Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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A partial EcoRI fragment of Bacillus coagulans DNA cloned in an Escherichia coli K12 bacteriophage lambda host-vector system was shown to direct the synthesis of a thermostable alpha-amylase whose activity could be detected in situ on petri plates using the iodine staining method. A 3.31 kb EcoRI fragment containing the active gene with its own promoter was subcloned in pBR322; in the new clone, called pAMY2, the amylase was shown to accumulate in the periplasmic space. The molecular weight of the enzyme, confirmed by in vivo labelling of plasmid products in minicells, was estimated to be 60000. The restriction map of the plasmid was determined for five restriction enzymes and two new plasmids with smaller DNA inserts were constructed, both directing the synthesis of amylase; one of them with a 2.2 kb PstI insert was shown to be responsible for the synthesis of a fused beta-lactamase-alpha-amylase protein with amylase activity.
Mol Gen Genet 1982
PMID:Cloning and expression of a Bacillus coagulans amylase gene in Escherichia coli. 618 47

The degree of activation of rat parotid gland cyclic AMP-dependent protein kinase (EC 2.7.1.37) was measured in tissue minces in vitro in order to assess the involvement of this enzyme in the parotid stimulus-secretion coupling mechanism. Kinase activation, determined by the activity ratio method, was measurably increased by isoproterenol, a beta-adrenergic agonist and a potent stimulator of alpha-amylase (EC 3.2.1.1) secretion. Muscarinic cholinergic and alpha-adrenergic stimulation, less effective in releasing amylase, did not affect protein kinase activation. Kinase activation closely paralleled the cyclic AMP concentration when the concentration of isoproterenol was varied. Amylase release exhibited a similar isoproterenol dose-dependence, except that amylase release was measurably increased at an isoproterenol concentration slightly lower than that required to increase detectably the cyclic AMP concentration or kinase activation. Partial dissociation between cyclic AMP levels, kinase activation, and secretion was seen when submaximal beta-adrenergic stimulation was combined with submaximal and supramaximal cholinergic stimulation. These results suggest an involvement of cyclic AMP-dependent protein kinase in beta-adrenergic-stimulated amylase release, but show that the extent of secretion is not rigidly coupled to the extent of kinase activation as determined by the activity ratio method. Protein kinase activation may function in concert with other factors in the regulation of exocytosis in this tissue.
Mol Pharmacol 1982 Jan
PMID:Rat parotid gland protein kinase activation. Relationship to enzyme secretion. 618 52

A 6.4 Kb HindIII fragment of Bacillus stearothermophilus DY-5 DNA cloned in Escherichia coli using pBR322 as a vector was shown to direct the synthesis of a thermophilic alpha-amylase. In attempts to reduce the size of the insert, the alpha-amylase gene was shown to be contained in a 3.1 Kb HindIII - BamHI fragment of the donor strain DNA. The alpha-amylase gene was stably maintained and expressed efficiently in E. coli. The enzymic properties of alpha-amylase produced in E. coli closely resembled those of the donor strain alpha-amylase and the temperature range for the maximal activity was from 65 degrees C to 80 degrees C. Nearly 100% of the activity remained after heating at 80 degrees C for 15 min. The alpha-amylase was shown to be accumulated in the periplasmic space. It was purified to a nearly homogenous protein with a molecular weight of 61,000, which was very similar in size to that produced by B. stearothermophilus DY-5.
Mol Gen Genet 1984
PMID:Cloning and expression of a thermophilic alpha-amylase gene from Bacillus stearothermophilus in Escherichia coli. 631 51

Several alpha-amylase clones were identified by screening a group of 345 cDNA clones derived from poly(A)+RNA from gibberellic acid (GA)-treated barley aleurone layer cells using an alpha-amylase cDNA clone that we had characterized previously as the hybridization probe. The alpha-amylase clones showed differences in the distribution of restriction enzyme sites, in the extent of cross hybridization, and in nucleotide sequence. There are at least three types of alpha-amylase clones in our collection, and the alpha-amylase cDNA clone reported by Rogers and Milliman [J. Biol. Chem. (1983) 258: 8169-8174] is a fourth type. The accumulation of alpha-amylase mRNAs in aleurones as a function of time after GA addition and of hormone concentration in the incubation medium was studied using different alpha-amylase cDNA clone probes. These studies indicate a differential control of the expression of the alpha-amylase genes by GA.
J Mol Appl Genet 1984
PMID:Expression and regulation of alpha-amylase gene family in barley aleurones. 633 20

We report the nucleotide sequence of mRNA for the common electrophoretic isozyme of human pancreatic alpha-amylase, Amy2 A. The sequence was derived from a nearly full-length complementary DNA (cDNA) isolated from a cloned cDNA library. The relatively short 5' untranslated region (15 nucleotides) was determined by primer-extension sequencing. The human Amy2 messenger codes for a 511-residue preamylase polypeptide. An amino-terminal signal peptide of 15 amino acids with an Ala X Gln cleavage site is proposed based on homology to mouse, dog and hog amylases. The Amy2 A mRNA sequence differs from a recently reported human Amy2 sequence. Differences were found at 31 nucleotide positions. The alpha-amylase proteins predicted by the two mRNAs differ at 17 amino acid positions. Relative to the known sequences of other mammalian amylases, most of the differences between the two human Amy2 sequences appear to have occurred as substitutions in the sequence reported by Nakamura et al. (1984). These substitutions predict a protein with a substantially greater net negative charge than that of Amy2 A. We suggest that the two sequences may represent either divergent Amy2 alleles or the expression of non-allelic pancreatic amylase genes.
Mol Biol Med 1984 Oct
PMID:A complementary DNA sequence that predicts a human pancreatic amylase primary structure consistent with the electrophoretic mobility of the common isozyme, Amy2 A. 633 37

The author found, in rat liver nuclei, a novel factor which exhibited a strong inhibitory effect on RNA chain initiation by various classes of RNA polymerases (I, II and III) using an exogenous DNA template. The molecular weight of this factor was estimated to be 70 K daltons, and its activity was not affected by treatment with trypsin, RNase A, lipase C, alpha-amylase and heat. However, its activity was inactivated by a digestion of glycosidases. The molecule is shown to contain a considerable amount of sugars by physicochemical analysis. In addition, it is elucidated that the factor is not heparin which has a similar biological activity.
Mol Biol Rep 1984 Jan
PMID:A novel factor for DNA-dependent RNA polymerase II in rat liver nuclei. 670 49

A new method for automatically assigning proton-proton NOESY spectra is described and demonstrated for simulated and experimental spectra of the proteins dendrotoxin K, alpha-amylase inhibitor tendamistat and the DNA-binding domain of the 434 repressor protein. The method assigns the NOESY spectrum and calculates three-dimensional protein structures simultaneously, using a list of proton chemical shifts and 3JNH alpha coupling constants. An ensemble of structures is iteratively calculated by self-correcting distance geometry from unambiguous and selected ambiguous NOESY cross peaks. New structure based filters recognize the correct constraints from the ambiguous cross peak list. For the first round of assignment neither a preliminary initial structure nor a sufficient set of unambiguous NOESY cross peaks is needed. The method can also be applied to cross peak lists containing hundreds of noise peaks. For an assumed tolerance of +/- 0.01 ppm in the chemical shifts of the peak positions, only about 10% of the NOESY cross peaks can be unambiguously assigned based on their chemical shifts alone. Our automated method assigned about 80% of all cross peaks with this chemical shift tolerance, and 95 to 99% of the assignments were correct. The average pairwise RMSD for the backbone atoms of the ten best final structures is about 1.5 A in all three proteins and the previously determined NMR solution structures are always embedded in this structure bundle. We regard our method as a highly practical tool for automatic calculation of three-dimensional protein structures from NMR spectra with minimal human interference.
J Mol Biol 1995 Dec 01
PMID:Automated assignment of simulated and experimental NOESY spectra of proteins by feedback filtering and self-correcting distance geometry. 749 Jul 63

The present differential scanning calorimetry and circular dichroism studies on the mechanism of protein stabilization by disulfide bonds were concerned with two questions: is the increase in unfolding entropy upon removal of disulfide links sufficient for the explantation of the general stability decrease of disulfide-deficient mutants? Is it immaterial by which residue cysteine residues are replaced when disulfide bridges are to be opened? To answer these questions we investigated two disulfide bridge mutants of the alpha-amylase inhibitor Tendamistat where the large loop (C45A/C73A) or the small loop (C11A/C27A) had been opened by recombinant DNA techniques, and we compared the stability of the mutated proteins with that of wild-type Tendamistat published previously. To elucidate the significance of the nature of the group that replaces Cys we introduced in position 27 of the small loop four different amino acids instead of Cys: Ala, Leu, Ser and Thr. Surprisingly, opening of the small loop (17 residues) causes larger destabilization than opening of the large loop comprising 29 residues. The thermodynamic parameters at pH 7.0 are: wild-type: t1/2 = 81.6 degrees C, delta Hcal = 296 kJ mol-1, large loop mutant (C45A/C73A): t1/2 = 58.6 degrees C, delta Hcal = 225 kJ mol-1 and small loop mutant (C11A/C27A): t1/2 = 42.7 degrees C, delta Hcal = 135 kJ mol-1. This finding is at variance with the entropy hypothesis. The relative contributions to stability of enthalpic and entropic terms can be varied by a proper choice of substitutions. While the destabilization originating from C45A/C73A exchanges in the large loop turns out to be purely entropic, the stability decreases of the small loop mutants are caused by changes in both enthalpic and entropic terms. Leu or Ser in position 27 leads to an overall enthalpic destabilization. Thr in position 27 increases the transition enthalpy of this mutant to the value of the wild-type protein but increases at the same time the value of the transition entropy with the result of an overall entropic destabilization. Finally, in the C11A/C27A small loop mutant of lowest stability a very large enthalpic destabilization occurs, which is, however, partly counterbalanced by a reduction in the transition entropy. The preferential perturbation of the native state by the mutations is manifest in the increase of the native state heat capacity relative to that of the wild-type protein and the identity of the heat capacity of the unfolded state.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1995 Dec 01
PMID:Mechanism of protein stabilization by disulfide bridges: calorimetric unfolding studies on disulfide-deficient mutants of the alpha-amylase inhibitor tendamistat. 749 Jul 64

The promoter (amyP) of the Bacillus subtilis alpha-amylase gene, which is recognized by E sigma A, has a three out of six match to the consensus promoter in both the -35 and -10 hexamers. Oligonucleotide-directed mutagenesis was used to identify important bases for promoter utilization in the spacer sequence between the hexamers. Mutations in the sequence TGTG extending from positions -18 to -15 (the -16 region) caused a 5-94-fold decrease in alpha-amylase production. A G-C transversion at position -15 was the most detrimental mutation: it essentially eliminated amyP utilization in B. subtilis and in Escherichia coli. Mutating the -35 and -10 hexamers to the E sigma A consensus promoter increased alpha-amylase production 56-fold in B. subtilis and fivefold in E. coli. Introducing the -15 G to C transversion into the consensus promoter reduced alpha-amylase production threefold, in contrast to the 94-fold reduction for the wild-type promoter in B. subtilis. The -15 G to C transversion did not reduce alpha-amylase synthesis directed by the consensus promoter in E. coli. The alpha-amylase gene is subject to two forms of transcriptional regulation: catabolite repression and temporal regulation. None of the mutants constructed in this study had any effect on either type of regulation. The -16 region, especially the G at position -15, appears to be moderately conserved in B. subtilis and in other Gram-positive organisms and weakly conserved in E. coli. The evidence suggests that the -16 region is an additional region of E sigma A promoters in B. subtilis and E sigma 70 promoters in E. coli, essential in some weak promoters such as the alpha-amylase promoter but, of little benefit to very strong promoters.
Mol Microbiol 1995 Jul
PMID:The -16 region, a vital sequence for the utilization of a promoter in Bacillus subtilis and Escherichia coli. 749 76

The alpha-amylase inhibitor Tendamistat (Hoe-467), a 74 amino acid beta-sheet protein from Streptomyces tendae has been expressed on the surface of the filamentous bacteriophage M13. Phage displaying Tendamistat inhibit the hydrolysis of starch by alpha-amylase, indicating that the displayed protein is functional. The displayed Tendamistat has been used as a molecular scaffold for the presentation of constrained random peptides. Two loops, comprising residues 38 to 40 and 60 to 65 of Tendamistat, were randomized using PCR mutagenesis. Libraries of approximately 10(8) different mutant Tendamistat molecules were tested for binding to monoclonal antibody A8, which recognizes endothelin. After three cycles of biopanning, phage were isolated that specifically bound the monoclonal antibody. Loop swapping and alanine replacement mutagenesis indicated that residues in the 60 to 65 loop are responsible for binding to the monoclonal antibody. This work demonstrates the use of relatively small non-antibody protein scaffolds for the presentation of constrained random peptide sequences to select for novel binding molecules.
J Mol Biol 1995 Jul 21
PMID:Tendamistat as a scaffold for conformationally constrained phage peptide libraries. 754 49


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