James C. Stewart

Antinutritive effects of wheat-germ agglutinin and other N-acetylglucosamine-specific lectins

Incorporation of N-acetylglucosamine-specific agglutinins from wheat germ (Triticum aestivum; WGA), thorn apple (Datura stramonium) or nettle (Urtica dioica) rhizomes in the diet at the level of 7 g/kg reduced the apparent digestibility and utilization of dietary proteins and the growth of rats, with WGA being the most damaging. As a result of their binding and endocytosis by the epithelial cells of the small intestine, all three lectins were growth factors for the gut and interfered with its metabolism and function to varying degrees. WGA was particularly effective; it induced extensive polyamine-dependent hyperplastic and hypertrophic growth of the small bowel by increasing its content of proteins, RNA and DNA. Furthermore, an appreciable portion of the endocytosed WGA was transported across the gut wall into the systemic circulation, where it was deposited in the walls of the blood and lymphatic vessels. WGA also induced the hypertrophic growth of the pancreas and caused thymus atrophy. Although the transfer of the gene of WGA into crop plants has been advocated to increase their insect resistance, as the presence of this lectin in the diet may harm higher animals at the concentrations required to be effective against most pests, its use in plants as natural insecticide is not without health risks for man.

A survey of the nutritional and haemagglutination properties of legume seeds generally available in the UK

Eighty-five samples from fifteen different legume seed lines generally available in the UK were examined by measurements of their net protein utilization by rats and by haemagglutination tests with erythrocytes from a number of different animal species. From these results the seeds were classified into four broad groups. Group a seeds from most varieties of kidney (Phaseolus vulgaris), runner (Phaseolus coccineus) and tepary (Phaseolus acutifolius) beans showed high reactivity with all cell types and were also highly toxic. Group b, which contained seeds from lima or butter beans (Phaseolus lunatus) and winged bean (Psophocarpus tetragonolobus), agglutinated only human and pronase-treated rat erythrocytes. These seeds did not support proper growth of the rats although the animals survived the 10 d experimental period. Group c consisted of seeds from lentils (Lens culinaris), peas (Pisum sativum), chick-peas (Cicer arietinum), blackeyed peas (Vigna sinensis), pigeon peas (Cajanus cajan), mung beans (Phaseolus aureus), field or broad beans (Vicia faba) and aduki beans (Phaseolus angularis). These generally had low reactivity with all cells and were non-toxic. Group d, represented by soya (Glycine max) and pinto (Phaseolus vulgaris) beans, generally had low reactivity with all cells but caused growth depression at certain dietary concentrations. This growth depression was probably mainly due to antinutritional factors other than lectins. Lectins from group a seeds showed many structural and immunological similarities. However the subunit composition of the lectin from the tepary bean samples was different from that of the other bean lectins in this or any other groups.

Isolation and characterization of a lectin from the seeds of Dioclea grandiflora (Mart.)

By a combination of solubility fractionation, affinity and molecular-sieve chromatography, a lectin preparation containing several closely related lectin components of different isoelectric point was isolated from the seeds of Dioclea grandiflora Mart. The lectins showed a carbohydrate specificty for D-mannose (D-glucose)-binding and had a requirement for the presence of Ca2+ and Mn2+. The results of preliminary characterization studies showed that the D. grandiflora lectins had similar properties to those of concanavalin A, the lectin from the seeds of Canavalia ensiformis, a plant also belonging to the tribe Diocleae. Thus the D. grandiflora lectins contained no covalently bound carbohydrate and had an amino-acid composition characterized by a low content of methionine and the virtual absence of cysteine. Above pH 4.8 they had molecular weight of about 100,000, while below pH 3.1 they were dissociated to half-molecules. Between these two pH values there was a fast association-dissociation equilibrium for the two species. In dissociating solvents, three subunits were obtained of the approximate size of 25–26,000, 13–14,000 and 8–9,000. The lectins from C. grandiflora similar to concanavalin A were more distantly related to the lectins obtained from the members of the tribe Vicieae although these were also specific for D-mannose (D-glucose)-binding.

Effect of the β‐adrenoceptor agonist clenbuterol and phytohaemagglutinin on growth, protein synthesis and polyamine metabolism of tissues of the rat

1 The kidney bean lectin, phytohaemagglutinin (PHA), induced a marked atrophy of skeletal muscle which was evident from the changes in tissue composition (protein, RNA, DNA and polyamine content) and from the reduction in weight and protein synthesis of hind leg muscles of rats fed on kidney bean‐diets for four days. The β‐adrenoceptor agonist, clenbuterol, induced skeletal muscle hypertrophy by transiently stimulating protein synthesis. As a consequence, the muscle loss caused by a short exposure to PHA was, in part, ameliorated by clenbuterol treatment. 2 Cardiac muscle was affected to a lesser extent than skeletal muscle by both clenbuterol and the lectin. However, there was evidence that protein synthesis in heart was reduced by PHA. 3 PHA had opposite effects on the gut, the lectin‐induced hyperplasia of the jejunum was accompanied by a large increase in protein synthesis. Clenbuterol alone had no effect on the jejunum whereas a combination of PHA and clenbuterol appeared to exacerbate the effect of the lectin on gut. 4 Both the lectin‐induced gut growth and the hypertrophy of skeletal muscle caused by clenbuterol were preceded by the accumulation of polyamines in the respective tissues. Of particular note was the observation that a significant increase in the proportion of the intraperitoneally injected 14C‐labelled spermidine or putrescine taken up by the growing tissues could be detected by the second day. Therefore, the measurement of uptake of labelled polyamines may be used as a sensitive indicator of early alterations in tissue metabolism.

Nutritional evaluation of the trypsin (EC 3.4.21.4) inhibitor from cowpea (Vigna unguiculata Walp.).

The effect of feeding rats purified cowpea (Vigna unguiculata Walp.) trypsin (EC 3.4.21.4) inhibitor in a semi-synthetic high-quality diet based on lactalbumin (10 g inhibitor/kg) for 10 d was a moderate reduction in the weight gain of rats in comparison with controls, despite an identical food intake in the two groups. The reduction in the growth rate was about 20% on a live weight basis. However, the corresponding value calculated from the weight of dry carcasses was less, only about 7%, probably because the water content of the body of the two groups of rats was different. Although most of the cowpea trypsin inhibitor (CpTI) was rapidly broken down in the digestive tract, its inclusion in the diet led to a slight, though significant, increase in the nitrogen content of faeces but not of urine. Accordingly, the net protein utilization of rats fed on inhibitor-containing diets was also slightly depressed while their energy expenditure was elevated. In agreement with results obtained for the protease inhibitors of soya bean, the slight anti-nutritional effects of CpTI were probably due mainly to the stimulation of the growth and metabolism of the pancreas. Thus, the nutritional penalty for increased insect-resistance after the transfer of the cowpea trypsin inhibitor gene into food plants is slight in the short-term.

A new type of Phaseolus vulgaris (cv. Pinto III) seed lectin: Isolation and characterization

Abstract From the seeds of Phaseolus vulgaris cv. ‘Pinto III’, previously regarded as a hemagglutinin-free bean, several glycoprotein lectins were purified by conventional methods, including solubility- and salt-fractionation, continuous high-voltage free-flow electrophoresis and molecular sieve chromatography. A preparation of similar lectins was also obtained by immunoaffinity chromatography on a Sepharose-4B column to which rabbit anti-Pinto seed lectin (conventionally purified) immunoglobulins had been attached. Both preparations gave one band of 28 000–29 000 subunit weight on SDS-polyacrylamide gel electrophoresis. They were however shown to contain components, with isoelectric points in the range of pH 4.7–5.0, by isoelectric focusing on polyacrylamide gels. The relative proportion of the individual lectin components was slightly different in the two preparations. Their sedimentation coefficient, 4.34 S, was the same; however, they had a slight difference in partial specific volume values; 0.691 ml/g for the conventional and 0.700 ml/g for the affinity preparation. The average molecular weight value ( M r,av 0 ) of 52 200 for the conventional preparation was significantly lower ( P > 0.1 ) than the value of 55 000 for the affinity-purified lectin. The ‘Pinto III’ seed lectin molecules contained two subunits only in place of the usual four subunits of the common P. vulgaris lectins. There was a slight immunochemical cross-reaction between the common P. vulgaris and the ‘Pinto III’ seed lectins. The ‘Pinto III’ seed lectins had low haemagglutinating activity when tested with rabbit erythrocytes, while their activity was high against pronase-treated rat cells. It is suggested that the low activity against rabbit cells might be the result of the lower affinity of the dimeric lectin for the exposed sugar structures on the red cell membrane.

Effect of Fasting and Refeeding on Basolateral Polyamine Uptake and Metabolism by the Rat Small Bowel

Fasting reduced the weight, protein, DNA, RNA and polyamine contents of the small intestine of rats, but its effects on the in vivo uptake of intraperitoneally injected 14C-spermidine through the basolateral membrane of the small intestine were small. The uptake of putrescine was nearly doubled by fasting for 48 h. Fasting for 48 h had reduced villus length but was without effects on the crypts. Refeeding for 6 h of rats fasted for 48 h led to hypertrophic growth: the length of both crypts and villi increased by about 50% without changes in cell number. The uptake of spermidine by the small intestine increased above not only that in fasted rats but also that in the controls fed ad libitum. The high putrescine uptake of rats fasted for 48 h was unchanged after refeeding for 6 h, but returned to control values after 12 h. Spermidine in the gut was well conserved, while most of the putrescine was transformed into non-polyamine metabolites. It is concluded that refeeding stimulates basolateral spermidine uptake, and this may be a general mechanism for polyamine accretion in adaptive growth of the small intestine.

Isolation of soybean trypsin inhibitors by affinity chromatography on anhydrotrypsin-Sepharose 4B

By repeated treatments of trypsin with phenylmethylsulfonyl fluoride (PMSF), followed by base elimination of PMS from the PMS-trypsin, a catalytically inactive anhydrotrypsin preparation of low (less than 1%) active trypsin content was obtained. Inactive material was removed by affinity chromatography on trypsin inhibitor-Sepharose 4B and the purified anhydrotrypsin with full binding capacity for trypsin inhibitors was coupled to cyanogen bromide-activated Sepharose 4B. When used below its maximum capacity for trypsin inhibitors the anhydrotrypsin-Sepharose-4B affinity column absorbed both classes of inhibitors present in soybean. When overloaded, the Kunitz type was bound preferentially. Based on this observation, conditions for the partial separation of the two types of inhibitors were worked out.

A novel method for the preparation of protein bodies by filtration in high (over 70% w/v) sucrose-containing media

Abstract A novel method for the preparation of protein bodies from the cotyledons of Phaseolus vulgaris is described in high (over 70% w/v) sucrose-containing media. Homogenates of cotyledons were filtered through no. 15 Ballotini beads and the filtrates were further purified by stepwise gradient centrifugation. The organelles harvested from the layer of density of 1.32 g/ml compared favourably in composition, size and yield with those prepared by other techniques and in iso-osmotic aqueous media.

Erythro- and lymphoagglutinins of Phaseolus acutifolius

Abstract A potent lymphoagglutinin which had low affinity for red cells or fetuin and another lectin which reacted strongly with red cells and fetuin but was a poor agglutinin for lymphocytes were isolated from seeds of Phaseolus acutifolius . A number of other lectin components with intermediate activity towards these cells was also isolated. All the lectins had very similar amino acid and carbohydrate composition, sedimentation patterns, partial specific volume and molecular weight values of about 116 600 and were thus smaller than the related Phaseolus vulgaris lectins (M r = 119 000). The lectins contained four subunits with only minor size and charge differences between the lympho- and erythroagglutinating subunits and their electrophoretic mobility in SDS gel electrophoresis was anomalously high. The existence of lympho- and erythroagglutinating subunits in two members of the genus Phaseolus supports their close morphological similarity.