Yehudith Birk

α-amylase inhibitors from wheat isolation and characterization

Abstract Two amylase inhibitors—AmI 1 and AmI 2 —have been isolated from an aqueous extract of wheat grains by ammonium sulfate fractionation followed by column chromatography on DEAE-cellulose and CM-cellulose. Both inhibitors are basic proteins with a molecular weight of 18 215 for AmI 1 and 26 200 for AmI 2 . Their purity and homogeneity have been established by ultracentrifugation, electrophoresis on paper, cellulose-acetate, and polyacrylamide gel. They differ markedly from each other in amino acid composition, and in specificity and affinity towards α-amylases from different origins. Of all the amylases examined, AmI 1 inhibits only Tenebrio molitor larval midgut amylase whereas the specificity of AmI 2 is wider. AmI 1 is more resistant to elevated temperatures than AmI 2 . Both inhibitors are inactivated by digestion with pepsin and pronase but are not affected by carboxypeptidase A and B or by 0.02 M Hcl. AmI 1 loses its activity when submitted to digestion with trypsin and chymotrypsin whereas AmI 2 is hardly affected by these enzymes. Both inhibitors are inactivated upon reduction with mercaptoethanol but do not lose activity when kept in 6.4 M urea. Chemical modifications, such as esterification and selective carboxymethylation, clearly indicate a stronger impairment of the inhibitory activity of AmI 2 towards salivary amylase than towards Tenebrio amylase. Mild treatments with CNBr resulted in an identical inactivation curve of the inhibitory activities of both AmI 1 and AmI 2 towards Tenebrio amylase. The two inhibitors are inactivated by treatment with fluorodinitrobenzene, and with nitrons acid but are not affected by carbamylation. The extent of inhibition of salivary α-amylase by AmI 2 is strongly dependent upon the order of addition of the reactants, the inhibitor being much more effective when preincubated with the enzyme. No similar phenomenon was found for inhibition of Tenebrio amylase by either AmI 1 or AmI 2 . The decrease in inhibition, resulting from inhibitor-substrate preincubation, has been attributed to the ability of AmI 2 to form complexes with polysaccharides, which might have resulted in masking AmI 2 s binding site for salivary amylase. It is suggested that AmI 2 is basically comprised of AmI 1 , which contains the active site against Tenebrio amylase but possesses another protein fragment with additional binding sites for salivary α-amylase and for the polysaccharidous substrate.

A new isoflavone from soya beans

Abstract A new isoflavone was isolated from soya beans and shown to be 7,4′-dihydroxy, 6-methoxyisoflavone, for which the name glycitein is proposed.

Comparative studies on proteolytic enzymes of Tenebrio molitor L.

Abstract 1. 1. Three distinct proteolytic components of Tenebrio molitor larvae are differentiated on the basis of selective inhibition by specific trypsin inhibitors: an endopeptidase—“Tenebrio trypsin”—and two exopeptidases—carboxypeptidase B and amino-tripeptidase. 2. 2. The exo- and endopeptidase activities are separated by column chromatography on ECTEOLA-cellulose, Tenebrio trypsin eluting freely. The latter is further purified by absorption on CM-cellulose and subsequent gradient elution. 3. 3. Sulphydryl or chelating compounds activate, stabilize and partially reactivate the exopeptidase activity. 4. 4. The relative proteolytic and esterolytic activities of both bovine and Tenebrio trypsins are similar, and esterolysis of carbobenzoxy-tyrosine nitrophenyl ester is completely inhibited by “crystalline soybean trypsin inhibitor”; this is in contrast to chymotrypsin. The stages of enzymatic hydrolysis of polylysine are strikingly similar for both trypsins.

Soya bean saponins—VII : A method for the determination of sapogenin and saponin contents in soya beans

Abstract A quantitative method has been devised for the determination of the saponin content in acid hydrolysates of soya beans, based on the use of a modified Lieberman-Burchard reagent and employing certain essential steps of purification from accompanying interfering materials. All five known soya sapogenins (soya sapogenols A, B, C, D and E) have been found to give the same colour yield per unit weight with this reagent. A 1:1 sapogenin:sugar ratio has been found to be typical for various soya bean saponin extracts prepared from different soya bean varieties, and has been used as a conversion factor of sapogenin into saponin content. The saponin content determined in six different soya bean varieties has been found to amount to ∼ 0·60 per cent of the defatted soya bean meal.

Comparison of a Vegetable-Based (Soya) and an Animal-Based Low-Protein Diet in Predialysis Chronic Renal Failure Patients

There is some experimental evidence to suggest that progression of chronic renal failure (CRF) is slower on diets based on soya protein than on diets based on animal protein. We have compared the effect of a soya-based vegetarian low-protein diet (VPD) and an animal-based low-protein diet (APD) in 15 patients with CRF. 15 patients with CRF (51Cr-EDTA-measured glomerular filtration rate 15–50 ml/min/1.73 m2) were studied. In a randomized crossover trial, the patients were given each diet (each containing 0.75 g protein and 32 kcal per kilogram body weight) for a 6-month period. Nine patients completed the trial, 2 others dropped out because they could not tolerate the VPD, 3 because of unrelated medical complications, and 1 for technical reasons. The caloric intake was higher and the protein, phosphate and essential amino acid intake lower on the VPD than on the APD. The compliance with the suggested caloric intake was better with the VPD than with the APD (97 vs. 88% of recommended intake), as was the compliance with the suggested protein intake (94 vs. 112% of recommended intake) and with the suggested phosphate intake (102 vs. 116%). The mean glomerular filtration rate, as judged by 51Cr-EDTA, was similar after 6 months on each diet and remained unchanged throughout the entire year of the study. The rate of fall of glomerular filtration, as measured by the slope of 1/serum creatinine was slowed by 73% during the 1-year study period as compared with the prestudy period. Nutritional status (as measured by body mass index, midarm circumference, and lean body mass and percent body fat), serum transferrin, cholesterol and albumin, and total lymphocyte count were similar on the two diets. The serum albumin level on both diets, however, was significantly higher on the two diets than during the prediet period. Blood urea nitrogen, urine urea nitrogen, protein catabolic rate, and 24-hour urine creatinine and phosphate were lower on the VPD than on the APD. The 24-hour protein excretion was similar on the two diets. The two low-protein diets resulted in a slowing in the progression of CRF. A VPD is well tolerated in CRF and is associated with lower protein and phosphate intakes and a higher caloric intake than an APD and may, therefore, be used as a safe alternative or partial substitute for the usual APD in CRF.

Studies on the midgut amylase activity of Tenebrio molitor L. larvae

Abstract The optimal conditions for in vitro amylase activity of the midgut of Tenebrio molitor L. larvae were determined as pH 5·4, 25°C for 10 min reaction, and 1% starch. The larval enzyme solution (LES) amylase, while stable for at least 20 hr at 5°C, loses 75 per cent of its activity on dialysis at this temperature, 25 per cent of its activity when kept at 25°C for 1 hr, and all its activity when kept at 35°C for 30 min. LES amylase is slightly activated by Ca ++ and Cl − , resembling in this respect α-amylase, and is inhibited by Hg ++ , Cu ++ , and ascorbic acid, which inhibit β-amylase. Both column chromatography and paper electrophoresis demonstrate the acidic nature of the enzyme. The soybean trypsin inhibitors and other soybean protein fractions assayed either activate LES amylase or exhibit self-amylase activity. It is suggested that the growth impairment of Tenebrio molitor larvae on raw soybean meal might be attributed to the sum of proteolytic inhibition on the one hand, and to amylolytic stimulation on the other, resulting in a metabolic imbalance more pronounced than that caused by proteolytic inhibition alone. Better carbohydrate utilization might result in reduced food consumption, which is correlated with the energy requirements of the organism, thereby lowering absolute protein consumption even more.

Studies on the proteolytic activity of the beetles Tenebrio and Tribolium

Abstract The optimal conditions for total proteolytic activity in vitro of the midgut as well as whole-larva homogenates of Tenebrio molitor L. were determined as pH 6·2–6·4, 37°C for 20 min reaction, and 0·5% casein. The same were found for both Tribolium confusum Duval and T. castaneum Herbst except for the pH, which was in the range of 6·5–6·9. Larvae of Tribolium grown under a fluctuating temperature, with a mean of 24·5°C, showed a remarkably increased proteolytic activity, as compared to those grown under a constant temperature of 28°C. The crystalline soybean trypsin inhibitor partially inhibited proteolysis in Tenebrio larvae, whereas the C1 fraction of soybeans inhibited some 70 per cent of this activity. Haemoglobin could not replace casein as a substrate. Moreover, when added to reaction mixtures containing optimal casein concentrations it inhibited proteolysis to a considerable extent. Likewise, larval growth of all three species was suppressed on basic diets supplemented with haemoglobin. The addition of methionine and/or isoleucine to the haemoglobin-supplemented diets could not overcome this suppression of growth. With a relative decrease in proteolytic activity during larval development there is a steady relative increase in amylolytic activity until both activities reach constant levels in the last instars.

Interaction of lucerne saponins with sterols

Abstract The structural features of sterols, which enable them to interact with lucerne saponins, were studied. It was found that an intact steroid ring structure having the conformation of cholestanol, to which a side chain characteristic to cholesterol or phytosterols is attached, is essential for the formation of a sterol-saponin addition product. Unlike the complex formed between digitonin and cholesterol, these addition products are extremely unstable and their formation is not dependent on the presence of a 3-β-hydroxyl group. In addition products formed with cholesterol or 7-dehydrocholesterol an approximately 1:5 molar ratio exists between saponin and sterol, whereas in the case of other sterols this ratio is approximately 1:8. Although lucerne saponins readily form addition products with a variety of sterols, a preferential affinity to cholesterol and 7-dehydrocholesterol was found when the addition product was formed while cholesterol and one of the other sterols were present simultaneously in the reaction mixture. These preferential affinities were demonstrated also by bioassays conducted with the fungus Sclerotium rolfsii Sacc.

Physiological aspects of host specificity in the Bruchidae—IV. Developmental incompatibility of soybeans for Callosobruchus

Abstract The developmental incompatibility of soybeans for Callosobruchus chinensis L. is partly attributed to the presence of soybean saponins. Larvae of Callosobruchus do not hydrolyse saponins in vitro . Higher animals which do so are known to be unaffected by physiological levels of these glycosides. These saponins may therefore be regarded as specific metabolic defence mechanisms of the soybean evolved against insects. All the known protease inhibitors in soybeans, and also urease, have no apparent effect on the development of this beetle nor do the constituents of soy saponins—their sapogenins and sugars. Saponin fraction c and urease are ovipositional attractants of Callosobruchus . The attractance of arabinose and deterrence of sapogenins for oviposition is of theoretical interest.

A basic trypsin- and chymotrypsin-inhibitor from groundnuts (Arachis hypogaea)

Abstract A trypsin- and chymotrypsin-inhibitor has been isolated by extraction of defatted groundnut meal at pH 5, by ammonium sulfate precipitation, and successive column chromatography on DEAE-cellulose, calcium phosphate, and CM-cellulose. The inhibitor was found to be homogeneous by ultracentrifugation, acrylamide gel electrophoresis, gel filtration on Sephadex G-50, and isoelectrofocusing in acrylamide gels. The pure inhibitor has an E1 cm1 % of 2.5 at 280 nm. Its isoelectric point lies between pH 8 and 9. Its molecular weight is about 7500. It is stable between pH 2 and 11 and stable in water when maintained at 100 °C for 15 min. The inhibitor forms stable complexes with trypsin and chymotrypsin at molar ratios of approximately 1:1. The phenomenon of temporary inhibition could not be observed. The complex of the inhibitor with either of the two enzymes fails to inhibit the other enzyme.