Sugar sensing and alpha-amylase gene repression in rice embryos

We used a transient expression system to study the mechanism by which carbohydrates repress a rice (Oryza sativa L.) alpha-amylase (EC 3.2.1.1) gene. Exogenously fed metabolizable carbohydrates are able to elicit repression of the alpha-amylase gene RAmy3D in the rice embryo, and our results indicate that repression is also triggered efficiently by endogenous carbohydrates. Glucose analogs that are taken up by plant cells but not phosphorylated by hexokinase are unable to repress the alpha-amylase gene studied, while 2-deoxyglucose, which is phosphorylable but not further metabolized, down-regulates RAmy3D promoter activity, indicating a role for hexokinase in the sugar-sensing mechanism triggering repression of the RAmy3D gene. We tested two different hexokinase inhibitors, mannoheptulose and glucosamine, but only the latter was able to relieve RAmy3D promoter activity from repression by endogenous carbohydrates. This correlates with the higher ability of glucosamine to inhibit the activity of rice hexokinases in vitro. The glucosamine-mediated relief of RAmy3D promoter activity from repression by endogenous carbohydrates does not correlate with a reduced rate of carbohydrate utilization.     

Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme

The gene encoding the Pyrococcus furiosus hyperthermophilic amylopullulanase (APU) was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a single 827-residue polypeptide with a 26-residue signal peptide. The protein sequence had very low homology (17 to 21% identity) with other APUs and enzymes of the alpha-amylase family. In particular, none of the consensus regions present in the alpha-amylase family could be identified. P. furiosus APU showed similarity to three proteins, including the P. furiosus intracellular alpha-amylase and Dictyoglomus thermophilum alpha-amylase A. The mature protein had a molecular weight of 89,000. The recombinant P. furiosus APU remained folded after denaturation at temperatures of < or = 70 degrees C and showed an apparent molecular weight of 50,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Denaturating temperatures of above 100 degrees C were required for complete unfolding. The enzyme was extremely thermostable, with an optimal activity at 105 degrees C and pH 5.5. Ca2+ increased the enzyme activity, thermostability, and substrate affinity. The enzyme was highly resistant to chemical denaturing reagents, and its activity increased up to twofold in the presence of surfactants.     

Acidostable and acidophilic proteins: the example of the alpha-amylase from Alicyclobacillus acidocaldarius

Acidophilic microorganisms grow optimally at pH values between 1-4. They have adapted to the acid condition by maintaining their cytoplasmic pH at a value close to neutrality. Hence, only those (macro)-molecules, which face the acid medium, have had to adapt to this extreme condition. Literature data show that several exoproteins from thermoacidophilic prokaryotes are characterized by a low charge density. It is proposed that this property contributes to the stability of these proteins both below and above the pKa-values of their glutamate and aspartate residues. As an example of an acidophilic protein, the alpha-amylase from the Gram-positive Alicyclobacillus acidocaldarius ATCC27009 was studied. The enzyme is thermoacidophilic, with optima of temperature and pH of 75 degrees C and pH 3, respectively. The nucleotide sequence of the cloned gene (8) indicates that the alpha-amylase belongs to a large family of starch-degrading enzymes with a characteristic catalytic (beta alpha)8-domain. Three essential and probably catalytic acidic residues have been conserved, suggesting that the acidophilic alpha-amylase degrades starch with essentially the same mechanism as do its neutrophilic relatives. Still, the acidophilic protein contains three exchanges in residues uniformally or almost uniformally conserved among all members of the enzyme family. In order to test whether these exchanges contribute to the acidic pH optimum, the alpha-amylase gene was expressed in Escherichia coli. Sonication of the enzyme-producing cells released alpha-amylase activity associated with a 140 kDa protein. The optima of temperature and pH for the protein produced in E. coli were similar to those of the native enzyme. Experiments are underway in which it is tested which residues contribute to the acid pH optimum of the alpha-amylase.     

alpha-Amylase as a marker for evaluating the stability of recombinant yeast in long-term cultivation

We describe here a simple method for analyzing the stability of recombinant yeast. The mouse salivary alpha-amylase gene was used in this system as a marker since the stability of recombinant yeast can be detected easily by a halo zone-for-ming assay on starch-supplemented plates. We used this method to evaluate the stability of recombinant yeast harbouring a 2 mu directed episomal plasmid and of yeast harbouring the r-DNA directed integrative vector. Our results demonstrated clearly that the alpha-amylase gene was a convenient and reliable marker for evaluating the stability of recombinant yeasts in long-term cultivation.     

Expression of the Streptomyces griseus alpha-amylase gene in Escherichia coli

The amy gene of Streptomyces griseus was not expressed in Escherichia coli cells due to the lack of recognition of the amy promoter by the E. coli RNA polymerase, as confirmed by using promoter-probe vectors. The expression of the amy gene in E. coli was detected only when the promoter-less gene was placed under the control of the lacZ promoter and was dependent on the level of IPTG added to the medium. The extracellular alpha-amylase detected in the culture broth seems to be released by cellular lysis. When the amy gene lacking both leader peptide and promoter was transcribed from the lacZ promoter, no alpha-amylase activity was detected but larger E. coli cells and inclusion bodies were observed.     

Metabolic regulation of alpha-amylase gene expression in transgenic cell cultures of rice (Oryza sativa L.)

Expression of two genes in the alpha-amylase gene family is controlled by metabolic regulation in rice cultured cells. The levels of RAmy3D and RAmy3E mRNAs in rice cultured cells are inversely related to the concentration of sugar in the culture medium. Other genes in the rice alpha-amylase gene family have little or no expression in cultured cells; these expression levels are not controlled by metabolic regulation. A RAmy3D promoter/GUS gene fusion was metabolically regulated in the transgenic rice cell line 3DG, just as the endogenous RAmy3D gene is regulated. An assay of GUS enzyme activity in 3DG cells demonstrated that RAmy3D/GUS expression is repressed when sugar is present in the culture medium and induced when sugar is removed from the medium. The 942 bp fragment of the RAmy3D promoter that was linked to the coding region of the GUS reporter gene thus contains all of the regulatory sequences necessary for metabolic regulation of the gene.     

The impact of female caregivers'' employment status on patterns of formal and informal eldercare

Expression of alpha-amylase genes during seedling development plays a key role in production of sugar from the starch stored in the cereal seed. Rice alpha-amylase Amy3D promoter/GUS constructs in transgenic rice cell lines were studied to identify cis elements in the promoter of this metabolite-regulated gene. Three sequences having the greatest effects on Amy3D gene expression included the amylase element (TATCCAT), the CGACG element, and a G box-related element (CTACGTGGCCA). These promoter cis elements are needed for high-level expression of Amy3D under conditions of sugar starvation. The involvement of G box cis-elements in environmental stress responses suggest a link between the nutrient stress and the environmental stress responses of the plant.     

Significance of HPr in catabolite repression of alpha-amylase

CcpA and HPr are presently the only two proteins implicated in Bacillus subtilis global carbon source catabolite repression, and the ptsH1 mutation in the gene for the HPr protein was reported to relieve catabolite repression of several genes. However, alpha-amylase synthesis by B. subtilis SA003 containing the ptsH1 mutation was repressed by glucose. Our results suggest HPr(Ser-P) may be involved in but is not required for catabolite repression of alpha-amylase, indicating that HPr(Ser-P) is not the sole signaling molecule for CcpA-mediated catabolite repression in B. subtilis.     

pBRINT-Ts: a plasmid family with a temperature-sensitive replicon, designed for chromosomal integration into the lacZ gene of Escherichia coli

A pBRINT-Ts family of integrative vectors for Escherichia coli was constructed by using a temperature-sensitive replicon derived from pSC101, a region of homology to the lacZ gene, and various antibiotic resistance markers (kanamycin, chloramphenicol and gentamycin) for selection of the integrants. The gene or group of genes to be integrated can be inserted into a multiple cloning site, flanked by an antibiotic resistance marker and lacZ sequences. A simple and rapid procedure was developed for the selection of cells where allelic exchange had occurred. With this procedure, the efficiency of integration of around 10-3 was observed, using several E. coli strains. From colonies with an integrated pBRINT-Ts plasmid, we detected an average allelic exchange event frequency of 7.5%. As a test for this system, we integrated the amy gene that codes for the alpha-amylase from B. stearothermophilus, into the lacZ gene of E. coli W3110. Production of alpha-amylase was found to be proportional to copy number; at up to 10 copies per chromosome.     

Characterization of a novel alpha-amylase from Lipomyces kononenkoae and expression of its gene (LKA1) in Saccharomyces cerevisiae

A highly active alpha-amylase (76,250 Da) secreted by the raw starch-degrading yeast Lipomyces kononenkoae strain IGC4052B was purified and characterized. Using high performance liquid chromatography (HPLC), end-product analysis indicated that the L. kononenkoae alpha-amylase acted by endo-hydrolysis on glucose polymers containing alpha-1,4 and alpha-1,6 bonds, producing mainly maltose, maltotriose and maltotetraose. The following NH2-terminal amino acids were determined for the purified enzyme: Asp-Cys-Thr-Thr-Val-Thr-Val-Leu-Ser-Ser-Pro- Glu-Ser-Val-Thr-Gly. The L. kononenkoae alpha-amylase-encoding gene (LKA1), previously cloned as a cDNA fragment, was expressed in Saccharomyces cerevisiae under the control of the PGK1 promoter. The native signal sequence efficiently directed the secretion of the glycosylated protein in S. cerevisiae. De-glycosylation of the enzyme indicated that post-translational glycosylation is different in S. cerevisiae from that in L. kononenkoae. Zymogram analysis indicated that glycosylation of the protein in S. cerevisiae had a negative effect on enzyme activity. Southern-blot analysis revealed that there is only a single LKA1 gene present in the genome of L. kononenkoae.     

Rice bifunctional alpha-amylase/subtilisin inhibitor: characterization, localization, and changes in developing and germinating seeds

A bifunctional alpha-amylase/subtilisin inhibitor (RASI) was purified to electrophoretic homogeneity from rice (Oryza sativa L.) bran. Its molecular mass was 21 kDa by SDS-PAGE and its isoelectric point was 9.05. Purified RASI inhibited subtilisin Carlsberg strongly and inhibited alpha-amylase from germinating rice seeds weakly. It inhibited rice alpha-amylase more than barley alpha-amylase, and the inhibition of rice alpha-amylase was greater at higher pHs. RASI did not inhibit trypsin, chymotrypsin, cucumisin, or mammalian alpha-amylase. The RASI was in the outermost part of the rice grain and its subcellular site seemed to be aleurone particles in aleurone cells. SDS-PAGE and western blotting showed that RASI was synthesized in the late milky stage in developing seeds, and it remained fairly constant during the first 7 days of germination.     

alpha-Amylases from Thermoactinomyces vulgaris: characteristics, primary structure and structure prediction

Two amylolytic active protein fractions (named alpha-amylase 1 and alpha-amylase 2) were isolated from the bacterium Thermoactinomyces vulgaris strain 94-2A. alpha-Amylase 1 had a molecular mass of 51.6 kDa, whereas alpha-amylase 2 consists of two fragments which have molecular masses of 17.0 and 34.6 kDa, respectively. These two fragments are products from a proteolytic cleavage of alpha-amylase 1 at amino acid position 303 (tryptophan) by a serine protease (thermitase) which is also produced by T. vulgaris. The purified alpha-amylase 1 and 2 follow the Michaelis-Menten kinetics in the presence of starch as substrate with Km values of 1.37 +/- 0.07 and 1.29 +/- 0.18 mg/mL, respectively. In effect they differ in their stability characteristics. The amino acid sequence of alpha-amylase from T. vulgaris derived from DNA sequence (1) was compared with those of other alpha-amylases. It reveals high homologies to alpha-amylases from other microorganisms (e.g. B. polymyxa, A. oryzae, S. occidentalis and S. fibuligera). A three-dimensional structure model for alpha-amylase 1 on the basis of the 3 A X-ray structure of Taka-amylase was constructed.     

Physicochemical and serological characterization of rice alpha-amylase isoforms and identification of their corresponding genes

We have identified, purified, and characterized 10 alpha-amylase isoforms from suspension-cultured rice (Oryza sativa L.) cells having different isoelectric point values. They had distinguishable optimum temperatures for enzymatic activity and molecular sizes. The results of immunoblotting indicated that polyclonal anti-A + B antibodies bound well to isoforms A, B, Y, and Z but weakly or not at all to E, F, G, H, I, and J. However, the anti-A + B antibodies inhibited the enzyme activities of only isoforms A and B. Polyclonal anti-H antibodies strongly bound to isoforms F, G, H, I, and J, whereas polyclonal anti-E antibodies preferentially recognized isoform E. A monoclonal antibody against isoform H (H-G49) inhibited the activities of isoforms E, G, H, I, and J, whereas it did not inhibit those of isoforms A, B, Y, and Z. Judging from their physicochemical and serological properties, we classified the rice alpha-amylase isoforms into two major classes, class I (A, B, Y, and Z) and class II (E, F, G, H, I, and J), and into four subgroups, group 1 (A and B), group 2 (Y and Z), group 3 (E), and group 4 (F, G, H, I, and J). Partial amino acid sequences for isoforms A, E, G, and H were also determined. In addition, the recombinant alpha-amylases expressed by plasmid pEno/103 containing the rice alpha-amylase gene RAmy1A in yeast were identified as both isoforms A and B. These analyses indicated that isoforms A and B were encoded by the gene RAmy1A, isoforms G and H were encoded by the gene RAmy3D, and isoform E was encoded by RAmy3E. The results strongly suggest that some isoforms within subgroups are formed by posttranslational modifications.     

Salivary alpha-amylase as a measure of endogenous adrenergic activity

This investigation was designed to evaluate the production rates and concentrations of salivary alpha-amylase as a measure of adrenergic activity under several conditions of stress in human subjects. Saliva and blood samples were simultaneously collected from men at four 15 min intervals both before and after regimens for exercise, a written examination, or a rest period. The regressions of salivary alpha-amylase on plasma norepinephrine (NE) concentrations were significant for both exercise (P < 0.001) and examination (P < 0.01) protocols. Aerobic exercise induced a 3-fold mean increase in alpha-amylase; both NE and epinephrine (EP) increased approximately 5-fold over control levels. Levels of alpha-amylase and NE returned to control levels within 30-45 min after exercise, but EP remained elevated by approximately 2-fold during the remaining hour of observation. During the written examination, alpha-amylase and NE, but not EP, concentrations increased in parallel. In further studies the effects of exercise and exposure to heat and cold on the relationship of salivary alpha-amylase to heart rate and body temperature were investigated. Greater intensities of exercise were associated with greater increases in alpha-amylase concentrations. During heat exposure in a sauna (66 degrees C for 40 min) amylase, heart rate and body temperature all increased progressively. However, during exposure to cold (4 degrees C for 40 min) amylase increased rapidly, though heart rate and body temperature remained unchanged. Salivary cortisol concentrations were unchanged during exposure to heat or cold. We conclude that salivary alpha-amylase concentrations are predictive of plasma catecholamine levels, particularly NE, under a variety of stressful conditions, and may be a more direct and simple end point of catecholamine activity than are changes in heart rate.