Identification of a novel bean alpha-amylase inhibitor with chitinolytic activity

Zabrotes subfasciatus is a devastating starch-dependent storage bean pest. In this study, we attempted to identify novel alpha-amylase inhibitors from wild bean seeds, with efficiency toward pest alpha-amylases. An inhibitor named Phaseolus vulgaris chitinolytic alpha-amylase inhibitor (PvCAI) was purified and mass spectrometry analyses showed a protein with 33330 Da with the ability to form dimers. Purified PvCAI showed significant inhibitory activity against larval Z. subfasciatus alpha-amylases with no activity against mammalian enzymes. N-terminal sequence analyses showed an unexpected high identity to plant chitinases from the glycoside hydrolase family 18. Furthermore, their chitinolytic activity was also detected. Our data provides compelling evidence that PvCAI also possessed chitinolytic activity, indicating the emergence of a novel alpha-amylase inhibitor class.     

Biosynthesis of alpha-amylase and protease by Streptomyces olivaceus 142. I. Regulation of alpha-amylase activity.

Streptomyces olivaceus 142 produces amylase in the logarithmic phase of growth of the culture. The synthesis of the enzyme is induced by maltose and starch. In the case of maltose the synthesis is induced by a contaminating compound, probably being a higher than maltose polymer of glucose. The synthesis of amylase is negatively controlled by catabolic repression. The level of the activity of the enzyme depends not only on the biosynthesis but also on it proteolytic degradation.     

Insecticidal activity of an alpha-amylase inhibitor-like protein resembling a putative precursor of alpha-amylase inhibitor in the common bean, Phaseolus vulgaris L.

alpha-Amylase inhibitor (alphaAI) in the common bean, Phaseolus vulgaris L., protects seeds from insect pests such as the cowpea weevil (Callosobruchus maculatus) and the azuki bean weevil (C. chinensis). Cultivars which lack alphaAI still show resistance to both bruchids. These cultivars have a glycoprotein that reacts with anti-alphaAI-1 antibodies. The glycoprotein with a molecular mass of 29 kDa (Gp29) was purified and the encoding gene was isolated. The primary structure of Gp29 is the same as alpha-amylase inhibitor-like protein (AIL) from which the encoding gene has already been isolated. AIL resembles a putative precursor of alphaAI, even though it does not form the active inhibitor. However, AIL has some inhibitory effect on the growth of C. maculatus but not C. chinensis. The presence of AIL alone is insufficient to explain the bruchid resistance of common bean cultivars lacking alpha-AI. Common bean seeds appear to contain several factors responsible for the bruchid resistance.     

Some aspects of the mechanism of complexation of red kidney bean alpha-amylase inhibitor and alpha-amylase

Bovine pancreatic alpha-amylase binds 1 mol of acarbose (a carbohydrate alpha-amylase inhibitor) per mol at the active site and also binds acarbose nonspecifically. The red kidney bean alpha-amylase inhibitor-bovine pancreatic alpha-amylase complex retained nonspecific binding for acarbose only. Binding of p-nitrophenyl alpha-D-maltoside to the final complex of red kidney bean alpha-amylase inhibitor and bovine pancreatic alpha-amylase has a beta Ks (Ks') value that is 3.4-fold greater than the Ks (16 mM) of alpha-amylase for p-nitrophenyl alpha-D-maltoside alone. The initial complex of alpha-amylase and inhibitor apparently hydrolyzes this substrate as rapidly as alpha-amylase alone. The complex retains affinity for substrates and competitive inhibitors, which, when present in high concentrations, cause dissociation of the complex. Maltose (0.5 M), a competitive inhibitor of alpha-amylase, caused dissociation of the red kidney bean alpha-amylase inhibitor--alpha-amylase complex. Interaction between red kidney bean (Phaseolus vulgaris) alpha-amylase inhibitor and porcine pancreatic alpha-amylase proceeds through two steps. The first step has a Keq of 3.1 X 10(-5) M. The second step (unimolecular; first order) has a forward rate constant of 3.05 min-1 at pH 6.9 and 30 degrees C. alpha-Amylase inhibitor combines with alpha-amylase, in the presence of p-nitrophenyl alpha-D-maltoside, noncompetitively. On the basis of the data presented, it is likely that alpha-amylase is inactivated by the alpha-amylase inhibitor through a conformational change. A kinetic model, in the presence and absence of substrate, is described for noncompetitive, slow, tight-binding inhibitors that proceed through two steps.     

Regulation by cations of adenylate cyclase activity in chicken tissues

The effects of magnesium and sodium ions on adenylate cyclase activity in plasma membranes from chicken heart and eggshell gland mucosa were studied. It was found that the increase in magnesium chloride concentration from 5 to 40 mM results in the stimulation (4.1-fold) of the adenylate cyclase activity. The increase in sodium chloride concentration up to 150 mM stimulated the enzyme activity 2-fold. The stimulation of adenylate cyclase by magnesium and sodium ions was less pronounced in the eggshell gland. GTP did not activate adenylate cyclase. The activating effect of magnesium and sodium ions was accompanied by the attenuation of the enzyme sensitivity to NaF, guanylyl imidodiphosphate and isoproterenol. Activation by guanylyl imidodiphosphate was completely abolished in the presence of 40 mM magnesium chloride. It is assumed that high concentrations of the salt promote subunit dissociation of the adenylate cyclase regulatory protein and its interaction with the catalytic subunit in the presence of endogenous nucleotides. The differences in the adenylate cyclase sensitivity to cations in chicken heart and eggshell gland mucosa correlate with the amount of pertussis toxin substrate.     

Protective mechanism of the Mexican bean weevil against high levels of alpha-amylase inhibitor in the common bean.

Alpha-amylase inhibitor (alpha AI) protects seeds of the common bean (Phaseolus vulgaris) against predation by certain species of bruchids such as the cowpea weevil (Callosobruchus maculatus) and the azuki bean weevil (Callosobruchus chinensis), but not against predation by the bean weevil (Acanthoscelides obtectus) or the Mexican bean weevil (Zabrotes subfasciatus), insects that are common in the Americas. We characterized the interaction of alpha AI-1 present in seeds of the common bean, of a different isoform, alpha AI-2, present in seeds of wild common bean accessions, and of two homologs, alpha AI-Pa present in seeds of the tepary bean (Phaseolus acutifolius) and alpha AI-Pc in seeds of the scarlet runner bean (Phaseolus coccineus), with the midgut extracts of several bruchids. The extract of the Z. subfasciatus larvae rapidly digests and inactivates alpha AI-1 and alpha AI-Pc, but not alpha AI-2 or alpha AI-Pa. The digestion is caused by a serine protease. A single proteolytic cleavage in the beta subunit of alpha AI-1 occurs at the active site of the protein. When degradation is prevented, alpha AI-1 and alpha AI-Pc do not inhibit the alpha-amylase of Z. subfasciatus, although they are effective against the alpha-amylase of C. chinensis. Alpha AI-2 and alpha AI-Pa, on the other hand, do inhibit the alpha-amylase of Z. subfasciatus, suggesting that they are good candidates for genetic engineering to achieve resistance to Z. subfasciatus.     

Regulation of alpha-amylase biosynthesis in Pichia burtonii B]

The synthesis of amylolytic enzymes by Pichia burtonii strain CBS 6141 requires the presence of etarch, maltose, and saccharose. Glucose exerts a strong repression which completely inhibited snzyme induction.     

Inhibition of human serum alpha-amylase activity

Amylase activity was measured in normal sera and serum of patients with acute pancreatitis and mumps. A significant decrease of enzyme activity in patients with acute pancreatitis was observed after incubation with specific antibody against hog pancreas amylase. A minimal effect of the antibody on normal serum and on that from mumps patients was noted. A comparable effect was observed in the presence of glyceraldehyde used as an amylase differentiating factor.     

Auxins induce alpha-amylase activity in pea cotyledons

The report documents how the development of α-amylase activity in detached cotyledons of Pisum sativum cv Alaska is accelerated 2- to 12-fold during incubation with 1 micromolar to 10 micromolar 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, or with 4-chloroindoleacetic acid, an endogenous auxin from Pisum sativum. It seems probable that auxin from the embryonic axis induces α-amylase in the attached cotyledons during germination.     

Regulation of alternative oxidase activity during phosphate deficiency in bean roots (Phaseolus vulgaris)

Cyanide-resistant respiration was studied in mitochondria isolated from the roots of bean plants ( Phaseolus vulgaris L. cv. Złota Saxa) grown hydroponically up to 16 days on a phosphate-sufficient (+P, control) or phosphate-deficient (−P) medium. Western blotting indicated that the alternative oxidase (AOX) was present only in its reduced (active) form, both in phosphate-sufficient and phosphate-deficient roots, but in the latter, the amount of AOX protein was greater. Addition of pyruvate to the isolation, washing and reaction media made mitochondria from +P roots cyanide-insensitive, similar to mitochondria from −P roots. The doubled activity of NAD-malic enzyme (NAD-ME) in −P compared with +P root mitochondria may suggest increased pyruvate production in −P mitochondria. Lower cytochrome c oxidase (COX) activity and no uncoupler effect on respiration indicated limited cytochrome chain activity in −P mitochondria. In −P mitochondria, the oxygen uptake decreased and the level of Q reduction increased from 60 to 80%. With no pyruvate present (AOX not fully activated), inhibition of the cytochrome pathway resulted in an increased level of the ratio of reduced ubiquinone (Qr) to total ubiquinone (Qt) (Qr/Qt) in +P mitochondria, but did not change Qr/Qt in −P mitochondria. When pyruvate was present, the kinetics for AOX were similar in mitochondria from −P and +P roots. It is suggested that AOX participation in −P respiration may provide an acclimation to phosphate deficiency. Stabilization of the ubiquinone reduction level by AOX might prevent the harmful effect of an increased formation of reactive oxygen species.     

Regulation of 3-hydroxyl-3-methylglutaryl-coenzyme A reductase activity in rat uterine tissues

Initiation of uterine DNA synthesis and mitosis in response to estrogen appears to depend upon the stimulation of protein synthesis. 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase could have a key function in controlling uterine mitosis through its control of mevalonic acid and cholesterol synthesis as the rate-limiting enzyme in their synthetic pathways. These studies were initiated to examine the kinetics of the uterine increases in HMG-CoA reductase activity in response to estradiol. In the uterus of the ovariectomized mature rat, estradiol increased levels of enzyme activity in both the luminal epithelium and stroma-myometrium up to 12 h after estradiol treatment. Levels of HMG-CoA reductase activity decreased after 12 h in the luminal epithelium and further increased in the stroma-myometrium. Previous studies have shown that estradiol does not increase DNA synthesis and mitosis in the stroma-myometrium of the uterus of the ovariectomized mature rat. Since estradiol increased HMG-CoA reductase activity in both the luminal epithelium and stroma-myometrium, we conclude that even though increased HMG-CoA reductase activity may be a prerequisite for increased DNA synthesis, increases in uterine HMG-CoA reductase activity are not necessarily followed by increased DNA synthesis.     

Regulation of the accumulation of mRNA for alpha-amylase in barley aleurone

The effect of gibberellic acid and Ca²⁺ on the accumulation of α-amylase mRNAs in aleurone layers of barley (Hordeum vulgare L. cv Himalaya) was studied using cDNA clones containing sequences of mRNAs for the high and low isoelectric point (pI) α-amylases. There is no significant hybridization between the two α-amylase cDNA clones under the hybridization and washing conditions employed. These clones were therefore used to monitor levels of mRNAs for high and low pI α-amylases. It is shown that although the synthesis of the high pI α-amylase proteins depends on the presence of Ca²⁺ in the incubation medium, the accumulation of mRNA for this group occurs to the same degree in the presence or the absence of Ca²⁺. The accumulation of low pI α-amylase mRNA is also not affected by the presence or absence of Ca²⁺ in the incubation medium. These results establish gibberellic acid, not Ca²⁺, as the principal regulator of α-amylase mRNA accumulation in barley aleurone, while Ca²⁺ controls high pI α-amylase synthesis at a later step in the biosynthetic pathway.     

Introducing transglycosylation activity in a liquefying alpha-amylase.

By mutating Ala-289 by Phe or Tyr in the Bacillus stearothermophilus alpha-amylase, we induced this enzyme to perform alcoholytic reactions, a function not present in the wild-type enzyme. This residue was selected from homology analysis with neopullulanase, where the residue has been implicated in the control of transglycosylation [Kuriki et al. (1996) J. Biol. Chem. 271, 17321-173291. We made some inferences about the importance of electrostatic and geometrical modifications in the active site environment of the amylase to explain the behavior of the modified enzyme.     

Mapping QTLs for alpha-amylase activity in rye grain

Genetic control of alpha-amylase activity in rye grain was investigated by QTL mapping based on DS2 x RXL10 intercross consisting of 99 F5-6 families propagated at one location during four vegetation seasons. A wide range of variation in alpha-amylase activity and transgression effects were found among families and parental lines. This variation was shown to be determined in 40.1% by 7 significant (LOD score not less than 2.5) and 2 putative QTLs (2 < LOD < 2.5) distributed on all rye chromosomes except 4R. Two significant QTLs located on 3RL and 5RL chromosome arms were expressed each year. The third significant QTL was detected in three years (1RL). The other four significant QTLs (2RL, 5RS, 6RL, 7RL) were found in one year of study. The number and composition of QTLs were specific for a given year varying from three to six. QTLs were not correlated with isoenzyme polymorphisms at the structural alpha-Amy1 loci. A QTL associated with a region containing the alpha-Amy3 locus was detected on chromosome 5RL. Both high- and low-activity QTL alleles were found in each parental line, which explains the appearance of transgressive recombinants in the segregating population.     

Regulation of cell wall synthesis by auxin and fusicoccin in different tissues of pea stem segments

Cell wall synthesis was studied by determining the incorporation of [¹⁴C]-glucose into epidermal and cortical cell walls of etiolated Pisum sativum L. cv. Alaska stem segments. Walls were fractionated into the matrix and cellulose components, and incorporation into these components assessed in terms of the total uptake of label into that tissue. When segments were allowed to elongate, the stimulation of total glucose uptake by indole-3-acetic acid (IAA) and fusicoccin (FC) was greater than their stimulation of incorporation. IAA and FC thus did not stimulate precursor incorporation in elongating segments. When elongation was inhibited by calcium, however, IAA and FC significantly promoted wall synthesis in the cortex and vasular tissue (which shows almost no growth or acidification response to auxin). In these tissues incorporation into matrix and cellulose was promoted approximately equally. In the epidermis (thought to be the tissue responsive to auxin in the control of growth), FC promoted a significant increase in wall synthesis, although less than that in the cortex, while there was some evidence of a similar promotion by IAA. Both IAA and FC had a greater effect on incorporation into the matrix component of the wall than into cellulose. The results that FC caused a substantial promotion of cell wall synthesis which was not due solely to elongation, and that the inner non-growth responsive cortical tissues can respond to IAA. Moreover, a comparison of the effects of IAA and FC on the different components of the wall suggests that the response in the epidermis differs from that in the other tissues.     

Chloroplastic regulation of apoplastic alpha-amylase activity in pea seedlings

Photobleaching of pea (Pisum sativum L.) seedling leaves by treatment with norflurazon (San 9789) and 7 days of continuous white light caused a 76- to 85-fold increase in the activity of the primary α-amylase, a largely apoplastic enzyme, over normally greening seedlings. Levels of chlorophyll were near zero and levels of plastid marker enzyme activities were very low in norflurazon-treated seedlings, indicating severe photooxidative damage to plastids. As levels of norflurazon or fluence rates were lowered, decreasing photobleaching of tissues, α-amylase activity decreased. Levels of leaf β-amylase and starch debranching enzyme changed very little in norflurazon-treated seedlings. Infiltration extraction of leaves of norflurazon-treated and normally greening seedlings indicated that at least 57 and 62%, respectively, of α-amylase activity was in the apoplast. α-Amylase activity recovered from the apoplast of photobleached leaves of norflurazon-treated seedlings was 18-fold higher than that for green leaves. Inhibitors of photosynthesis (DCMU and atrazine) and an inhibitor of chlorophyll accumulation that does not cause photooxidation of plastid components (tentoxin) had little effect on levels of α-amylase activity, indicating norflurazon-caused loss of chlorophyll and lack of photosynthesis did not cause the large induction in α-amylase activity. An inhibitor of both abscisic acid and gibberellin synthesis (paclobutrazol [PP333]) and an analog of norflurazon which inhibits photosynthesis but not carotenoid synthesis (San 9785) caused only moderate (about five-fold) increases in α-amylase activity. Lincomycin and chloramphenicol increased α-amylase activity in light grown seedings to the same magnitude as norflurazon, indicating that the effect of norflurazon is probably through the destruction of plastid ribosomes. It is proposed that chloroplasts produce a negative signal for the regulation of the apoplastic α-amylase in pea.     

Regulation by nicotinic acid of malate dehydrogenase activity in tissues of Black Sea mussels

Nicotinic acid was studied for its effect on the malate dehydrogenase activity from mussels' tissues and on its ability to link substrate and coenzyme. NADH and nicotinic acid in high concentrations are shown to produce an inhibiting effect on the reverse malate dehydrogenase reaction which is determined by the nonspecific action either of the vitamin or its metabolites. When pH of the medium is shifted toward the acid zone the affinity of the enzyme to the coenzyme decreases. This phenomenon may be one of the mechanisms of the mussel organism adaptation to anaerobiosis.