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Query: UNIPROT:P06889 (Mol)
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Unfolding of the small alpha-amylase inhibitor tendamistat (74 residues, 2 disulfide bridges) has been characterized thermodynamically by high sensitivity scanning microcalorimetry. To link the stability parameters with structural information we use heat capacity group parameters and water accessible surface areas to calculate the change in heat capacity on unfolding of tendamistat. Our results show that both the group parameter and surface area approaches provide a reasonable, though not perfect, basis for delta Cp calculations. When using the experimentally determined temperature-independent heat capacity increase of 2.89 kJ mol-1 K-1 tendamistat exhibits convergence of thermodynamic parameters at about 140 degrees C, in agreement with recent predictions of the temperature at which the hydrophobic hydration is supposed to disappear. Despite the apparent support of this new view of the hydrophobic effect, there are inconsistencies in the interpretation of the thermodynamic parameters and these are addressed in the Discussion. The specific stability of tendamistat is similar to that of modified bovine pancreatic trypsin inhibitor, with only two of the native three disulfide bridges intact. This observation confirms our previous conclusion that disulfide bridges affect significantly the enthalpy and entropy of unfolding. The recent study by Doig & Williams provides additional convincing support for this conclusion. The predictive scheme proposed by these authors permits a fair estimate of the Gibbs free energy and enthalpy changes of these two proteins.
J Mol Biol 1992 Feb 05
PMID:Thermodynamics of unfolding of the alpha-amylase inhibitor tendamistat. Correlations between accessible surface area and heat capacity. 154 17

A method is described for obtaining transgenic plants with a high level of expression of the introduced gene. Tobacco protoplasts were transformed with an expression construct containing a translational fusion between mature alpha-amylase from Bacillus licheniformis and the signal peptide of the tobacco PR-S protein. A total number of 5200 transformed protoplasts was cultured to microcalli and screened for alpha-amylase expression by incubation on media containing starch followed by staining with iodine. The calli were divided into four classes, based on the resulting halo sizes on the plates. The halo sizes were found to correlate with the expression levels in transgenic plants regenerated from the calli. The expression levels varied between 0 and 0.5% of soluble leaf protein in the regenerated transgenic plants. Wider implications of this method are discussed.
Plant Mol Biol 1992 Apr
PMID:Direct screening for high-level expression of an introduced alpha-amylase gene in plants. 160 Jan 49

The Brassica napus cDNA clone A9 and the corresponding Arabidopsis thaliana gene have been sequenced. The B. napus cDNA and the A. thaliana gene encode proteins that are 73% identical and are predicted to be 10.3 kDa and 11.6 kDa in size respectively. Fusions of an RNase gene and the reporter gene beta-glucuronidase to the A. thaliana A9 promoter demonstrated that in tobacco the A9 promoter is active solely in tapetal cells. Promoter activity is first detectable in anthers prior to sporogenous cell meiosis and ceases during microspore premitotic interphase. The deduced A9 protein sequence has a pattern of cysteine residues that is present in a superfamily of seed plant proteins which contains seed storage proteins and several protease and alpha-amylase inhibitors.
Plant Mol Biol 1992 Jul
PMID:The isolation and characterisation of the tapetum-specific Arabidopsis thaliana A9 gene. 162 74

The pehA gene encoding an endopolygalacturonase (pectinase) of Erwinia carotovora subsp. carotovora has been cloned previously [Saarilahti et al., Mol. Microbiol. 4 (1990) 1037-1044]. We expressed pehA in Bacillus subtilis using a secretion vector based on the promoter and signal sequence of the alpha-amylase (Amy)-encoding gene, amyE, from Bacillus amyloliquefaciens. To test whether the location of the junction between the secretion vector and pehA affects the protein yield, we made four different junctions. Two constructs contained an intact Amy signal sequence, whereas the other two were fusions between the Amy signal sequence and the polygalacturonase (PG) signal sequence. There was approximately fourfold variation in the production efficiency of B. subtilis strains carrying the different constructs. The most efficient construct contained the N-terminal and hydrophobic regions of the Amy signal peptide joined to the C terminus of PG signal peptide. This construct produced, in a shake flask culture, 0.8 g of polygalacturonase per liter of growth medium. In a pulse-chase experiment, the signal peptide of the most efficient construct was rapidly cleaved while cleavage was slow in the other constructs. Our results suggest that fusions containing intact signal peptides, which are common when producing foreign proteins, are not necessarily the most efficient.
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PMID:Expression of the Erwinia carotovora polygalacturonase-encoding gene in Bacillus subtilis: role of signal peptide fusions on production of a heterologous protein. 162 41

Rice genomic clones containing eight different alpha-amylase genes have been previously classified into five groups based on DNA hybridization studies and restriction site mapping. This report describes the clustering of three Group 3 genes (RAmy3A, RAmy3B and RAmy3C) within 28 kb of genomic DNA. The genes are separated from each other by about 5 kb and transcribed in the same direction. At the protein level, RAmy3B and RAmy3C are 95% homologous while each is 78% homologous to RAmy3A. All three genes have relatively small introns in the first and third positions. RAmy3A; however, has an additional 409 bp intron in the second intron insertion site. Nucleotide sequence comparisons of the coding and 3' flanking regions suggest that clustering of the RAmy3 genes occurred by gene duplication resulting from unequal crossing-over at repetitive sequences. A comparison of the 5' flanking regions revealed several sequences that may be involved in transcription. Expression of RAmy3B/C first appears in the germinating seed after two days and at a higher level after four days. Quantitative primer extension analysis indicates that RAmy3B and RAmy3C contribute 25% and 75%, respectively, of the transcripts from this cluster at four days of germination. No primer extension band specific to RAmy3A transcripts could be detected at this time point. However, RAmy3A PCR products could be amplified from RNA isolated from embryo-derived callus tissue.
Plant Mol Biol 1991 Apr
PMID:Characterization of an alpha-amylase multigene cluster in rice. 171 18

A bifunctional alpha-amylase/trypsin inhibitor that has two binding sites has been purified from ragi. The inhibitor has been crystallized from its ammonium sulphate solution by the vapour diffusion method. The crystals belong to the orthogonal space group P2(1)2(1)2(1) with unit cell dimensions a = 30.49 A, b = 56.30 A, c = 73.65 A and Z = 4.
J Mol Biol 1991 Nov 05
PMID:Preliminary X-ray investigation of a bifunctional inhibitor from Indian finger millet (ragi). 171 18

The primary structure of the insect alpha-amylase inhibitor CMa of barley seeds was deduced from a full-length cDNA clone pc43F6. Analysis of RNA from barley endosperm shows high levels 15 and 20 days after flowering. The cDNA predicts an amino acid sequence of 119 residues preceded by a signal peptide of 25 amino acids. Ala and Leu account for 55% of the signal peptide. CMa is 60-85% identical with alpha-amylase inhibitors of wheat, but shows less than 50% identity to trypsin inhibitors of barley and wheat. The 10 Cys residues are located in identical positions compared to the cereal inhibitor family with a Pro-X-Cys motif present in all.
Plant Mol Biol 1992 Jan
PMID:Nucleotide sequence of a cDNA coding for the barley seed protein CMa: an inhibitor of insect alpha-amylase. 173 2

Five different mutations were introduced into the leader peptide region of the alpha-amylase gene of Streptomyces griseus IMRU 3570. A mutation which increased the positive charge of the N-terminal region of the leader peptide enhanced the secretion of alpha-amylase by two- to threefold. Replacement of the native promoter of the amylase gene by the promoter of the Tn5 neo gene or by the promoter of the saf gene resulted in a 16-fold increase in alpha-amylase secretion. The enhanced secretion of alpha-amylase obtained by using the most efficient promoters was due to a correlated increase in the amount of transcript formed. The translation and secretion processes in S. lividans are not a bottleneck for enzyme secretion even at very high transcription rates, since stimulation of transcription of the alpha-amylase gene results in a proportionate increase in secretion of the enzyme.
Mol Gen Genet 1991 Dec
PMID:Effects of replacement of promoters and modification of the leader peptide region of the amy gene of Streptomyces griseus on synthesis and secretion of alpha-amylase by Streptomyces lividans. 175 48

Agglutination of washed rabbit erythrocytes caused by pancreatic acinar cell extract was inhibited by glucose, maltose and cellobiose. Process of elimination and purification divulged that the acinar cell enzyme alpha-amylase was responsible for attributing the agglutinin activity. Assay of enzyme and agglutinin activity data of different fractions of re-purified alpha-amylase eluted from HPLC column showed that both the activity resides in the same fraction which suggests that the enzyme binds to the glucosyl residues of the rabbit erythrocytes via the carbohydrate binding/catalytic sites. Similar properties of other glycosidases were also noted.
Mol Cell Biochem 1991 Dec 11
PMID:Studies on the agglutinating activity of pancreatic extracts and its relevance to function. 177 63

Gibberellic acid (GA3) and abscisic acid (ABA) control the transcription of alpha-amylase genes in barley aleurone cells. This control is likely to be exerted through cis-acting hormone-responsive elements in the promoter region of the gene. In order to further define these elements, we have developed procedures for obtaining transient expression of chimaeric genes in protoplasts prepared from mature barley aleurone layers. Constructs with heterologous constitutive promoters and with heterologous and homologous GA3- and ABA-regulated promoters were expressed specifically by these cells. This system would appear to offer great potential in gene regulation studies especially for hormonally regulated homologous genes. Functional analysis of a barley alpha-amylase gene has been performed using this system. A 2050 bp fragment from a high-pI alpha-amylase gene was fused to a reporter gene (GUS) and control of its expression was examined. Deletion analysis of this promoter fragment showed that major GA- and ABA-responsive elements occurred between 174 and 41 bp upstream from the transcription initiation site.
Plant Mol Biol 1991 Apr
PMID:Control of transient expression of chimaeric genes by gibberellic acid and abscisic acid in protoplasts prepared from mature barley aleurone layers. 183 Oct 55


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