Purification of bovine and porcine enterokinase by affinity chromatography with immobilized kidney bean enterokinase inhibitor

A specific enterokinase inhibitor isolated from kidney bean (Phaseolus vulgaris) was immobilized on Affigel-10. Solubilized preparation of bovine and porcine enterokinases were bound to this matrix at pH 7.5 and the complex was dissociated by elution with l0 mM HCl, resulting in the isolation of the enzymes in homogeneous form as judged by gel chromatography on Sephadex G-200, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. However, human enterokinase could not be purified by this method in sufficient yield since it did not bind strongly to the insolubilized inhibitor.     

The release of proteinase inhibitors from legume seeds during germination

The seeds of twelve common species of legumes were examined for the release of proteinase inhibitor activity during germination. All species released inhibitory activity against bovine trypsin (EC 3.4.21.4), ranging from 1.0 unit per g dry wt. of seed in 24 hr for soybean (Glycine max), to 0.07 unit per g for broad beans (Vicia faba) and sugar pod peas (Pisum sativum). This release corresponds to approximately 1–13 % of the total trypsin inhibitory activity of the seed, with lentils (Lens culinaris) releasing the greatest percentage, and the scarlet runner bean (Phaseolus coccineus) the least. In most species the amount of inhibitor released increases until 24–48 hr of germination, and then remains roughly the same or decreases slightly by 72 hr of germination. Five species of legumes were also examined for the release of inhibitory activity against bovine chymotrypsin (EC 3.4.21.1). In each case chymotryptic inhibitory activity was released in a manner similar to the trypsin inhibitor.     

A specific inhibitor of Colletotrichum lindemuthianum protease from kidney bean (Phaseolus vulgaris) seeds

A specific protein—an inhibitor of Colletotrichum lindemuthianum protease—was isolated from kidney bean seeds in a homogeneous form. The purification procedure included gel filtration, isoelectric focusing and affinity chromatography on trypsin-Sepharose column. The latter was introduced to separate the fungal protease inhibitor from closely related trypsin and chymotrypsin inhibitors present in kidney bean seeds.     

Enzyme inhibitors from plants. Isolation and characterization of a protease inhibitor from arrow root (Maranta arundinaceae) tuber

A protease inhibitor from arrow root (Maranta arundinaceae) tuber has been isolated in a homogeneous form. The inhibitor has a Mr of 11,000-12,000; it inhibited bovine trypsin, bovine enterokinase, bovine α-chymotrypsin and the proteolytic activity of human and bovine pancreatic preparations. The inhibitor is resistant to pepsin, and elastase. It could withstand heat treatment at 100°C for 60 min and exposure to a wide range of pH (1.0–12.5) for 72 h at 4°C without loss of activity. Arginyl groups are essential for the action of the inhibitor. Preincubation of the inhibitor at pH 3.7 with trypsin or chymotrypsin caused nearly a two-fold increase in inhibitor potency     

Leaf Spot of Alternanthera sessilis and Its Implications for Food Crops

A leaf spot of Alternanthera sessilis caused by Fusarium pallidoroseum (syn. F. semitectum) is described. During pathogenicity tests, the fungus caused leaf spots of Amarantbus sp. Abelmoschus esculentus (okra), Celosia sp. and Sorghum bicolor. It also caused rots of tubers of Dioscorea rotundata, D. alata, Solanum tuberosum, corms of Colocasia esculenta, bulbs of Allium cepa and roots of Daucus carota when wounded. Unwounded tubers, corms, bulbs and roots of these named plants were not infected except roots of Daucus carota which disintegrated between 4 and 6 days after inoculation.     

Proteolytic enzymes; further studies on protein,polypeptide, and other inhibitors of serum proteinase,leucoproteinase, trypsin,and papain

1. Serum proteinase precursor was found in plasma protein fractions I and III of Cohn. Inhibitors of serum proteinase, leucoproteinase, trypsin, and papain were found in fractions IV-1 and IV-4, and to a lesser extent in fractions V and I. 2. Pancreatic, soy bean, lima bean, and egg white inhibitors inhibited trypsin stoichiometrically. Pancreatic inhibitor had comparable inhibitory activity against serum proteinase; soy bean inhibitor had somewhat less, lima bean inhibitor even less, and egg white inhibitor very little. None of these inhibitors appreciably inhibited leucoproteinase or papain. 3. Serum and fractions IV - 1 and IV - 4 had marked inhibitory activity against trypsin and leucoproteinase, and somewhat less against serum proteinase and papain. The inhibitory activity of the plasma proteins against trypsin and leucoproteinase was due almost entirely to fractions IV - 1 and IV - 4; against serum proteinase and papain fraction V was slightly more important. The "reconstituted plasma proteins" accounted for 8 to 25 per cent of the proteinase-inhibitory activity of whole serum or plasma. 4. The proteinase-inhibitory activity of serum, plasma protein fractions, and soy bean inhibitor was heat labile, while that of pancreatic, lima bean, and egg white inhibitors was relatively heat stable. 5. Reducing and oxidizing agents, in very high concentration, inhibited serum proteinase, as well as trypsin and leucoproteinase. These proteinases were not influenced by mercurial sulfhydryl inhibitors, indicating that free sulfhydryl groups do not play an important part in their activity.     

Aphid population abundance and pestiferous effect on various bean plant species

The population abundance, infestation, and harmful effects of the aphid Aphis craccivora Koch (Hemiptera: Aphididae) were studied on four bean plant species, namely the country bean (Lablab purpureus var. BARI Seem 1), the yard‐long bean (Vigna sesquipedalis var. BARI Borboti 1), the hyacinth bean (Dolichos lablab var. BARI Seem 6), and the bush bean (Phaseolus vulgaris var. BARI Jar Seem 3). Aphid abundance and infestation on the leaves, inflorescences, flowers, and pods differed significantly among the bean plant species, with P. vulgaris and V. sesquipedalis having the lowest and highest results, respectively. Aphid severity grade and the number of trichomes of the bean plant species were negatively correlated. The duration of the growth stages among the bean plant species were significantly different, with V. sesquipedalis having the shortest durations. Aphid abundance and infestation significantly affected the physical and phytochemical characteristics of the bean plant species. The highest reduction of number of leaves, flower inflorescences, and pod inflorescences per plant, and moisture and chlorophyll content in the leaves was found in L. purpureus. The results for V. sesquipedalis revealed the highest reduction in plant height, seed weight, and pH, while those of D. lablab showed the highest reduction in leaf area.     

Chymotrypsin inhibitors in mosquitoes: Activity profile during development and after blood feeding

Chymotrypsin and trypsin inhibitors persist throughout all developmental instars of Aedes aegypti. After a blood meal, inhibitor activity against chymotrypsin was more than double that of sugar-fed females, but only weak activity was detected in midguts where proteinase inhibitors has been thought to regulate proteinases during blood digestion. A fourfold increase in the ratio of abdominal/thoracic inhibitor activity after the blood meal strongly suggested that fat body, or other abdominal tissues, represent the major source of inhibitor. Chymotrypsin inhibitor activity was deposited in maturing oocytes. Similar results were obtained with blood-fed Anopheles albimanus. Chymotrypsin inhibitor was active against different mosquito proteinases and against bovine α-chymotrypsin and trypsin, but not against subtilisin, pancreatic elastase, or fungal proteases; chymotrypsin inhibitors did not interfere with bacterial growth. The hypothesis on the regulation of blood digestion through the action of proteinase inhibitors during the gonotrophic cycle was abandoned and its involvement in the phenoloxidase cascade in the mosquito egg chorion is suggested instead. Arch. Insect Biochem. Physiol. 36:315–333, 1997. © 1997 Wiley-Liss, Inc.     

Protease inhibitors from jackfruit seed (Artocarpus integrifolia)

Protease inhibitory activity in jackfruit seed (Artocarpus integrifolia) could be separated into 5 fractions by chromatography on DEAE-cellulose at pH 7.6. A minor fraction (I) that did not bind to the matrix, had antitryptic, antichymotryptic and antielastase activity in the ratio 24:1.9:1.0. Fraction II bound least tightly to the ion exchanger eluting with 0.05 M NaCl and could be resolved into an elastase/chymotrypsin inhibitor and a chymotrypsin/trypsin inhibitor by chromatography on either immobilized trypsin or phenyl Sepharose CL-4B. Fractions III and IV eluted successively with 0.10 M NaCl and 0.15 M NaCl from DEAE-cellulose, inhibited elastase, chymotrypsin and trypsin in the ratio 1.0: 0.53:0.55 and 1.0:8.9:9.8 respectively. Fraction V, most strongly bound to the matrix eluting with 0.3 M NaCl and was a trypsin/chymotrypsin inhibitor accounting for 74% of total antitryptic activity. This inhibitor was purified further. The inhibitor with a molecular weight of 26 kd was found to be a glycoprotein. Galactose, glucose, mannose, fucose, xylose, glucosamine and uronic acid were identified as constitutent units of the inhibitor. Dansylation and electrophoresis in the presence of mercaptoethanol indicated that the inhibitor is made up of more than one polypeptide chain. The inhibitor combined with bovine trypsin and bovine α-chymotrypsin in a stoichiometric manner as indicated by gel chromatography. It had very poor action on subtilisin BPN′, porcine elastase, pronase,Streptomyces caespitosus protease andAspergillus oryzae protease. It powerfully inhibited the caseinolytic activities of rabbit and horse pancreatic preparations and was least effective on human and pig pancreatic extracts. Modification of amino groups, guanido groups and sulphydryl groups of the inhibitor resulted in loss of inhibitory activity. Reduction of disulphide bridges, reduction with sodium borohydride and periodate oxidation also decreased the inhibitory activity.     

Characterization of digestive proteases in the weevil Aubeonymus mariaefranciscae and effects of proteinase inhibitors on larval development and survival

The major digestive proteinase activities of a new sugar beet pest, Aubeonymus mariaefranciscae Roudier (Coleoptera: Curculionidae), were characterized. Both larvae and adults of A. mariaefranciscae were found to use a complex proteolytic system for protein digestion based on at least trypsin-, chymotrypsin-, elastase-, cathepsin D, leucine aminopeptidase-, carboxypeptidase A- and carboxypeptidase B-like activities. An azocaseinolytic activity at pH 5.0–7.0 was identified, that was not affected by specific inhibitors and activators, making its classification in any of the mechanistic classes established not possible. According to this proteolytic profile, several serine proteinase inhibitors were tested in vitro and in vivo to establish their potential as resistance factors against A. mariaefranciscae. Larvae fed from neonate to pupation on diets containing 0.2% (w/w) soybean Bowman-Birk trypsin-chymotrypsin inhibitor, soybean Kunitz trypsin inhibitor, turkey egg white trypsin inhibitor, or lima bean trypsin inhibitor endure lower survival rates and display significant delays in the developmental time to pupation and to adult emergence. Interestingly, the most significant levels of mortality (about 90%) occurred with larvae fed on diets containing a combination of two or three inhibitors, suggesting a synergistic toxicity.     

Localization of ubiquitin to differentiating vascular tissues

The selective degradation of proteins, an essential process of any developmental program, may entail conjugation of the protein to be destroyed to the polypeptide ubiquitin. Experiments were designed to localize ubiquitin as a first step in determining whether this molecule is crucial for certain developmental processes in plant tissues and cells. Antibodies to ubiquitinated protein were detected on tissue prints of cross sections of bean petioles (Phaseolus vulgaris, Fabaceae), cotton hypocotyls (Gossypium hirsutum, Malvaceae), and Coleus stems (Coleus x hybridus, Lamiaceae). For most of the material investigated, there appears to be an accumulation of ubiquitin antibodies in vascular tissues, but not preferentially in the abscission zone of bean petioles. Vascular localization was confirmed using immunohistochemical methods on fixed and sectioned internodal tissues of Coleus. Antibodies to ubiquitin are detected in parenchyma cells of the cortex and pith, but are most concentrated in the xylem, especially secondary xylem, and in the cambial region, and in the phloem. Thus, ubiquitin accumulates in certain vascular tissues, some of which may be undergoing programmed cell death. Ubiquitin can also be detected in nondifferentiating cells, and its level is elevated in rapidly dividing cambial cells.     

Effects of bean and wheat α-amylase inhibitors on α-amylase activity and growth of stored product insect pests

Insect α-amylase inhibiting and/or growth inhibiting activities of proteinaceous inhibitors from red kidney bean (Phaseolus vulgaris) and hard red winter wheat (Triticum aestivum) were examined. The bean inhibitor was most effectivein vitro against α-amylases from the red flour beetle (Tribolium castaneum) and the confused flour beetle (T. confusum), followed by those from the rice weevil (Sitophilus oryzae) and yellow mealworm (Tenebrio molitor). The insect enzymes were from two- to 50-fold more susceptible than human salivary α-amylase. When the inhibitors were added at a 1% level to a wheat flour plus germ diet, the growth of red flour beetle larvae was slowed relative to that of the control group of larvae, with the bean inhibitor being more effective than the wheat inhibitor. Development of both the red flour beetle and flat grain beetle (Cryptolestes pusillus) was delayed by 1% bean inhibitor, but development of the sawtoothed grain beetle (Oryzaephilus surinamensis) and lesser grain borer (Rhyzopertha dominica) was not affected by either the bean or wheat inhibitor at the 1% level. Rice weevil adults fed a diet containing 1% bean or wheat inhibitor exhibited more mortality than weevils fed the control diet. When the wheat amylase inhibitor was combined with a cysteine protease inhibitor, E-64, and fed to red flour beetle larvae, a reduction in the growth rate and an increase in the time required for adult eclosion occurred relative to larvae fed either of the inhibitors separately. The bean inhibitor was just as effective alone as when it was combined with the protease inhibitor. These results demonstrate that plant inhibitors of insect digestive enzymes act as growth inhibitors of insects and possibly as plant defense proteins, and open the way to the use of the genes of these inhibitors for genetically improving the resistance of cereals to storage pests. Cooperative investigation between the Agricultural Research Service, the University of California, San Diego, and the Kansas Agricultural Experiment Station (Contribution no. 94-416-J). Supported in part by a grant from the Ministry of Education and Science, Spain-Fulbright Program to J.J.P. Mention of a proprietary product does not constitute a recommendation or endorsement by the USDA. The USDA is an equal opportunity/affirmative action employer and all agency services are available without discrimination.     

Novel proteinase inhibitors in seeds of sunflower (Helianthus annuus L.): polymorphism, inheritance and properties

A highly sensitive gelatin overlay procedure was used to identify inhibitors of serine proteinases and of the cysteine proteinase ficin in seeds and leaves of sunflower. One major and two minor groups of trypsin inhibitors were identified in seeds, the former having a high pI (@10) and also inhibiting chymotrypsin. Three groups of trypsin/subtilisin inhibitors were also present in seeds, together with three inhibitors of ficin. All groups showed polymorphism between lines of Helianthus annuus, while the trypsin and trypsin/subtilisin inhibitors also varied between wild species of Helianthus, with no apparent relationship to growth type (annual or perennial), genome constitution or ploidy level. Genetic analysis showed that the major trypsin inhibitor and three groups of trypsin/subtilisin inhibitors are each controlled by single Mendelian loci, with the three loci for trypsin/subtilisin inhibitors showing recombination values of 0.23–0.40. Purification by RP-HPLC allowed the M _r of two trypsin inhibitors to be determined by SDS-PAGE to be about 1,500 and 2,500, while the three trypsin/subtilisin inhibitors varied in M _r from about 1,500 to 6,000. Received: 7 March 1999 / Accepted: 18 March 1999     

New Techniques for the Estimation of Naturally Occurring Brassinosteroids

We have developed enzyme-linked immunosorbent assays (ELISAs) for measuring 24-epicastasterone and related brassinolide analogs, with detection ranges of 0.005 to 50 pmoles. Polyclonal antibodies used in these assays were raised against 24-epicastasterone carboxymethyloxime-bovine serum albumin conjugates and were found to have high specificity for 24-epibrassinosteroids. Natural brassinosteroids (BRs), such as brassinolide and 24-epibrassinolide, exhibited relatively high cross-reactivities with the generated antibodies, whereas other BR analogs with β-oriented hydroxyl groups at C-2, C-3, C-22, and C23 lacked immunoreactivity. Through the use of internal standardization, dilution assays, recovery of authentic [³H]24-epicastasterone, and immunohistograms, the ELISAs have been shown to be applicable for estimating 24-epibrassinosteroid levels in crude plant extracts. To analyze brassinosteroids in tissues from young bean (Phaseolus vulgaris L., cv. Pinto), Daucus carota ssp.sativus plants and Arabidopsis thaliana L. Heynh. seedlings, and rape (Brassica napus L.) pollen, the extracts were fractionated by high performance liquid chromatography (HPLC) and the resulting fractions were analyzed by the ELISA method. Immunohistogram ELISA analysis of HPLC fractions indicated that major peaks of immunoreactivity co-chromatographed with the labeled and unlabeled 24-epibrassinolide. A highly sensitive electrospray ionization mass spectrometry (MS) technique (LOD: 50 fmol) was also developed and the results obtained by the HPLC-ELISA and HPLC-MS approaches were compared.     

Attachment of bean rust cell wall material to host and non-host plant tissue

A preparation of bean rust (Uromyces phaseoli) germ tube walls, consisting of short, filamentous particles, was labeled with fluorescein iso-thiocyanate. Freeze sections of host and non-host tissue were incubated in the labeled preparation. Maximum staining was observed in host plant tissue (Phaseolus vulgaris), in which bean rust regularly forms haustoria. In tissue of the non-host plantsVigna sinensis andPhaseolus lunatus, where fewer haustoria were formed, staining was only weak. However, no staining was observed in the non-host tissue ofPhaseolus aureus, Helianthus annuus, Brassica oleracea andHordeum vulgare in which the infection hypha did not form haustoria. This would appear to indicate that formation of haustoria is induced by a specific attachment of the hyphal wall to the host wall. The possibility that elicitors attach in a similar way, is discussed.Abbreviations FITC Fluorescein iso-thiocyanate - cv cultivar     

Microelectrophoretic method for the detection of proteinase inhibitors

A microelectrophoretic method for the detection of proteinase inhibitors is deseribed. Microscope slides covered with agar gel containing casein were used for the electrophoretic separation of proteins from bean seeds. Subsequently, the slides were covered with a filter paper strip saturated with a proteinase solution of a known concentration. After 60 min of digestion, the filter paper strips were removed, and the slides were stained with amido black. Blue spots appeared where the casein substrate was protected from digestion by the presence of proteinase inhibitors. Single seed or leaf extracts can be studied by this method. Different trypsin inhibition patterns were observed for samples of different bean varieties. An inhibitor for subtilisin was detected in all the Ph. vulgaris samples studied.     

On the Relationship between the Activity of O-Methyltransferase and the Content of Lignin in Various Organs of Kidney Bean

The activity of O-methyltransferase (OMT) and the content of lignin in various organs of 12- to 14-day-old seedlings of kidney bean (Phaseolus vulgarisL., cv. Saksa) were determined. The epicotyl and hypocotyl were characterized by the highest OMT activity, and the leaves, by the lowest OMT activity. At the same time, the lignin content gradually increased in the following order: leaves < epicotyl < hypocotyl < roots. Thus, in kidney bean seedlings, there is no direct relationship between the OMT activity and the lignin content. It is suggested that lignin biosynthesis is limited by biosynthetic steps other than O-methylation of caffeic and 5-hydroxyferulic acids affected by OMT.     

Two kunitz-type proteinase inhibitors from potato tubers

Two proteinase inhibitors have been isolated from tubers of potato (Solanum tuberosum). Based on N-terminal amino acid sequence homologies, they are members of the Kunitz family of proteinase inhibitors. Potato Kunitz inhibitor-1 (molecular weight 19,500, isoelectric point 6.9) is a potent inhibitor of the animal pancreatic proteinase trypsin, and its amino terminus has significant homology to a recently characterized cathepsin D Kunitz inhibitor from potato tubers (Mares et al. [1989] FEBS Lett 251:94-98). Potato Kunitz inhibitor-2 (molecular weight 20,500, isoelectric point 8.6) is an inhibitor of the microbial proteinase subtilisin Carlsberg; its amino terminus is almost identical to an abundant 22 kilodalton protein from potato tubers (Suh et al. [1990] Plant Physiol 94:40-45) and has significant homology to other Kunitz-type subtilisin inhibitors from small grains. Both Kunitz inhibitors are abundant proteins of the cortex of potato tubers.     

Purification of a trypsin inhibitor secreted by embryogenic carrot cells

A protease inhibitor with a molecular weight of about 12,800 was purified to electrophoretic homogeneity from Daucus carota cells. The protease inhibitor was heat stable and inhibited trypsin but had no activity toward chymotrypsin or subtilisin. Nonembryogenic as well as embryogenic strains contained the inhibitor in similar amounts, but in the embryogenic strains the trypsin inhibitor was released from the cells and as a result accumulated in high concentrations in the culture medium, whereas no release of the trypsin inhibitor was found during cultivation of the nonembryogenic strains. Very low amounts of acid phosphatase or α-mannosidase activity were found in the culture filtrate of both embryogenic and nonembryogenic strains, which suggest that the release of the inhibitor from embryogenic strains was not due to cell lysis.     

Pectin esterase in relation to leaf abscission in coleus and phaseolus

Pectin esterase (PE) activities in abscission zones, other portions of leaves, and adjacent stem tissues were compared in attached leaves and abscissing petioles (previously debladed) of Coleus blumei Benth. and Phaseolus vulgaris L., cv. Canadian Wonder. Earlier findings of Osborne in bean were confirmed and changes in PE activity in coleus were shown to resemble those in bean in some respects. In both plants PE was lower in the distal portion of abscission zones of abscissing petioles than in that portion of attached leaves but this difference was not as large or as consistently clear-cut in coleus as in bean. The general level of PE activity was an order of magnitude lower and changes associated with abscission were smaller in coleus than in bean. Auxin treatment of debladed petioles of coleus prevented abscission and resulted in small increases in PE activity in abscission zones and most of the other regions sampled. The largest increase was observed in the stem tissue adjacent to the attached leaf opposite the debladed, auxin treated one.