entailed extraction, affinity chromatography on Affi-gel blue gel and ion exchange chromatography on CM-Sepharose. The protein exhibited a molecular weight of 38 kDa in SDS-polyacrylamide gel electrophoresis. It possessed an N-terminal amino acid sequence which showed similarity to those of miraculin (a sweet protein) and pisavin (a ribosome-inactivating protein from Pisum sativum var arvense Poir manifesting similarity to miraculin). Unlike pisavin, however, sativin demonstrated negligible ribonuclease activity and inhibited translation in a rabbit reticulocyte lysate system with a very low potency (IC50= 14 microM). Sativin exerted antifungal activity against Fusarium oxysporum, Coprinus comatus and Pleurotus ostreatus but not against Rhizoctonia solani.

Structurally dissimilar proteins with antiviral and antifungal potency from cowpea (Vigna unguiculata) seeds.

Evidence is presented for the existence of multiple proteins with antifungal and antiviral potency in cowpea seeds. The two proteins, designated alpha- and beta-antifungal proteins in accordance with their order of elution from the CM-Sepharose column, were capable of inhibiting human immunodeficiency virus (HIV) reverse transcriptase and one of the glycohydrolases associated with HIV infection, alpha-glucosidase, but beta-glucuronidase was not repressed. The ability of the proteins in retarding mycelial growth of a variety of fungi was also demonstrated with alpha-antifungal protein being more potent in most of the cases. Beta-antifungal protein was more active in only one instance. Both antifungal proteins had low cell-free translation-inhibitory activity. The proteins were adsorbed on Affi-gel blue gel-and CM-Sepharose but could be separated from one another during chromatography on the latter medium by means of a linear NaCl concentration gradient. Different molecular weights were exhibited by the proteins, being 28 kDa and 12 kDa respectively for alpha- and beta- antifungal proteins. Alpha-antifungal protein was characterized by an N-terminal sequence showing close resemblance to sequences of chitinases. Beta-antifungal protein exhibited an N-terminal sequence hitherto unknown in the literature.

Isolation of lactoperoxidase, lactoferrin, α-lactalbumin, β-lactoglobulin B and β-lactoglobulin A from bovine rennet whey using ion exchange chromatography

A mild and rapid method is described for isolating various milk proteins from bovine rennet whey. beta-Lactoglobulin from bovine rennet whey was easily adsorbed on and desorbed from a weak anion exchanger, diethylaminoethyl-Toyopearl. However, alpha-lactalbumin could not be adsorbed onto the resin. alpha-Lactalbumin and beta-lactoglobulin from rennet whey could also be adsorbed and separated using a strong anion exchanger, quaternary aminoethyl-Toyopearl. The rennet whey was passed through a strong cation exchanger, sulphopropyl-Toyopearl, to separate lactoperoxidase and lactoferrin. alpha-Lactalbumin and beta-lactoglobulin were adsorbed onto quaternary aminoethyl-Toyopearl. alpha-Lactalbumin was eluted using a linear (0-0.15 M) concentration gradient of NaCl in 0.05 M Tris-HCl buffer (pH 8.5). Subsequently, beta-lactoglobulin B and beta-lactoglobulin A were eluted from the column with 0.05 M Tris-HCl (pH 6.8), using a linear (0.1-0.25 M) concentration gradient of NaCl. The yields were 1260 mg alpha-lactalbumin, 1290 mg beta-lactoglobulin B and 2280 mg beta-lactoglobulin A from 1 l rennet whey.

Isolation of a novel peroxidase from French bean legumes and first demonstration of antifungal activity of a non-milk peroxidase.

A novel antifungal protein with its N-terminal sequence bearing similarity to the C-terminal sequences of peroxidases was isolated from French bean legumes. The protein, which possessed a molecular weight of 37 kDa, was adsorbed on Affi-gel blue gel and CM-Sepharose. The protein exhibited peroxidase activity with a Km of 58 microM and a Vmax of 3.36 U/nmol. Optimal peroxidase activity was found at 22 degrees C and pH 4. It exerted antifungal activity against a variety of fungal species including Coprinus comatus, Mycosphaerella arachidicola, Fusarium oxysporum and Botrytis cinerea. It inhibited the activities of alpha-glucosidase and beta-glucosidase but was without any inhibitory effect on HIV-1 reverse transcriptase.

Isolation of a new cyclophilin-like protein from chickpeas with mitogenic, antifungal and anti-HIV- 1 reverse transcriptase activities

A protein designated chickpea cyclophilin-like antifungal protein (CLAP) was isolated from seeds of the chickpea (Cicer arietinum). Chickpea CLAP was characterized by a molecular weight of 18 kDa and an N-terminal sequence homologous to cyclophilins. The protein was isolated with a procedure involving affinity chromatography on Affi-gel blue gel and ion exchange chromatography on CM-Sepharose. In addition to an inhibitory effect on the growth of fungi including Rhizoctonia solani, Mycosphaerella arachidicola and Botrytis cinerea, the protein was capable of inhibiting human immunodeficiency virus-1 reverse transcriptase. Chickpea CLAP did not possess lectin and ribonuclease activities but it weakly inhibited translation in a rabbit reticulocyte lysate system. The protein stimulated 3H-thymidine incorporation by mouse splenocytes.

Cicerin and arietin, novel chickpea peptides with different antifungal potencies

Two antifungal peptides with novel N-terminal sequences, designated cicerin and arietin were isolated from seeds of the chickpea (Cicer arietinum), respectively. Both peptides were adsorbed on Affi-gel blue gel and CM-Sepharose and exhibited a molecular weight of approximately 8.2 and 5.6 kDa, respectively. Arietin was more strongly adsorbed on CM-Sepharose than cicerin and manifested a higher translation-inhibiting activity in a rabbit reticulocyte lysate system and a higher antifungal potency toward Mycosphaerella arachidicola, Fusarium oxysporum and Botrytis cinerea. Both were devoid of mitogenic and anti-HIV-1 reverse transcriptase activities.

Ribonuclease, cell-free translation-inhibitory and superoxide radical scavenging activities of the iron-binding protein lactoferrin from bovine milk

The purpose of this study was to characterize the ribonuclease (RNase) and cell-free translation-inhibitory activities of lactoferrin isolated from bovine milk. It was found that bovine lactoferrin exhibited ribonucleolytic activity toward yeast transfer RNA in a dose-dependent manner. The pH optimum for this RNase activity was in the vicinity of 7.5. Lactoferrin exerted RNase activity on poly C with an activity of 2.15 U/mg. No activity was detected toward poly A, poly G, and poly U. The milk protein inhibited cell-free translation in rabbit reticulocyte lysate with an IC50 of 9.6 microM. The protein was devoid of N-glycosidase activity characteristic of ribosome inactivating proteins which also possess RNase and cell-free translation-inhibitory activities. It inhibited superoxide radical formation.

Inhibitory effects of antifungal proteins on human immunodeficiency virus type 1 reverse transcriptase, protease and integrase.

A variety of antifungal proteins were isolated from seeds of leguminous plants including French bean, cowpea, field bean, mung bean, peanut and red kidney bean. They were assayed for ability to inhibit human immunodeficiency virus type I (HIV-1) reverse transcriptase, protease and integrase, enzymes essential to the life cycle of HIV-1 . It was found that the cowpea beta-antifungal protein had a high potency in inhibiting HIV-1 protease and HIV-1 integrase. Cowpea alpha-antifungal protein was potent in inhibiting HIV-1 reverse transcriptase and HIV-1 integrase. Peanut antifungal protein was characterized by a high inhibitory activity against HIV-1 integrase and an intermediate potency in inhibiting HIV- I reverse transcriptase and HIV- I protease. French bean thaumatin-like protein expressed low HIV- I protease inhibitory activity and red kidney bean lectin inhibited HIV- I integrase by only a very small extent. Antifungal proteins from the field bean and mung bean had an intermediate potency in inhibitory HIV-1 protease and integrase. However, mung bean antifungal protein was not capable of inhibiting HIV-1 reverse transcriptase. The results indicate that nearly all leguminous antifungal proteins examined were able to inhibit HIV-1 reverse transcriptase, protease and integrase to some extent.

Inhibition of human immunodeficiency virus type 1 reverse transcriptase, protease and integrase by bovine milk proteins

Different proteins have been isolated from bovine milk including lactoferrin, lactoperoxidase, glycolactin, angiogenin-1, lactogenin, alpha-lactalbumin, lactoglobulin and casein. These proteins have been assayed for inhibitory activity against human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, protease and integrase, enzymes crucial to the HIV-1 life cycle. It was found that different milk proteins inhibited the three aforementioned HIV enzymes to different extents. Lactoferrin strongly inhibited HIV-1 reverse transcriptase but only slightly inhibited HIV-1 protease and integrase. On the other hand, alpha-lactalbumin, beta-lactoglobulin and casein inhibited HIV-1 protease and integrase to an appreciable extent but did not inhibit HIV-1 reverse transcriptase. Glycolactin and angiogenin-1 suppressed the activity of HIV-1 reverse transcriptase by a moderate extent but more powerfully inhibited HIV-1 protease and integrase. In comparison with the other milk proteins glycolactin was a strong inhibitor of HIV-1 protease and integrase and a moderate inhibitor of HIV-1 reverse transcriptase. Lactogenin was a strong inhibitor of HIV-1 integrase, a moderate inhibitor of HIV-1 reverse transcriptase and a weak inhibitor of HIV-1 protease.

A novel and potent ribonuclease from fruiting bodies of the mushroom Pleurotus pulmonarius.

A ribonuclease (RNase), with an N-terminal sequence different from those of ribonucleases from the mushrooms Irpex lacteus, Lentinus edodes, Pleurotus ostreatus, Pleurotus tuber-regium, and Volvariella volvacea, was purified from fruiting bodies of the edible mushroom Pleurotus pulmonarius. The N-terminal sequence of P. pulmonarius RNase manifested homology to a portion of the sequences of ribosome inactivating protein abrin-b, abrin-c, and abrin-d, and Bacillus subtilis transcriptional regulator. The ribonuclease was adsorbed on Affi-gel blue gel, CM-Sepharose, and Mono S. It displayed a molecular mass of 14.4 kDa in both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration on Superdex 75. The ribonuclease exhibited an activity of 25 114 U/mg on yeast tRNA. The highest ribonucleolytic activity was demonstrated toward poly C, followed by poly A, and then by poly G. There was no activity toward poly U. The optimal pH for its activity was 7 and the optimal temperature was 55 degrees C. It inhibited cell-free translation in a rabbit reticulocyte lysate with an IC50 of 0.33 nM.