Genes encoding α-amylase inhibitors are located in the short arms of chromosomes 3B, 3D and 6D of wheat (Triticum aestivum L.)

Summary Three -amylase inhibitors, designated Inh. I, II and III have been purified from the 70% ethanol extract of hexaploid wheat (Triticum aestivum L.) and characterized by amino acid analysis, N-terminal amino acid sequencing and enzyme inhibition tests. Inhibitors I and III have identical N-terminal sequences and inhibitory properties to those of the previously described 0.19/0.53 group of dimeric inhibitors. Inhibitor II has an N-terminal sequence which is identical to that of the previously described 0.28 monomeric inhibitor, but differs from it in that in addition to being active against -amylase from Tenebrio molitor, it is also active against mammalian salivary and pancreatic -amylases. Compensating nulli-tetrasomic and ditelosomic lines of wheat cv. Chinese Spring have been analysed by two-dimensional electrophoresis, under conditions in which there is no overlap of the inhibitors with other proteins, and the chromosomal locations of the genes encoding these inhibitors have been established: genes for Inh. I and Inh. III are in the short arms of chromosomes 3B and 3D, respectively, and that for Inh. II in the short arm of chromosome 6D.     

Plant Proteinase inhibitors: A defense against herbivorous insects?

Using the tomato plant, Lycopersicon esculentum L., and the beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), we have demonstrated that insect herbivory induces a rapid decline in plant quality. This decline in plant quality manifests itself by a highly significant reduction in rate of larval growth on a medium containing foliage from insect-damaged as opposed to undamaged tomato plants. The induction of tomato proteinase inhibitors, as a result of larval feeding, is invoked as a factor that systemically reduces leaf quality.

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Zusammenfassung Eine zentrale Theorie in der Erforschung von Insekten-Wirtspflanzen-Wechselbeziehungen ist, dass sich bestimmte natürlich vorkommende Pflanzeninhaltsstoffe in Pflanzen zur Abwehr herbivorer Insekten und anderer Parasiten entwickelt haben. Zur Zeit herrschende Überlegungen beinhalten auch das Konzept, dass herbivore Insekten das Potential besitzen, Veränderungen in der Abwehrund/oder Nahrungsqualität von Wirtspflanzen hervorzurufen, die den herbivoren Insekten schaden. Gegenwärtig is nur wenig über die Ursachen dieser von Insekten induzierten Veränderungen der Wirtspflanzenqualität bekannt. Jedoch werden häufig unterschiedliche Gehalte der Pflanzen an Phenolderivaten, Protein und/oder Proteinaseinhibitoren mit den Abwehrmechanismen in Zusammenhang gebracht. Diese Arbeit untersucht das Potential von Pflanzenproteinaseinhibitoren, als induzierbare Abwehr gegen herbivore Noctuidenlarven zu wirken.Tomatenpflanzen enthalten Proteinaseinhibitoren (PIs), die durch Verletzen der Pflanze induziert werden. Es besteht die Hypothese, dass diese Synthese von PIs einen Abwehrmechanismus gegen blattfressende Insekten darstellt. Diese Hypothese ist niemals angemessen in planta getestet worden und wird von uns anhand von Spodoptera exigua und Tomatenpflanzen, Lycopersicon esculentum, getestet.Wieterhin sollte festgestellt werden, ob eine Beziehung zwischen der PI-Konzentration im Blatt und dem Wachstum von Blattmaterial fressenden Larven besteht. Anstelle lebender Pflanzen wurde dazu eine feste, Blattmaterial enthaltende Diät (15% gefriergetrocknetes Blattmaterial angemischt in Agarlösung und Sorbinsäure) benutzt, was die Verwending einer unverletzten Kontrolle ermöglichte. Diese künstliche Diät wurde S. exigua angeboten. Die Ergebnisse des Fütterungsversuches (Fig. 2) zeigen, dass eine signifikante inverse Beziehung (r²=0.81, p=0.05) zwischen der PI-Konzentration im Blattgewebe und den mittleren Gewichten von Larven von S. exigua besteht. Nicht dargestellt ist die nicht signifikante Korrelation zwischen mittlerem Gewicht der Larven und den Gehalten an Blattprotein (r²=0.47, p>0.25) und Phenolderivaten (r²=0.50, p=0.25).Diese Ergebnisse veranlassen uns zu dem Rückschluss, dass Tomatenproteinaseinhibitoren als systemisch induzierbare antibiotische chemische Abwehr gegen herbivore Insekten wie Noctuidenlarven anzusehen sind, und dass PIs einen Faktor darstellen, der zur systemischen Reduktion der Blattqualität von Tomaten führt.

     

Partial characterization of a major gut thiol proteinase from larvae of Callosobruchus maculatus F.

Much of the proteolytic activity in the digestive tract of Callosobruchus maculatus larvae can be attributed to a thiol proteinase(s) that hydrolyzes [3H]methemoglobin optimally at pH 5.0. Maximal hydrolysis of [3H]methemoglobin, [3H]alpha-casein, and N-benzoyl-DL-arginine napthylamide-(BANA) required the presence of thiol reducing agents. Larval gut proteinase activity was strongly inhibited by p-hydroxymercuribenzoic acid (pHMB), Nethylmaleimide (NEM), and iodoacetic acid (IAA) but was unaffected by the Bowman-Birk and Kunitz proteinase inhibitors from soybeans or by lima bean trypsin inhibitor. L-Trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane (E-64), a specific inhibitor of thiol proteinases, potently inhibited proteolysis of [3H]methemoglobin by larval gut homogenates. Proteolytic activity in the larval gut was located in the lumen contents and thus appears to play a major role in extracellular digestion. The pH of the larval midgut is slightly acidic, and midgut contents exhibit a negative redox potential, conditions supporting the activity of a thiol proteinase. The significance of these findings is discussed with reference to the vulnerability of this digestive proteinase as a target for existing or genetically engineered plant chemical defenses.     

Effect of some metabolic inhibitors on citric acid production by Aspergillus niger

Stationary cultures of Aspergillus niger grown on a synthetic medium have been used to study the effect of some metabolic inhibitors on citric acid production. Addition of 0.05 to 1 mM sodium malonate or 0.01 to 0.1 mM potassium ferricyanide, iodoacetate, sodium azide, sodium arsenate or sodium fluoride stimulated citric acid production (3.6 to 45%), but not total titratable acids. Addition of higher concentrations (0.2 to 10 mM) of later inhibitors caused a marked inhibition of fungal growth and citric acid production. The implications of these preliminary findings are discussed.     

The permeability of the cell-to-cell membrane channel and its regulation in mammalian cell junctions

Summary Mammalian cell-to-cell channels show polar permselective properties discriminating against negatively charged 14 ?-wide molecules and are more restrictive than the channels of insect cell junctions. The channel permeability is modulated by conditions affecting the concentration of intracellular ionic Ca: elevation of the external Ca load (B cells), treatment of cell cultures with Ca-transporting ionophore (in the presence of external Ca, but not in its absence), treatment with a combination of cyanide and iodoacetate, or with high levels of carbon dioxide, all cause depression of channel permeability. Treatment of cell cultures with cyclic AMP or its more permeable derivative, dibutyryl cyclic AMP, produces increase in permeability. A similar channel up regulation is observed upon elevation of the endogenous level of cyclic AMP by serum deprivation or lowering of cell density. Presented in the symposium on Molecular and Morphological Aspects of Cell-Cell Communication at the 31st Annual Meeting of the Tissue Culture Association, St. Louis, Missouri, June 1–5, 1980. This symposium was supported in part by Contract 263-MD-025754 from the National Cancer Institute and the Fogarty International Center. This work was supported by grant number 5 R01 CA14464, awarded by the National Cancer Institute, DHEW.     

Nitrogen leaching losses from conventional and new nitrogenous fertilizers in low-land rice culture

Summary A pot-culture experiment was conducted to assess the leaching losses of N from the conventional and new nitrogen fertilizers under low-land rice culture. Leaching losses of N were generally less than 20% of applied N with sources other than sodium nitrate and these could be reduced by blending urea with nitrification inhibitor N-Serve or coating withneem cake or by using urea super granules or slow-release N fertilizer sulphur coated urea. These new nitrogen fertilizers were more effective than urea for rice.     

Peptides isolated from human liver with specific inhibitory effects on reassociation/reactivation of in vitro dissociated lactic dehydrogenase (LDH-M4 and −H4) isozymes

Two different peptides have been purified from human liver, similar to those previously reported (Schoenenberger, G.A., and Wacker, W.E.C. (1966) Biochemistry 5, 1375–1379) to be present in human urine, which may serve as metabolic regulators of lactate dehydrogenase (EC 1.1 1.27) isoenzymes (LDH-M₄ = muscle type; LDH-H₄ = heart type). By trichloroacetic acid precipitation, ultrafiltration, Sephadex G-25 and Bio-Gel P-2 columns, affinity chromatography on immobilized LDH-isozymes and HPLC two peptides which differed with respect to molecular weight, retention on the affinity columns and amino acid composition were isolated. No effect was observed when native, tetrameric lactate dehydrogenase was incubated with these peptides. However, when lactate dehydrogenase was dissociated to monomers at low pH and allowed to reassociate by adjusting the pH to 7.5 complete inhibition of the reactivation occurred when the inhibitors were incubated together with respective reassociating monomeric isozymes. The two peptides showed no cross-specificity, i.e. each peptide exhibited inhibitory activity only on one of the two isozymes LDH-M₄ or LDH-H₄. From the amino acid analyses, gel-filtration and PAGE + SDS, molecular weight of 1800 for the M₄ and ≈2700 for the H₄ inhibitor were calculated. An apparent K_i of ≈3 × 10^?5 mM for the H₄ and ≈7 × 10^?5 mM for the H₄ inhibitor was estimated. The interaction of the inhibitors with the enzyme system showed strong cooperativity with Hill coefficients of 2.9 (LDH-M₄-specific) and 2.4 (LDH-H₄-specific). Mathematical modelling of the reassociation and reactivation of lactate dehydrogenase and its specific inhibition by the peptides led to the conclusion that the peptides reacts with monomers, dimers or a transition state during the tetramerisation process. k₁ for the dimerisation step of M₄ = 2.0 × 10⁵ M^?1 · s^?1 and of H₄ = 8.2 × 10⁴ M^?1 · s^?1; k₂ for the tetramerisation step of M₄ = 2.8 × 10⁵ M^?1 · s^?1 and of H₄ = 1.2 × 10⁵ · M^?1 · s^?1, were calculated, the second step still being the faster one.     

Protease inhibitor system in horses: classification and detection of a new allele

A method of horizontal thin layer polyacrylamide gel electrophoresis at acid pH has been developed for the separation of the prealbumins in equine plasma. Using this method, it has been possible to split the S allele into two, S₁ and S₂, bringing the total number of prealbumin alleles in Thoroughbred horses to eight. The gene frequencies of these eight alleles in Australian Thoroughbreds are presented. All eight prealbumin types exhibit antiprotease activity and therefore, it is suggested that the name prealbumin (Pr) should be abandoned in favour of protease inhibitor (Pi) although at this stage it is not known whether this incorporates the Pi1 and Pi2 described by Juneja et al. (1979).     

Trypsin inhibitors in plants

Trypsin inhibitors were found in several food plants. Potato and sweet corn were the most inhibitory, while fruits had negligible activity. Intermediate in activity were sweet potato, spinach, broccoli, Brussels sprouts and cucumber. The trypsin inhibitor of sweet corn was isolated by extraction in dilute salt solution, ammonium sulfate fractionation, chromatography with Sephadex G75 and CM-cellulose and lyophilization. Two components were demonstrated by disc gel electrophoresis. The inhibitor was heat stable. It had little inhibitory activity against papain but was moderately active against chymotrypsin.     

Growth-inhibitory volatile aromatic compounds produced by Solanum tuberosum tubers

Some of the aromatic compounds evolved by stored potato tubers have been identified by combined GLC-MS. Of the identified compounds, benzothiazole, 1,4-dimethylnaphthalene and 1,6-dimethylnaphthalene are comparatively potent inhibitors of sprout growth in the potato tuber. The growth suppressing activity of the two dimethylnaphthalenes is comparable with that of isopropyl-(N-3-chlorophenyl)-carbamate, which is used commercially in potato storage.