A metabolite profiling approach to follow the sprouting process of mung beans (Vigna radiata)

A metabolite profiling methodology based on capillary gas chromatography/mass spectrometry (GC/MS) was employed to investigate time-dependent metabolic changes in the course of the sprouting of mung beans (Vigna radiata). Intact mung beans and sprout samples taken during the germination process were subjected to an extraction and fractionation procedure covering a broad spectrum of lipophilic (e.g. fatty acid methyl esters, hydrocarbons, fatty alcohols, sterols) and hydrophilic (e.g. sugars, acids, amino acids, amines) low molecular weight constituents. Investigation of the obtained fractions by GC resulted in the detection of more than 450 distinct peaks of which 146 were identified by means of MS. Statistical assessment of the metabolite profiling data via principal component analysis demonstrated that the metabolic changes during the sprouting of mung beans are reflected by time-dependent shifts of the scores which were comparable for two spouting processes independently conducted under the same conditions. Analysis of the loadings showed that polar metabolites were major contributors to the separation along the first principal component. The dynamic changes of single metabolites revealed significantly increased levels of monosaccharides, organic acids and amino acids and a decrease in fatty acid methyl esters in mung bean sprouts.     

Characterization of the cultivable microbiota of sprouts and their potential for application as protective cultures

The microbiota of ten seeds and ready-to-eat sprouts produced thereof was characterized by bacteriological culture and denaturing gradient gel electrophoresis (DGGE) of amplified DNA fragments of the 16S rRNA gene. The predominant bacterial biota of hydroponically grown sprouts mainly consisted of enterobacteria, pseudomonades and lactic acid bacteria (LAB). For adzuki, alfalfa, mung bean, radish, sesame and wheat, the ratio of these bacterial groups changed strongly in the course of germination, whereas for broccoli, red cabbage, rye and green pea the ratio remained unchanged. Within the pseudomonades, Pseudomonas gesardii and Pseudomonas putida have been isolated and strains of the potentially pathogenic species Enterobacter cancerogenes and Pantoea agglomerans were found as part of the main microbiota on hydroponically grown sprouts. In addition to the microbiota of the whole seedlings, the microbiota of root, hypocotyl and seed leafs were examined for alfalfa, radish and mung bean sprouts. The highest and lowest total counts for aerobic bacteria were found on seed leafs and hypocotyls, respectively. On the other hand, the highest numbers for LAB on sprouts were found on the hypocotyl. When sprouting occurred under the agricultural conditions, e.g. in soil, the dominating microbiota changed from enterobacteria to pseudomonades for mung beans and alfalfa sprouts. No pathogenic enterobacteria have been isolated from these sprout types. Within the pseudomonades group, Pseudomonas jessenii and Pseudomonas brassicacearum were found as dominating species on all seedling parts from soil samples. In practical experiments, a strain of P. jessenii was found to exhibit a potential for use as protective culture, as it suppresses the growth of pathogenic enterobacteria on ready-to-eat sprouts.     

Computational Screening of Tip and Stalk Cell Behavior Proposes a Role for Apelin Signaling in Sprout Progression

Angiogenesis involves the formation of new blood vessels by sprouting or splitting of existing blood vessels. During sprouting, a highly motile type of endothelial cell, called the tip cell, migrates from the blood vessels followed by stalk cells, an endothelial cell type that forms the body of the sprout. To get more insight into how tip cells contribute to angiogenesis, we extended an existing computational model of vascular network formation based on the cellular Potts model with tip and stalk differentiation, without making a priori assumptions about the differences between tip cells and stalk cells. To predict potential differences, we looked for parameter values that make tip cells (a) move to the sprout tip, and (b) change the morphology of the angiogenic networks. The screening predicted that if tip cells respond less effectively to an endothelial chemoattractant than stalk cells, they move to the tips of the sprouts, which impacts the morphology of the networks. A comparison of this model prediction with genes expressed differentially in tip and stalk cells revealed that the endothelial chemoattractant Apelin and its receptor APJ may match the model prediction. To test the model prediction we inhibited Apelin signaling in our model and in an in vitro model of angiogenic sprouting, and found that in both cases inhibition of Apelin or of its receptor APJ reduces sprouting. Based on the prediction of the computational model, we propose that the differential expression of Apelin and APJ yields a “self-generated” gradient mechanisms that accelerates the extension of the sprout.     

The Appearance of New Active Forms of Trypsin Inhibitor in Germinating Mung Bean (Vigna radiata) Seeds

Ungerminated seeds of mung bean contain a single major species (F) of trypsin inhibitor with five minor species (A-E) separable on diethylaminoethyl-cellulose. During germination the level of trypsin inhibitory activity decreases from 1.8 units/grams dry weight in ungerminated cotyledons to 1.2 units/grams in cotyledons from seeds germinated 5 days. This decrease is accompanied by major changes in the distribution of inhibitory activity among the inhibitor species. By 48 hours of germination, inhibitor F has largely disappeared with an accompanying rapid increase in inhibitor C. Similarly, though less rapidly, inhibitor E decreases while inhibitor A increases. A similar sequence of changes is found in vitro when purified inhibitor F is incubated with extracts from seeds germinated 96 hours. The combined in vivo and in vitro data suggest a conversion sequence of: F → E → C → A. The in vitro conversion is inhibited by phenylmethyl sulfonyl fluoride but not by iodoacetamide, indicating that at least the initial phases of inhibitor conversion are not catalyzed by the mung bean vicilin peptidohydrolase.     

Internalization of bioluminescent Escherichia coli and Salmonella Montevideo in growing bean sprouts

AIMS: Investigate the interaction of bioluminescent Escherichia coli and Salmonella Montevideo with germinating mung bean sprouts. METHODS AND RESULTS: E. coli or Salm. Montevideo introduced on mung beans became established both internally and externally on sprouts after the initial 24 h germinating period. In both cases the inoculated bacterium formed the predominant microflora on the sprouted beans throughout. From the bioluminescent profile of inoculated sprouting beans, bacterial growth was found to be in close proximity to the roots but not on the hypocotyls. Clumps (biofilms) of cells with low viability were observed within the grooves between epidermal cells on hypocotyls. Treatment with 20,000 ppm sodium hypochlorite removed the majority of bacteria from the surface of hypocotyls although nonviable single cells were occasionally observed. However, viable bacteria were recovered from the apoplastic fluid, and extracts of surface-sterilized sprouts indicating that the internal bacterial populations had been protected. This was confirmed using in situ beta-glucuronidase staining of surface-sterilized sprouts where cleaved enzyme substrate (by the action of internalized E. coli) was visualized within the plant vascular system. CONCLUSIONS: E. coli or Salmonella present on seeds become internalized within the subsequent sprouts and cannot be removed by postharvest biocidal washing. SIGNIFICANCE AND IMPACT OF THE STUDY: Mung bean production should be carefully controlled to prevent contamination occurring in order to minimize the health risk associated with raw bean sprouts.     

Evaluation of Aerated Steam Treatment of Alfalfa and Mung Bean Seeds To Eliminate High Levels of Escherichia coli O157:H7 and O178:H12, Salmonella enterica,and Listeria monocytogenes

Sprouts contaminated with human pathogens are able to cause food-borne diseases due to the favorable growth conditions for bacteria during germination and because of minimal processing steps prior to consumption. We have investigated the potential of hot humid air, i.e., aerated steam, to treat alfalfa and mung bean seeds which have been artificially contaminated with Escherichia coli O157:H7, Salmonella enterica subsp. enterica serovar Weltevreden, and Listeria monocytogenes Scott A. In addition, a recently collected E. coli O178:H12 isolate, characterized by a reduced heat sensitivity, was exposed to the treatment described. Populations of E. coli O157:H7 and S. enterica on alfalfa and mung bean seeds could be completely eliminated by a 300-s treatment with steam at 70 ± 1°C as revealed by enrichment studies. L. monocytogenes and E. coli O178:H12 could not be completely eliminated from artificially inoculated seeds. However, bacterial populations were reduced by more than 5 log CFU/g on alfalfa and by more than 4 log CFU/g on mung bean seeds. The germination rate of mung beans was not affected by the 300-s treatment compared to the germination rate of untreated seeds whereas that of alfalfa seeds was significantly lower by 11.9%. This chemical-free method is an effective alternative to the 20,000-ppm hypochlorite treatment presently recommended by the U.S. Food and Drug Administration (FDA).     

Evaluation of different leguminous seeds as food sources for the bean bug Riptortus pedestris

This study was conducted to determine the effects of six different leguminous seeds (cowpea, white kidney bean, soybean cultivars Cheongja and Daepung, mung bean, and azuki bean) on the life history traits of the bean bug Riptortus pedestris (F.) (Hemiptera: Alydidae). The total developmental time from the first instar nymph to adult ranged from 18 to 25 d; R. pedestris fed with white kidney beans were the slowest to develop. In addition, bugs fed with white kidney beans had the highest mortality (93%) and the shortest longevity (18 d). R. pedestris fed with cowpeas, soybeans, or azuki beans had high reproductive success, with the highest egg hatching success recorded in bugs fed cowpeas. The results suggest that cowpea may be a beneficial food source for the development and reproduction of bean bugs.     

The role of cotyledon cell structure during in vitro digestion of starch in navy beans

Studies on the physico-chemical, microstructural characteristics and in vitro (under simulated gastric and small intestine conditions) starch digestibility of navy beans were carried out. The microstructure of raw and cooked beans observed through scanning electron microscopy (SEM) showed the presence of hexagonal or angular shaped cotyledon cells (50-100 μm size) containing starch granules with a size ranging between 10 and 50 μm. The extent of starch hydrolysis (%) after 120 min of in vitro gastro-intestinal digestion differed between whole navy beans (∼60%) and milled bean flour and bean starch (85-90%) after they were cooked under similar conditions. Starch hydrolysis (%) increased significantly when the cotyledon cells in the cooked whole navy beans were disrupted using high pressure treatment (French press). The storage of freshly cooked whole beans resulted in a lower (40-45%) starch hydrolysis whereas re-heating increased the same to 70-80% during in vitro small intestinal digestion. The SEM pictures of cooked navy bean digesta after different intervals of in vitro gastric and small intestinal digestion showed that the cotyledon cell structure is maintained well throughout the digestion period. However cotyledon cells appear shrunken and developed wrinkles during in vitro digestion. Particle size analysis of cooked bean paste taken before and after the in vitro gastro-intestinal digestion showed similar particle size distributions.     

beta-Glucoside Activators of Mung Bean UDP-Glucose: beta-Glucan Synthase : II. Comparison of Effects of an Endogenous beta-Linked Glucolipid with Synthetic n-Alkyl beta-d-Monoglucopyranosides

n-Alkyl (C₆-C₁₂) β-d-monoglucopyranosides have been found to be highly potent activators of mung bean β-glucan synthase in vitro, increasing the V_max of the enzyme as much as 60-fold and with K_a values as low as 10 micromolar. Activation is highly specific for the β-linked terminal glucose residue; other alkyl glycosides such as, octyl-α-glucoside, dodecyl β-maltoside, 6-lauryl sucrose, 6-lauryl glucose, which lack this structure, are ineffective as activators. Based on the similarities in their structure and effects on β-glucan synthesis under a variety of conditions, it is proposed that the alkyl β-glucosides are structural analogs of the native glucolipid activator of β-glucan synthase isolated from mung bean extracts.     

Low-Income US Women Under-informed of the Specific Health Benefits of Consuming Beans

Background

Bean consumption can reduce chronic disease risk and improve nutrition status. Consumer knowledge of bean health benefits could lead to increased intakes. Low-income women have poorer health and nutrition, but their level of knowledge about bean health benefits is unknown. Beans are a familiar food of reasonable cost in most settings and are cultural staples for Hispanics and other ethnicities. Study objectives were to assess awareness of bean health benefits among low-income women, and to evaluate any differences by acculturation status for Hispanic women in the Southwestern United States.

Methods

A convenience sample of 406 primarily Mexican-origin (70%) low-income women completed a survey on knowledge of bean health benefits and general food behaviors. Principal components analysis of responses identified two summary scale constructs representing “bean health benefits” and “food behaviors.” Acculturation level was the main independent variable in chi-square or ANOVA.

Results

The survey completion rate was 86% (406/471). Most women agreed or strongly agreed that beans improved nutrition (65%) and were satiating (62%). Over 50% answered ‘neutral’ to statements that beans could lower LDL cholesterol (52%), control blood glucose (56%) or reduce cancer risk (56%), indicating indifference or possible lack of knowledge about bean health benefits. There were significant differences by acculturation for beliefs that beans aid weight loss and intestinal health. Scores on the bean health benefits scale, but not the food behavior scale, also differed by acculturation.

Conclusions

Limited resource women have a favorable view of the nutrition value of beans, but the majority did not agree or disagreed with statements about bean health benefits. Greater efforts to educate low-income women about bean health benefits may increase consumption and improve nutrition.     

芽用绿豆品种子粒性状及其豆芽生理特性研究

以21份芽用绿豆品种为材料,研究了种皮色泽、种皮比重等种皮性状、百粒重等物理特性、发芽期间的生物产量变化规律、种子吸水速率等生理特性以及绿豆芽产量、豆芽形态特征和感官品质等芽用特性指标,分析了各项物理生理特性指标与芽用特性指标的相关性。结果表明,芽用绿豆品种的产出比、下胚轴粗和下胚轴长等特性指标范围分别为111.7-143.2、7.21-11.79和0.245-0.353;品种的种皮色泽与绿豆芽生物产量、绿豆芽下胚轴粗及下胚轴长无显著相关,而与品种的百粒重呈显著相关性（P<0.05）;百粒重与绿豆芽生物产量呈显著负相关（P<0.05）;下胚轴粗与绿豆芽生物产量呈极显著负相关（P<0.05）;初步建立了适用于评价绿豆品种芽用特性的指标：豆芽产量、下胚轴粗和下胚轴长,筛选出具有较好芽用特性的绿豆品种MB01、MB07、MB19和MB45。     

Inactivation of Escherichia coli O157:H7 and Salmonella on artificially or naturally contaminated mung beans (Vigna radiata L) using a stabilized oxychloro-based sanitizer

AIMS: To evaluate the efficacy of a stabilized oxychloro-based (SOC) sanitizer to decontaminate mung beans artificially or naturally contaminated with Escherichia coli O157:H7 or Salmonella. METHODS AND RESULTS: Naturally contaminated beans were produced by introducing a five-strain cocktail of E. coli O157:H7 or Salmonella onto the flowers of growing mung bean plants. Escherichia coli O157:H7 was only sporadically recovered from sprout lots (three testing positive from 10 tested) derived from harvested beans. In contrast, Salmonella was recovered from 18 of 20 lots screened. Pathogens present on naturally contaminated seed could be successfully inactivated with SOC applied at 200 ppm for 24 h at 28 degrees C. SOC treatment could also decontaminate artificially inoculated mung bean batches containing different levels of contaminated seed. SOC inactivated E. coli O157:H7, but not Salmonella introduced onto damaged (scarified) beans. CONCLUSIONS: SOC sanitizer could inactivate Salmonella or E. coli O157:H7 naturally or artificially introduced onto mung beans. However, the SOC treatment failed to inactivate Salmonella introduced onto damaged mung beans. SIGNIFICANCE AND IMPACT OF THE STUDY: SOC sanitizer represents an effective method for decontaminating undamaged mung beans.     

Rapid degradation and limited synthesis of phospholipids in the cotyledons of mung bean seedlings

Seedling growth of mung bean is accompanied by the rapid catabolism of the three major phospholipids in the cotyledons (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol). The decline starts 24 hours after the beginning of imbibition and by the 4th day of growth more than 50% of the phospholipids have been catabolized. Extracts of cotyledons of 24-hour-imbibed beans contain enzymes capable of degrading membrane-associated phospholipids in vitro. This degradation involves phospholipase D and phosphatase activity.     

Partial Purification of an Ethylene-binding Component from Plant Tissue

An ethylene binding component(s) has been partially purified from mung bean sprouts. Tissue was homogenized in 0.3 molar sucrose and 0.2 molar potassium phosphate buffer (pH 7.0). The homogenate was centrifuged, and resuspended fractions were assayed by incorporating them onto cellulose fibers (0.7 grams per milliliter). These were exposed to [¹⁴C]ethylene (3.7 × 10⁻² microliters per liter of 120 millicurie per millimole) in the presence or absence of 1000 microliters per liter unlabeled ethylene. The cellulose was transferred to separate containers and the [¹⁴C]ethylene was absorbed in mercury perchlorate and counted. Distribution of ethylene binding to various fractions was: 0 to 3,000g, 3%; 3,000 to 12,000g; 4%; 12,000 to 100,000g, 69%; cellular debris, 24%; 100,000g supernatant, 0%. Adjustment of the pH to 4.0 precipitates the ethylene-binding component. Neutralization, addition of Triton X-100, and readjustment of the pH to 4.0 “solubilized” most of the binding component. Further purification was obtained by chromatography on CM-Sephadex in 10 millimolar potassium acetate buffer, (pH 5.0) containing 1% Triton X-100. Elution was with 200 millimolar potassium phosphate (pH 6.0) containing 1% Triton X-100. Upon treatment of the Triton “solubilized” component with cold acetone, over 90% of the binding capacity was lost. Extraction of the acetone-precipitated residue with 2% Triton X-100 restored some of the binding capacity which was found in the soluble fraction. The pH optimum for binding is 6.0. Passing the Triton X-100 extract of the acetone powder through Sepharose 6B provides considerable purification. The binding component moved ahead of most of the protein.     

Studies of Cream Seeded Carioca Beans (Phaseolus vulgaris L.) from a Rwandan Efficacy Trial: In Vitro and In Vivo Screening Tools Reflect Human Studies and Predict Beneficial Results from Iron Biofortified Beans

Iron (Fe) deficiency is a highly prevalent micronutrient insufficiency predominantly caused by a lack of bioavailable Fe from the diet. The consumption of beans as a major food crop in some populations suffering from Fe deficiency is relatively high. Therefore, our objective was to determine whether a biofortified variety of cream seeded carioca bean (Phaseolus vulgaris L.) could provide more bioavailable-Fe than a standard variety using in-vivo (broiler chicken, Gallus gallus) and in-vitro (Caco-2 cell) models. Studies were conducted under conditions designed to mimic the actual human feeding protocol. Two carioca-beans, a standard (G4825; 58μg Fe/g) and a biofortified (SMC; 106μg Fe/g), were utilized. Diets were formulated to meet the nutrient requirements of Gallus gallus except for Fe (33.7 and 48.7μg Fe/g, standard and biofortified diets, respectively). In-vitro observations indicated that more bioavailable-Fe was present in the biofortified beans and diet (P<0.05). In-vivo, improvements in Fe-status were observed in the biofortified bean treatment, as indicated by the increased total-body-Hemoglobin-Fe, and hepatic Fe-concentration (P<0.05). Also, DMT-1 mRNA-expression was increased in the standard bean treatment (P<0.05), indicating an upregulation of absorption to compensate for less bioavailable-Fe. These results demonstrate that the biofortified beans provided more bioavailable Fe; however, the in vitro results revealed that ferritin formation values were relatively low. Such observations are indicative of the presence of high levels of polyphenols and phytate that inhibit Fe absorption. Indeed, we identified higher levels of phytate and quercetin 3–glucoside in the Fe biofortified bean variety. Our results indicate that the biofortified bean line was able to moderately improve Fe-status, and that concurrent increase in the concentration of phytate and polyphenols in beans may limit the benefit of increased Fe-concentration. Therefore, specific targeting of such compounds during the breeding process may yield improved dietary Fe-bioavailability. Our findings are in agreement with the human efficacy trial that demonstrated that the biofortified carioca beans improved the Fe-status of Rwandan women. We suggest the utilization of these in vitro and in vivo screening tools to guide studies aimed to develop and evaluate biofortified staple food crops. This approach has the potential to more effectively utilize research funds and provides a means to monitor the nutritional quality of the Fe-biofortified crops once released to farmers.     

A stochastic model for the study of oviposition evolution of the pest callosobruchus maculatus on mung beans, Phaseolus aureus

Mitchell (1975) experimentally investigated the oviposition behavior of bruchid beetles on mung beans. In this paper, we develop a stochastic model to study the oviposition tactics of the bean weevils on beans. This model identifies an avoidance probability that is invariant among weevils, and explains the nuances in egg laying behavior of different weevils by varying the rate parameter λ alone.     

UDP-Glucose: (1-->3)-beta-Glucan Synthases from Mung Bean and Cotton: Differential Effects of Ca and Mg on Enzyme Properties and on Macromolecular Structure of the Glucan Product

A re-examination of the kinetic properties of UDP-glucose: (1→3)-β-glucan (callose) synthases from mung bean seedlings (Vigna radiata) and cotton fibers (Gossypium hirsutum) shows that these enzymes have a complex interaction with UDP-glucose and various effectors. Stimulation of activity by micromolar concentrations of Ca²⁺ and millimolar concentrations of β-glucosides or other polyols is highest at low (<100 micromolar) UDP-glucose concentrations. These effectors act both by raising the V_max of the enzyme, and by lowering the apparent K_m for UDP-glucose from >1 millimolar to 0.2 to 0.3 millimolar. Mg²⁺ markedly enhances the affinity of the mung bean enzyme for Ca²⁺ but not for β-glucoside; with saturating Ca²⁺, Mg²⁺ only slightly stimulates further production of glucan. However, the presence of Mg²⁺ during synthesis, or NaBH₄ treatment after synthesis, changes the nature of the product from dispersed, alkali-soluble fibrils to highly aggregated, alkali-insoluble fibrils. Callose synthesized in vitro by the Ca²⁺, β-glucoside-activated cotton fiber enzyme, with or without Mg²⁺, is very similar in size to callose isolated from cotton fibers, but is a linear (1→3)-β-glucan lacking the small amount of branches at C-0-6 found in vivo. We conclude that the high degree of aggregation of the fibrils synthesized with Mg²⁺in vitro is caused either by an alteration of the glucan at the reducing end or, indirectly, by an effect of Mg²⁺ on the conformation of the enzyme. Rate-zonal centrifugation of the solubilized mung bean callose synthase confirms that divalent cations can affect the size or conformation of this enzyme.     

Serine Hydroxymethyltransferase from Mung Bean (Vigna radiata) Is Not a Pyridoxal-5'-Phosphate-Dependent Enzyme

Serine hydroxymethyltransferase from mammalian and bacterial sources is a pyridoxal-5′-phosphate-containing enzyme, but the requirement of pyridoxal-5′-phosphate for the activity of the enzyme from plant sources is not clear. The specific activity of serine hydroxymethyltransferase isolated from mung bean (Vigna radiata) seedlings in the presence and absence of pyridoxal-5′-phosphate was comparable at every step of the purification procedure. The mung bean enzyme did not show the characteristic visible absorbance spectrum of a pyridoxal-5′-phosphate protein. Unlike the enzymes from sheep, monkey, and human liver, which were converted to the apoenzyme upon treatment with l-cysteine and dialysis, the mung bean enzyme similarly treated was fully active. Additional evidence in support of the suggestion that pyridoxal-5′-phosphate may not be required for the mung bean enzyme was the observation that pencillamine, a well-known inhibitor of pyridoxal-5′-phosphate enzymes, did not perturb the enzyme spectrum or inhibit the activity of mung bean serine hydroxymethyltransferase. The sheep liver enzyme upon interaction with O-amino-d-serine gave a fluorescence spectrum with an emission maximum at 455 nm when excited at 360 nm. A 100-fold higher concentration of mung bean enzyme-O-amino-d-serine complex did not yield a fluorescence spectrum. The following observations suggest that pyridoxal-5′-phosphate normally present as a coenzyme in serine hydroxymethyltransferase was probably replaced in mung bean serine hydroxymethyltransferase by a covalently bound carbonyl group: (a) inhibition by phenylhydrazine and hydroxylamine, which could not be reversed by dialysis and or addition of pyridoxal-5′ phosphate; (b) irreversible inactivation by sodium borohydride; (c) a spectrum characteristic of a phenylhydrazone upon interaction with phenylhydrazine; and (d) the covalent labeling of the enzyme with substrate/product serine and glycine upon reduction with sodium borohydride. These results indicate that in mung bean serine hydroxymethyltransferase, a covalently bound carbonyl group has probably replaced the pyridoxal-5′-phosphate that is present in the mammalian and bacterial enzymes.     

DNA content of free living rhizobia and bacteroids of various Rhizobium-legume associations

The DNA content of bacteroids from 22 different Rhizobium-legume associations was compared to that of the corresponding free living Rhizobium species using laser flow microfluorometry. In all 18 effective associations, the bacteroids had either similar or higher DNA content than the free living rhizobia. Bacteroid populations isolated from effective clover (Trifolium repens) and alfalfa (Medicago sativa) nodules had an average DNA content of >1.5-fold higher than free living R. trifolii and R. meliloti. These populations also contained a significant number of bacteroids with more than 3-fold the DNA content of the free living rhizobia. Populations isolated from effective nodules of winged beans (Psophocarpus tetragonolobus), peas (Pisum sativum), and mung beans (Phaseolus aureus) had an average DNA content of 1.1- to 1.5-fold higher than free living R. “cowpeas” and R. leguminosarum. Bacteroids from nodules of lupins (Lupinus angustifolius and L. minaretta), kidney beans (Phaseolus vulgaris), and soybeans (Glycine max), however, had similar DNA content to the free living forms. Two of the four associations which formed ineffective nodules contained bacteroids with lower DNA content than the free living rhizobia. The other two associations contained bacteroids with slightly higher or similar DNA content to the free living rhizobia. Nodules of the ineffective associations also did not contain leghemoglobin.     

Characterization of the Proteinase that Initiates the Degradation of the Trypsin Inhibitor in Germinating Mung Beans (Vigna radiata)

The proteinase (proteinase F) responsible for the initial proteolysis of the mung bean (Vigna radiata) trypsin inhibitor (MBTI) during germination has been purified 1400-fold from dry beans. The enzyme acts as an endopeptidase, cleaving the native inhibitor, MBTI-F, to produce the first modified inhibitor form, MBTI-E. The cleavage of the Asp76-Lys77 peptide bond of MBTI-F occurs at a pH optimum of 4.5, with the tetrapeptide Lys-Asp-Asp-Asp being released. Proteinase F exhibited no activity against the modified inhibitor forms MBTI-E and MBTI-C. Vicilin, the major storage protein of the mung bean, does not serve as a substrate for proteinase F between pH 4 and 7. Proteinase F is inhibited by phenylmethylsulfonyl fluoride, chymostatin, p-hydroxymercuribenzoate, and p-chlorophenylsulfonate, but not by iodoacetate and CuCl₂. It is not activated by dithiothreitol, and is stable for extended periods of time (10 months, 4°C, pH 4.0) in the absence of reducing agents. An apparent molecular weight of 65,000 was found for proteinase F by gel filtration. Subcellular fractionation in glycerol suggests that greater than 85% of the proteinase F activity is found in the protein bodies of the ungerminated mung bean. The same studies indicate that at least 56% of the MBTI of the seed is also localized in the protein bodies.