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PubMed1991

Alterations of amylase secretion in the chronically reserpinized rat: an acetylcholine-mediated phenomenon.

Chronic reserpine treatment of animals, an experimental model for cystic fibrosis (CF), results in generalized exocrinopathy, impaired secretion, and decreased pancreatic content of amylase. However, the mechanisms of altered acinar pancreatic function in both CF patients and reserpine-treated rats are still unknown. The present study was designed to investigate the secretory response of the rat pancreas after reserpine treatment. Rats were given reserpine (1 mg kg-1 day-1 ip) or vehicle, and killed 2, 4, or 7 days later. To distinguish between specific effects of reserpine and those related to secondary malnutrition caused by the drug, secretory response of a group of pair-fed animals to reserpine was also investigated. During reserpine treatment, body weight and food intake were significantly reduced from the 2nd day on. Amylase release from dispersed pancreatic acini, prepared from control pair-fed and from reserpine-treated rats were used to evaluate functional secretory capacity. After 7 days, both chronic reserpine and pair-feeding significantly decreased pancreatic amylase concentration to 75 and 50% of controls, respectively. Reserpine caused desensitization of the pancreatic acini secretory response to carbamylcholine, without altering maximal amylase release. Desensitization occurred gradually and reached a maximum after 7 days. Secretory responses to caerulein and to the phorbol ester 12.0-tetradecanoyl phorbol-13 acetate (TPA) were not altered. These findings indicate that desensitization of amylase release after reserpine was specific to this muscarinic agonist. It may result from increased endogenous acetylcholine release in the course of treatment.

Acetylcholine↗
PubMed1993

Effect of verapamil on the cyclic AMP-mediated pathway for amylase secretion in rat pancreatic acini.

In dispersed acini from rat pancreas, verapamil (a phenylalkylamine calcium channel blocker) potentiated amylase secretion stimulated by vasoactive intestinal peptide (VIP), secretin, peptide histidine isoleucine, helodermin, forskolin, and 8-bromocyclic AMP. The action of verapamil on VIP-stimulated amylase secretion was detectable at 10 microM verapamil and maximal at 100 microM verapamil. Verapamil did not alter binding of 125I-VIP, basal cAMP, the increase in cAMP caused by VIP, or the increase in cAMP-dependent protein kinase caused by VIP. The effects of verapamil on stimulated amylase secretion were fully reversible and could be reproduced by nicardipine (a 1,4-dihydropyridine calcium channel blocker) and diltiazem (a benzothiazepine calcium channel blocker), but not by cinnarizine (a piperazine calcium channel blocker). Although 300 microM verapamil increased outflux of 45Ca, 100 microM verapamil, the concentration that produced maximal potentiation of VIP-stimulated amylase secretion, did not alter 45Ca outflux. Our results indicate that the action of verapamil to potentiate amylase secretion stimulated by secretagogues that activate the cAMP pathway occurs at a step that is distal to the activation of cAMP-dependent protein kinase.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1996

Carboxylic ester hydrolase and amylase in ischemic pancreatitis in the guinea pig.

The observation that an elevated level of pancreatic carboxylic ester hydrolase (CEH) in serum is a more sensitive and specific marker of acute pancreatitis than is elevated serum amylase activity prompted us to explore whether these findings could be confirmed in an experimental model and, if so, to find the explanation behind this difference. We therefore developed a model for ischemic pancreatitis in the guinea pig and a sandwich enzyme-linked immunosorbent assay for determination of CEH in this species. There was a strong correlation between duration of ischemia and severity of pancreatic inflammation and between severity of inflammation and serum CEH level. In contrast, serum amylase was elevated only in animals with the most severe grade of inflammation. Amylase was, however, increased in urine in animals with mild inflammation, but the level did not increase with severity of inflammation. Only one of 31 animals had detectable CEH in urine. In animals with intermediate serum CEH levels the serum and biliary concentrations correlated, indicating that CEH may be cleared by the liver. Amylase was detectable in bile only in animals with high serum levels. The results confirm our observations made in previous clinical studies. A likely explanation for differences in serum levels of CEH and amylase is clearance from the circulation at different rates and, at least partly, via different routes, e.g., the liver and kidney, respectively.

Amylases↗
PubMed1997

A comparison of two methods to measure amylase, lipase, trypsin, and chymotrypsin activity and the effect of freezing and thawing on enzyme activities in pancreatic juice.

An experiment was conducted to compare two methods, referred to as A and B, for measuring amylase, lipase, trypsin, and chymotrypsin activities in samples of pancreatic juice and to investigate the effect of freezing and thawing on enzyme activities. Three growing pigs were used in this study. A catheter was surgically inserted into the pancreatic duct and a simple T-cannula was placed in the duodenum to allow the return of collected pancreatic juice. Pancreatic juice was collected for a total of 24 h and 27 pooled 8-h samples were used in the analyses. Following collection and pooling, the samples were frozen at -80 degrees C, due to the large number of samples and analyses, and stored for 2 weeks. The samples were thawed and enzyme activities were determined with Method A. The samples were frozen again at -80 degrees C, stored for 2 weeks, and rethawed and enzyme activities were determined with Methods A and B. There was a direct relationship between Method A and Method B for measuring amylase, trypsin, and chymotrypsin activity (R2 = 0.95, 0.81, and 0.90, respectively). However, a poor relationship was observed between a titrimetric and a photometric method for determining lipase activity (R2 = 0.42). A second freezing, storage, and thawing did not affect (p > 0.3) the concentration of protein or amylase activity in pancreatic juice. However, the activities of chymotrypsin, trypsin, and, especially, lipase were decreased by 29.0% (p < 0.11), 40.4% (p < 0.01), and 82.9% (p < 0.001), respectively. In conclusion, either Method A or Method B can be used to measure amylase, trypsin, and chymotrypsin activities in pancreatic juice but a titrimetric method should be used to determine lipase activity. Furthermore, if the activities of lipase, trypsin, and chymotrypsin are to be determined, the sample should only be frozen and thawed once. If amylase activity will be determined, the sample can be repeatedly frozen and thawed.

Amylases↗
PubMed1988

Monitoring urinary amylase and pH in pancreas transplantation with urinary drainage in rats.

Whole pancreas isografts or allografts (ACI donors, RT1a) with bladder drainage of exocrine secretions were performed in Lewis rats (RR1(1] with streptozotocin-induced diabetes. Urinary amylase, pH, and volume and serum glucose were measured daily. They were analyzed alone, or in combination, to determine patterns in deviations from normal values, from isograft control values, or from a posttransplant baseline in relation to rejection (defined as reversion of plasma glucose of greater than 200 mg/dl) in nonimmunosuppressed recipients. Also studied were the sensitivity and specificity by which such deviations predicted rejection. Functioning grafts were associated with increased urinary amylase and pH compared with normal or diabetic controls; urinary volume was less than that of diabetic rats, but greater than that of normal rats. In nonimmunosuppressed allograft recipients (n = 9), rejection occurred at a mean (+/- SD) of 7.78 +/- 0.44 days. Serum glucose rose to above normal (greater than 134 mg/dl) 1 day before rejection in 3 animals (sensitivity 33%, false negative rate 66%; false positive rate in 9 isograft recipients, 44%). Urinary volume dropped below 3 ml at a mean of 3.17 +/- 0.98 days (range 2-5 days) before rejection in 6 animals (sensitivity 66%, false negative rate 33%; false positive rate 0%). Urinary pH fell below 7.25 at a mean of 3.13 +/- 1.81 days (range 1-5 days) before rejection in 8 rats (sensitivity of 89%, false negative rate 11%; false positive rate 29%). Urinary amylase dropped from a posttransplant peak at a mean of 3.56 +/- 1.42 days (range 1-6 days) before rejection in 9 animals (sensitivity 100%, false negative rate 0%; false positive rate 43%), and dropped below 1500 units per 24 hr at a mean of 2.00 +/- 1.32 days (range 1-5 days) before rejection in 8 animals (sensitivity 89%, false negative rate 11%; false positive rate 0%). A drop in urinary amylase combined with a drop in urinary volume or pH occurred at a mean of 3.22 +/- 1.48 days (range 1-5 days) before rejection in 9 rats (sensitivity 100%, false negative rate 0%; false positive rate 0%). In a separate group of 10 allograft recipients, immunosuppression with prednisone and cyclosporine was begun concomitant with, or within 2 days of, the drop in urinary amylase from the peak value; rejection did not occur in 3 animals and was delayed to a mean of 12.0 +/- 5.0 days posttransplant in 7 animals (P less than .05 compared with the nonimmunosuppressed group).(ABSTRACT TRUNCATED AT 400 WORDS)

Amylases↗
PubMed1987

Protein processing to form extracellular thermostable alpha-amylases from a gene fused in a Bacillus subtilis secretion vector.

A thermostable alpha-amylase gene (amyT631) from Bacillus stearothermophilus A631 was cloned into pBR322 and recloned into pUB110: the resulting plasmid was designated pTUB607. To investigate the processing from preproenzyme to mature enzyme, amyT631 from pTUB607, after digestion with BAL31, was introduced into the B. subtilis alpha-amylase secretion vector pTUB285. Three chimaeric plasmids, pTUB613, pTUB616, and pTUB617, were isolated. The fused alpha-amylases expressed from the three plasmids seemed to be synthesized as preproenzymes. From analysis of the NH2-terminal amino acid sequences of purified extracellular alpha-amylases, the precursors of the fused enzymes appeared to be cleaved at first between amino acids 31 and 32 from the translation initiator Met (positions -11 and -10 with respect to the beginning of the mature enzyme), and processed to mature extracellular enzymes in which the NH2-terminal amino acid sequences were the same as that of the parental pTUB607 alpha-amylase, in spite of the lengths of the prosequences and the amino acid composition near the secondary cleavage sites being different in each enzyme.

Amino Acid Sequence↗
PubMed1989

Cloning and expression of the Clostridium thermohydrosulfuricum alpha-amylase-pullulanase gene in Escherichia coli.

An alpha-amylase-pullulanase gene from Clostridium thermohydrosulfuricum DSM 3783 was cloned in Escherichia coli on a 7.0 kb EcoRI fragment using a lambda vector. The gene produced, from an indigenous promoter, active thermostable alpha-amylase-pullulanase, seemingly mostly a soluble intracellular enzyme in E. coli. Gel filtration separated the active enzyme produced into three peaks, each having both alpha-amylase and pullulanase activities. Immunoblotting after SDS-PAGE revealed more than ten alpha-amylase-pullulanase specific polypeptides; the biggest of these had an Mr of about 165,000, whereas the smallest enzymically active polypeptide had an Mr of about 100,000. Despite the marked degeneration of its constituent polypeptides, the apparent temperature optimum of the enzyme (80-85 degrees C) was only some 5 degrees C lower and the heat stability the same as that of the extracellular alpha-amylase-pullulanase produced by the native host. Oligonucleotide probes prepared according to the NH2-terminal amino acid sequences of the enzyme and its satellite polypeptide (a polypeptide associated with the extracellular enzyme of the native host) hybridized to different regions of the 7.0 kb DNA insert.

Bacterial Proteins↗
PubMed1990

Artificial insertion of peptides between signal peptide and mature protein: effect on secretion and processing of hybrid thermostable alpha-amylases in Bacillus subtilis and Escherichia coli cells.

To study the effect of inserted peptides on the secretion and processing of exported proteins in Bacillus subtilis and Escherichia coli, pBR322-derived DNA fragments coding for small peptides were inserted between the DNA coding for the 31 amino acid B. subtilis alpha-amylase signal peptide and that coding for the mature part of the extracellular thermostable alpha-amylase of B. stearothermophilus. Most of the inserted peptides (21 to 65 amino acids) decreased the production of the enzyme in B. subtilis and E. coli, the effect of each peptide being similar in the two strains. In contrast, with one peptide (a 21 amino acid sequence encoded by the extra DNA in pTUBE638), the production of alpha-amylase was enhanced more than 1.7-fold in B. subtilis in comparison with that of the parent strain. The molecular masses of the thermostable alpha-amylases in the periplasm of the E. coli transformants varied for each peptide insert, whereas those in the culture supernatants of the B. subtilis transformants had molecular masses similar to that of the mature enzyme. Based on the NH2-terminal amino acid sequence of the hybrid protein from pTUBE638, it was shown that in E. coli, the NH2-terminally extended thermostable alpha-amylase was translocated and remained in the periplasm after the 31 amino acid signal sequence was removed. In the case of B. subtilis, after the removal of a 34-amino acid signal sequence, the hybrid protein was secreted and processed to the mature form.

Amino Acid Sequence↗
PubMed1992

Molecular cloning, characterization and nucleotide sequence of the gene for secreted alpha-amylase from Xanthomonas campestris pv. campestris.

alpha-Amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) of apparent molecular mass 45 kDa was secreted by Xanthomonas campestris pv. campestris grown in medium containing starch or maltose. We isolated its structural gene from a recombinant lambda library and located it on a 2.7 kb DNA fragment. Nucleotide sequencing of the fragment revealed a potential ORF encoding a protein of 475 amino acid residues, including a potential signal sequence of 35 amino acids. The signal processing site was confirmed by N-terminal amino acid sequence analysis of the exported alpha-amylase. The deduced amino acid sequence of the mature protein is very similar to that of the alpha-amylase of Aeromonas hydrophila. It also contains all four amino acid sequences highly conserved in the alpha-amylases from a wide range of organisms. Expression of the amy gene in Escherichia coli was poor from its own promoter, but was enhanced by the upstream promoter on the vector. The alpha-amylase synthesized in E. coli was located in the periplasm.

Amino Acid Sequence↗
PubMed1990

Regulation of amylase gene expression in diabetic mice is mediated by a cis-acting upstream element close to the pancreas-specific enhancer.

We localized the cis-acting sequences that mediate the regulation of a pancreatic amylase gene, Amy-2.2, in diabetic mice. We constructed three hybrid genes containing sequences from the 5'-flanking region of the amylase gene upstream of the heterologous elastase promoter linked to the CAT structural gene. These constructs were transferred to the germ line of transgenic mice by microinjection of fertilized eggs. The amylase sequences had two effects on expression of the elastase promoter: Basal expression was increased, and expression in diabetic animals was reduced to approximately 2% of basal levels. A 30-bp amylase fragment was sufficient to transfer both of these regulatory functions to the elastase promoter. Sequences within this 30-bp fragment are included in the binding site for the pancreatic nuclear protein PTF1. The close association of the PTF1-binding site and the regulatory functions is consistent with a mechanism based on interference with activation by PTF1 in diabetic pancreas. PTF1-binding activity is not reduced in diabetic pancreas. The data presented here demonstrate that the 5'-flanking region of the pancreatic amylase gene contains a novel insulin-dependent element (IDE) that mediates the loss of expression in diabetic animals.

Amylases↗
PubMed2002

The alpha-amylase induction in endosperm during rice seed germination is caused by gibberellin synthesized in epithelium.

We recently isolated two genes (OsGA3ox1 and OsGA3ox2) from rice (Oryza sativa) encoding 3beta-hydroxylase, which catalyzes the final step of active gibberellin (GA) biosynthesis (H. Itoh, M. Ueguchi-Tanaka, N. Sentoku, H. Kitano, M. Matsuoka, M. Kobayashi [2001] Proc Natl Acad Sci USA 98: 8909-8914). Using these cloned cDNAs, we analyzed the temporal and spatial expression patterns of the 3beta-hydroxylase genes and also an alpha-amylase gene (RAmy1A) during rice seed germination to investigate the relationship between GA biosynthesis and alpha-amylase expression. Northern-blot analyses revealed that RAmy1A expression in the embryo occurs before the induction of 3beta-hydroxylase expression, whereas in the endosperm, a high level of RAmy1A expression occurs 1 to 2 d after the peak of OsGA3ox2 expression and only in the absence of uniconazol. Based on the analysis of an OsGA3ox2 null mutant (d18-Akibare dwarf), we determined that 3beta-hydroxylase produced by OsGA3ox2 is important for the induction of RAmy1A expression and that the OsGA3ox1 product is not essential for alpha-amylase induction. The expression of OsGA3ox2 was localized to the shoot region and epithelium of the embryo, strongly suggesting that active GA biosynthesis occurs in these two regions. The synthesis of active GA in the epithelium is important for alpha-amylase expression in the endosperm, because an embryonic mutant defective in shoot formation, but which developed epithelium cells, induced alpha-amylase expression in the endosperm, whereas a mutant defective in epithelium development did not.

Blotting, Northern↗
PubMed2004

Signal peptide-dependent targeting of a rice alpha-amylase and cargo proteins to plastids and extracellular compartments of plant cells.

alpha-Amylases are important enzymes for starch degradation in plants. However, it has been a long-running debate as to whether alpha-amylases are localized in plastids where starch is stored. To study the subcellular localization of alpha-amylases in plant cells, a rice (Oryza sativa) alpha-amylase, alphaAmy3, with or without its own signal peptide (SP) was expressed in transgenic tobacco (Nicotiana tabacum) and analyzed. Loss-of-function analyses revealed that SP was required for targeting of alphaAmy3 to chloroplasts and/or amyloplasts and cell walls and/or extracellular compartments of leaves and suspension cells. SP was also required for in vitro transcribed and/or translated alphaAmy3 to be cotranslationally imported and processed in canine microsomes. alphaAmy3, present in chloroplasts of transgenic tobacco leaves, was processed to a product with Mr similar to alphaAmy3 minus its SP. Amino acid sequence analysis revealed that the SP of chloroplast localized alphaAmy3 was cleaved at a site only one amino acid preceding the predicted cleavage site. Function of the alphaAmy3 SP was further studied by gain-of-function analyses. beta-Glucuronidase (GUS) and green fluorescence protein fused with or without the alphaAmy3 SP was expressed in transgenic tobacco or rice. The alphaAmy3 SP directed translocation of GUS and green fluorescence protein to chloroplasts and/or amyloplasts and cell walls in tobacco leaves and rice suspension cells. The SP of another rice alpha-amylase, alphaAmy8, similarly directed the dual localizations of GUS in transgenic tobacco leaves. This study is the first evidence of SP-dependent dual translocations of proteins to plastids and extracellular compartments, which provides new insights into the role of SP in protein targeting and the pathways of SP-dependent protein translocation in plants.

Cell Wall↗
PubMed1995

Sugar-inducible expression of a gene for beta-amylase in Arabidopsis thaliana.

The levels of beta-amylase activity and of the mRNA for beta-amylase in rosette leaves of Arabidopsis thaliana (L.) Heynh. increased significantly, with the concomitant accumulation of starch, when whole plants or excised mature leaves were supplied with sucrose. A supply of glucose or fructose, but not of mannitol or sorbitol, to plants also induced the expression of the gene for beta-amylase, and the induction occurred not only in rosette leaves but also in roots, stems, and bracts. These results suggest that the gene for beta-amylase of Arabidopsis is subject to regulation by a carbohydrate metabolic signal, and expression of the gene in various tissues may be regulated by the carbon partitioning and sink-source interactions in the whole plant. The sugar-inducible expression of the gene in Arabidopsis was severely repressed in the absence of light. The sugar-inducible expression in the light was not inhibited by 3(3,4-dichlorophenyl)-1,1-dimethylurea or by chloramphenicol, but it was inhibited by cycloheximide. These results suggest that a light-induced signal and de novo synthesis of proteins in the cytoplasm are involved in the regulation. A fusion gene composed of the 5' upstream region of the gene for beta-amylase from Arabidopsis and the coding sequence of beta-glucuronidase showed the sugar-inducible expression in a light-dependent manner in rosette leaves of transgenic Arabidopsis.

Amino Acid Sequence↗
PubMed1997

Characterization of a maize beta-amylase cDNA clone and its expression during seed germination.

A maize (Zea mays L.) cDNA clone (pZMB2) encoding beta-amylase was isolated from a cDNA library prepared from the aleurone RNA of germinating kernels. The cDNA encodes a predicted product of 488 amino acids with significant similarity to known beta-amylases from barley (Hordeum vulgare), rye (Secale cereale), and rice (Oryza sativa). Glycine-rich repeats found in the carboxyl terminus of the endosperm-specific beta-amylase of barley and rye are absent from the maize gene product. The N-terminal sequence of the first 20 amino acids of a beta-amylase peptide derived from purified protein is identical to the 5th through 24th amino acids of the predicted cDNA product, indicating the absence of a conventional signal peptide in the maize protein. Recombinant inbred mapping data indicate that the cDNA clone is single-copy gene that maps to chromosome 7L at position 83 centimorgans. Northern blot analysis and in vitro translation-immunoprecipitation data indicate that the maize beta-amylase is synthesized de novo in the aleurone cells but not in the scutellum during seed germination.

Amino Acid Sequence↗
PubMed2000

Glucose and disaccharide-sensing mechanisms modulate the expression of alpha-amylase in barley embryos.

The aim of this study was to investigate the sugar-sensing processes modulating the expression of alpha-amylase in barley (Hordeum vulgaris L. var Himalaya) embryos. The results highlight the existence of independent glucose (Glc) and disaccharides sensing. Glc treatment destabilizes the alpha-amylase mRNA. Non-metabolizable disaccharides repress alpha-amylase induction, but have no effects on transcript stability. Structure-function analysis indicates that a fructose (Fru) moiety is needed for disaccharide sensing. Lactulose (beta-galactose [Gal][1-->4]Fru), palatinose (Glc[1-->6]Fru), and turanose (Glc[1-->3]Fru) are not metabolized but repress alpha-amylase. Disrupting the fructosyl moiety of lactulose and palatinose, or replacing the Fru moiety of beta-Gal[1-->4]Fru with Glc or Gal results in molecules unable to repress alpha-amylase. Comparison of the molecular requirements for sucrose transport with those for disaccharide sensing suggests that these sugars are perceived possibly at the plasma membrane level independently from sucrose transport.

Blotting, Northern↗
PubMed2002

Three novel MYB proteins with one DNA binding repeat mediate sugar and hormone regulation of alpha-amylase gene expression.

The expression of alpha-amylase genes in cereals is induced by both gibberellin (GA) and sugar starvation. All alpha-amylase genes isolated from cereals contain a TATCCA element or its variants at positions approximately 90 to 150 bp upstream of the transcription start sites. The TATCCA element was shown previously to be an important component of the GA response complex and the sugar response complex of alpha-amylase gene promoters. In the present study, three cDNA clones encoding novel MYB proteins with single DNA binding domains were isolated from a rice suspension cell cDNA library and designated OsMYBS1, OsMYBS2, and OsMYBS3. Gel mobility shift experiments with OsMYBSs showed that they bind specifically to the TATCCA element in vitro. Yeast one-hybrid experiments demonstrated that OsMYBS1 and OsMYBS2 bind to the TATCCA element and transactivate a promoter containing the TATCCA element in vivo. Transient expression assays with barley half-seeds showed that OsMYBS1 and OsMYBS2 transactivate a promoter containing the TATCCA element when sugar is provided, whereas OsMYBS3 represses transcription of the same promoter under sugar starvation. Transient expression assays also showed that these three OsMYBSs cooperate with a GA-regulated transcription factor, HvMYBGa, in the transactivation of a low-pI barley alpha-amylase gene promoter in the absence of GA. Two-hybrid experiments with barley half-seeds showed that OsMYBS1 is able to form a homodimer. The present study demonstrates that differential DNA binding affinity, promoter transactivation ability, dimerization, and interactions with other protein factors determine the biological function of OsMYBSs. This study also suggests that common transcription factors are involved in the sugar and hormonal regulation of alpha-amylase gene expression in cereals.

Amino Acid Sequence↗
PubMed2006

Interaction between rice MYBGA and the gibberellin response element controls tissue-specific sugar sensitivity of alpha-amylase genes.

Expression of alpha-amylase genes during cereal grain germination and seedling growth is regulated negatively by sugar in embryos and positively by gibberellin (GA) in endosperm through the sugar response complex (SRC) and the GA response complex (GARC), respectively. We analyzed two alpha-amylase promoters, alphaAmy3 containing only SRC and alphaAmy8 containing overlapped SRC and GARC. alphaAmy3 was sugar-sensitive but GA-nonresponsive in both rice (Oryza sativa) embryos and endosperms, whereas alphaAmy8 was sugar-sensitive in embryos and GA-responsive in endosperms. Mutation of the GA response element (GARE) in the alphaAmy8 promoter impaired its GA response but enhanced sugar sensitivity, and insertion of GARE in the alphaAmy3 promoter rendered it GA-responsive but sugar-insensitive in endosperms. Expression of the GARE-interacting transcription factor MYBGA was induced by GA in endosperms, correlating with the endosperm-specific alphaAmy8 GA response. alphaAmy8 became sugar-sensitive in MYBGA knockout mutant endosperms, suggesting that the MYBGA-GARE interaction overrides the sugar sensitivity of alphaAmy8. In embryos overexpressing MYBGA, alphaAmy8 became sugar-insensitive, indicating that MYBGA affects sugar repression. alpha-Amylase promoters active in endosperms contain GARE, whereas those active in embryos may or may not contain GARE, confirming that the GARE and GA-induced MYBGA interaction prevents sugar feedback repression of endosperm alpha-amylase genes. We demonstrate that the MYBGA-GARE interaction affects sugar feedback control in balanced energy production during seedling growth and provide insight into the control mechanisms of tissue-specific regulation of alpha-amylase expression by sugar and GA signaling interference.

Gene Expression Regulation, Plant↗
PubMed2000

Structure of the bifunctional inhibitor of trypsin and alpha-amylase from ragi seeds at 2.2 A resolution.

The crystal structure of a bifunctional inhibitor of alpha-amylase and trypsin (RATI) from ragi seeds (Indian finger millet, Eleusine coracana Gaertneri) has been determined by X-ray diffraction at 2.2 A resolution. The inhibitor consists of 122 amino acids, with five disulfide bridges, and belongs to the plant alpha-amylase/trypsin inhibitor family. The crystals were grown by the microdialysis method using ammonium sulfate as a precipitating agent. The structure was determined by the molecular-replacement method using as models the structures of Corn Hageman factor inhibitor (CHFI) and of RATI at 2.9 A resolution determined previously. It has been refined to an R factor of 21.9%. The structure shows an r.m.s. deviation for C(alpha) atoms of 2.0 A compared with its own NMR structure, whereas the corresponding value compared with CHFI is found to be 1.4 A. The r.m.s. difference for C(alpha) atoms when compared with the same protein in the structure of the complex with alpha-amylase is 0.7 A. The conformations of trypsin-binding loop and the alpha-amylase-binding N-terminal region were also found to be similar in the crystal structures of native RATI and its complex with alpha-amylase. These regions differed considerably in the NMR structure.

Amino Acid Sequence↗