Proteinase inhibitors I and II in fruit of wild tomato species: Transient components of a mechanism for defense and seed dispersal

The juice of unripe fruit from a wild species of tomato, Lycopersicon peruvianum (L.) Mill., LA 107, contains over 50% of its soluble proteins as the sum of two proteinase inhibitors. These are the highest levels of proteinase inhibitors and highest percentage of soluble proteins as proteinase inhibitors of any plant or animal tissue found to date. Fruit of the modern tomato, L. esculentum Mill., contains only negligible quantities of the two inhibitors. The two proteinase inhibitors in the fruit of L. peruvianum are members of the Inhibitor I and II families previously found in potato tubers and in leaves of wounded potato and tomato plants. The levels of the two inhibitors in the unripe fruit decrease significantly during ripening. Unripe fruit from other wild Lycopersicon species such as L. parviflorum Rick, Kesicki, Fobes et Holle, L. hirsutum Humb. et Bonpe., L. pimpinellifolium Mill., and other lines of L. peruvianum contain moderate levels of the inhibitors that also decrease during ripening. Another wild tomato species, L. pennellii Corr., is similar to L. esculentum in not containing the two proteinase inhibitors in either unripe or ripe fruit. The transient levels of the inhibitors in fruit of wild species indicate that they are present in unripe fruit as defensive chemicals against insects, birds or small mammals and their disappearance during ripening may render them edible to facilitate seed dispersal. High levels of mRNAs coding for Inhibitors I and II in unripe fruit of L. peruvianum, LA 107, indicate that strong promoters may regulate the developmentally expressed proteinase-inhibitor genes in tomato fruit that may have a substantial potential for use in genetic-engineering experiments to enhance the production of large quantities of proteinase inhibitors or other proteins in field tomatoes.Abbreviations poly(A)⁺ mRNA polyadenylated mRNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide electrophoresis Project 1791, College of Agriculture and Home Economics Research Center, Washington, State University     

Relation between the anion exchange protein in kidney medullary collecting duct cells and red cell band 3

Summary A membrane protein that is immunochemically similar to the red cell anion exchange protein, band 3, has been identified on the basolateral face of the outer medullary collecting duct (MCD) cells in rabbit kidney. In freshly prepared separated rabbit MCD cells, M.L. Zeidel, P. Silva and J.L. Seifter (J. Clin. Invest. 77:1682–1688, 1986) found that Cl^–/HCO ₃ ^- exchange was inhibited by the stilbene anion exchange inhibitor, DIDS (4,4-diisothiocyano-2,2-disulfonic stilbene), with aK ₁ similar to that for the red cell. We have measured the binding affinities of a fluorescent stilbene inhibitor, DBDS (4,4-dibenzamido-2,2-disulfonic stilbene), to MCD cells in 28.5 mM citrate and have characterized both a high-affinity site (K ₁ ^s =93±24 mM) and a lower affinity site (K ₂ ^s =430±260 nM), which are closely similar to values for the red cell of 110±51 nM for the high-affinity site and 980±200 nM for the lower affinity site (A.S. Verkman, J.A. Dix & A.K. Solomon,J. Gen. Physiol. 81:421–449, 1983). When Cl^– replaces citrate in the buffer, the two sites collapse into a single one withK ₁ ^s =1500±400 nM, similar to the singleK ₁ ^s =1200±200 nM in the red cell (J.A. Dix, A.S. Verkman & A.K. Solomon,J. Membrane Biol. 89:211–223, 1986). The kinetics of DBDS binding to MCD cells at 0.25 M^–1 are characterized by a fast process, =0.14±0.03 sec, similar to =0.12±0.03 sec in the red cell. These similarities show that the physical chemical characteristics of stilbene inhibitor binding to MCD cell band 3 closely resemble those for red cell band 3, which suggests that the molecular structure is highly conserved.     

X-ray and model-building studies on the specificity of the active site of proteinase K

Proteinase K, the extracellular serine endopeptidase (E.C. 3.4.21.14) from the fungus Tritirachium album limber, is homologous to the bacterial subtilisin proteases. The binding geometry of the synthetic inhibitor carbobenzoxy-Ala-Phechloromethyl Ketone to the active site of proteinase K was the first determined from a Fourier synthesis based on synchrotron X-ray diffraction data between 1.8 Å and 5.0 Å resolution. The protein inhibitor complexes was refined by restrained least-squares minimization with the data between 10.0 and 1.8 Å. The final R factor was 19.1% and the model contained 2,018 protein atoms, 28 inhibitors atoms, 125 water molecules, and two Ca²⁺ ions. The peptides portion of the inhibitor is bound to the active center of proteinase K by means of a three-stranded antiparallel pleated sheet, with the side chain of the phenylalanine located in the P1 site. Model building studies, with lysine replacing phenylalanine in the inhibitor, explain the relatively unspecific catalytic activity of the enzyme.     

Respiratory oxidation of reduced sulfur compounds by intact cells of Thiobacillus tepidarius (type strain)

Thiobacillus tepidarius (type strain) was grown in microaerophilic conditions, on tetrathionate, thiosulfate or crystalline S^o. The rates of tetrathionate, thiosulfate, elemental sulfur (S^o) and sulfite oxidation of the different cultures were measured respirometrically, using exponentially growing cells, with an oxygen electrode. Cells growing on the three different sulfur compounds retain thiosulfate-, tetrathionate, and S^o-oxidizing activities (SOA), but lack respiratory sulfite-oxidizing activity. The SOA for all the cultures was almost totally inhibited by 50 M myxothiazol, an inhibitor of the quinone-cytochrome b region, and by 10 M of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). Tetrathionate- and thiosulfate-oxidizing activities were moderately and weakly inhibited by 50 M totally inhibited (>95%) all respiratory activities. This study suggests that electrons released by S^o oxidation enter the respiratory chain in the quinone-cytochrome b region.Abbreviation SOA sulfur-oxidizing activity     

The role of ethylene and reducing agents on anther culture response of tetraploid potato (Solanum tuberosum L.)

Summary The role of ethylene in embryogenesis of cultured potato anthers was studied indirectly by testing various substances known to affect ethylene formation. The reducing agents ascorbic acid and L-cysteine prevented browning of anther cultures and significantly stimulated embryogenesis. Embryogenesis was also promoted by the use of the ethylene inhibitors AgNO₃ and n-propyl-gallate and by the polyamines spermidine and putrescine. The use of the ethylene releasing compound ethrel significantly inhibited embryogenesis.Abbreviations MS Murashige & Skoog - PVP polyvinylpyrrolidone - MW molecular weight - ACC 1-aminocyclopropane-1-carboxylic acid - ethrel 2-chloroethylphosphonic acid (ethephon)     

Interaction of MAR-sequences with nuclear matrix proteins.

The recent discovery of DNA sequences responsible for the specific attachment of chromosomal DNA to the nuclear skeleton (MARs/SARs) was an important step towards our understanding of the functional and structural organization of eukaryotic chromatin [Mirkovitch et al.: Cell 44:273-282, 1984; Cockerill and Garrard: Cell 44:273-282, 1986]. A most important question, however, remains the nature of the matrix proteins involved in the specific binding of the MARs. It has been shown that topoisomerase II and histone H1 were capable of a specific interaction with SARs by the formation of precipitable complexes [Adachi et al.: EMBO J8:3997-4006, 1989; Izaurralde et al.: J Mol Biol 210:573-585, 1989]. Here, applying a different approach, we were able to "visualize" some of the skeletal proteins recognizing and specifically binding MAR-sequences. It is shown that the major matrix proteins are practically the same in both salt- and LIS-extracted matrices. However, the relative MAR-binding activity of the individual protein components may be different, depending on the method of matrix preparation. The immunological approach applied here allowed us to identify some of the individual MAR-binding matrix proteins. Histone H1 and nuclear actin are shown to be not only important components of the matrix, but to be involved in a highly efficient interaction with MAR-sequences as well. Evidence is presented that proteins recognized by the anti-HMG antibodies also participate in MAR-interactions.     

Indirect chiral separation and analyses in human biological fluids of the stereoisomers of a thienothiopyran-2-sulfonamide (TRUSOPT), a novel carbonic anhydrase inhibitor with two chiral centers in the molecule.

The indirect chiral separation of the four stereoisomers (1)-(4) of a novel carbonic anhydrase inhibitor with two chiral centers in the molecule is reported. The method is based on chemical derivatization of the secondary amino group of the inhibitor with chiral isocyanate, formation of diastereomeric urea derivatives, each with three chiral centers in the molecule, and their separation under nonchiral HPLC conditions. The attempts to separate racemic mixture (1) + (2) from its diastereomeric counterpart (3) + (4) under nonchiral conditions, and to separate enantiomers (1) and (2) directly on a chiral stationary phase (CSP) are also reported. The indirect method was utilized for the assessment of an in vivo inversion of configuration at either one or both chiral centers of the molecule of (1). Analyses of selected whole blood and urine samples from human subjects after multiple bilateral topical ocular dosing with (1) did not reveal the presence of any of the three possible stereoisomers (2)-(4) of (1) indicating that the inversion of configuration at neither one nor two chiral centers of (1) occurs in vivo.     

Dietary modulation of α-amylase activity in eight geographical strains of Sitophilus oryzae and Sitophilus zeamais

Rank transformation of specific activity values of -amylase across four strains of Sitophilus oryzae (L.) and four strains of S. zeamais Motschulsky indicates that levels of these predominant enzymes are highest in adults feeding on hulled barley or long-grain brown rice. Intermediate activity levels are found in weevils feeding on yellow corn (maize) and lowest levels are found in wheat-fed weevils. Although extracts prepared from barley contain inhibitory activity against two purified isoamylases from S. oryzae, levels of the naturally-occurring -amylase inhibitors against these two enzymes are about 2.2-fold and 6.1-fold, respectively, more concentrated in wheat. Ingestion of these amylase inhibitors and formation of an inactive enzyme:inhibitor complex with previously secreted amylase may account for the lower activity of amylase in weevils of both species feeding on wheat. Amylase levels across all strains feeding on a given diet are about 2-fold higher in S. oryzae than in S. zeamais. Significant differences in activity levels were also found between strains in both species. Since -amylase is a predominant digestive hydrolase in these species, the degree to which cereal diets affect amylase levels may indicate their suitability as potential hosts.

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Résumé La transformation de rang des valeur d'activité spécifique de l'-amylase de 4 souches de S. oryzae et de 4 souches de S. zeamais montre que les niveaux les plus élevés de ces enzymes prédominantes s'observent chez les adultes nourris d'orge mondé ou de riz brun á grains longs. Des niveaux intermédiaires d'activité ont été obtenus chez les insectes élevés sur maïs jaune, et les niveaux les plus faibles chez ceux élevés sur blé. Bien que les extraits préparés à partir d'orge présentent une activité inhibitrice de deux isoamylases purifiées de S. oryzae, les niveaux des inhibiteurs naturels -amylase de ces deux enzymes sont environ respectivement 2,2 et 6,1 fois plus concentrés dans le blé. L'ingestion de ces inhibiteurs d'amylase et la formation d'un complexe enzyme inactive/inhibiteur avec l'amylase secrétée antérieurement, peut rendre compte de la plus faible activité de l'amylase chez les charançons consommant du blé. Le niveau d'amylase de S. oryzae est 2 fois plus élevé que celui de S. zeamais pour toutes les souches élevées sur un régime donné. Des niveaux d'activité significativement différents ont été trouvés suivant les souches pour chacune des deux espèces. Puisque l'amylase est la principale hydrolase digestive de ces espèces, l'intensité de la modification des teneurs en amylase par la consommation de céréales peut indiquer leur adéquation comme hôtes potentiels.

     

Extracellular Fluid Proteins of Goldfish Brain: Evidence for the Presence of Proteases and Esterases

Preparations of enriched fractions of extracellular fluid (ECF) proteins from goldfish brain were found to contain protease(s) and esterase(s). The N-substituted furanacryloyl (FA) peptides FA-Phe-Gly-Gly and FA-Phe-OMe were used as model substrates for determining protease and esterase activity, respectively, in a spectrophotometric assay. Studies of the profile of substrate specificity and identification of the types of compounds that were effective as inhibitors showed that these ECF enzymes have some distinctive properties. GSH, but not GSSG, and EDTA inhibited the protease(s) without influencing the esterase(s), whereas L-1-tosylamide-2-phenylethylchloromethyl ketone blocked both protease and esterase activities of ECF. Most of the protease and esterase properties of ECF could be bound to concanavalin A-Sepharose affinity chromatographic columns in association with ependymin--a brain extracellular protein. These observations indicate that ECF may contain a metalloprotease(s) and raise the possibility that the ependymins might be a substrate for these ECF enzymes.     

Uptake of glycine by excised pulvini of Mimosa pudica in relation to proton pump activity

Uptake of [³H]-glycine by sections of Mimosa pudica L. pulvini is pH dependent (maximum at pH 5.5) and exhibits biphasic saturation kinetics in the range of concentrations tested (1–75 m M ). Effects of compounds which increase [fusicoccin (FC)] or decrease (uncouplers, ATPase inhibitors) the proton-motive force were tested both on the pH variations induced in the incubation medium and on glycine uptake by the pulvinar tissues: there is a close relationship between the time required for the effect of these compounds on the acidification (for FC) and the pH rise (for the inhibitors) of the medium and that needed respectively for promotion and inhibition of glycine uptake. Experiments with sulfhydryl-reacting compounds show that N-ethylmaleimide induces a large rise in pH in the incubation medium and strongly inhibits glycine uptake, whereas p -chloromercuribenzenesulfonic acid has less effect on these processes. These results argue for a proton-glycine symport mechanism in the pulvinar tissue and thus support the previously postulated involvement of a proton pump in the regulation of pulvinar movement.