Post-translational processing of two alpha-amylase inhibitors and an arcelin from the common bean, Phaseolus vulgaris.
(9/1178)
Mass spectrometric methods were used to investigate the proteolytic processing and glycopeptide structures of three seed defensive proteins from Phaseolus vulgaris. The proteins were the alpha-amylase inhibitors alphaAI-1 and alphaAI-2 and arcelin-5, all of which are related to the seed lectins, PHA-E and PHA-L. The mass data showed that the proteolytic cleavage required for activation of the amylase inhibitors is followed by loss of the terminal Asn residue in alphaAI-1, and in all three proteins, seven or more residues were clipped from the C-termini, in the manner of the seed lectins. In most instances, individual glycoforms could be assigned at each Asn site, due to the unique masses of the plant glycopeptides. It was found that alphaAI-1 and alphaAI-2 differed significantly in their glycosylation patterns, despite their high sequence homology. These data complement the previous X-ray studies of the alpha1-amylase inhibitor and arcelin, where many of the C-terminal residues and glycopeptide residues could not be observed. (+info)
Tunicamycin-resistant mutants and chromosomal locations of mutational sites in Bacillus subtilis.
(10/1178)
The types of tunicamycin-resistant mutants of Bacillus subtilis were analyzed, and their mutational sites on the chromosome were mapped. A type 1 mutation that simultaneously expressed hyperproductivity of extracellular alpha-amylase was located close to amy E. Type 2 mutations were near aroI. (+info)
Antibody to glucosyltransferase induced by synthetic peptides associated with catalytic regions of alpha-amylases.
(11/1178)
We examined the immunogenicity and induction of inhibitory activity of 19-mer synthetic peptides which contained putative catalytic regions that were associated with the beta5 (EAW) and beta7 (HDS) strand elements of the suggested (beta,alpha)8 catalytic barrel domain of Streptococcus mutans glucosyltransferase (GTF). Both peptides readily induced serum immunoglobulin G (IgG) and salivary IgA antipeptide activity which was reactive both with the inciting peptide and with intact S. mutans GTF. Antisera to each peptide construct also inhibited the ability of S. mutans GTF to synthesize glucan. These observations support the existence of catalytic subdomains containing glutamate and tryptophan (EAW) or aspartate and histidine (HDS) residues, each of which have been suggested to be involved with the catalytic activity of GTF. Furthermore, the epitopes defined in these sequences have significant immunogenicity and can induce immune responses which interfere with GTF-mediated glucan synthesis. (+info)
A temperature-dependent switch from chaperone to protease in a widely conserved heat shock protein.
(12/1178)
Misfolding or unfolding of polypeptides can occur as a consequence of environmental stress and spontaneous mutation. The abundance of general chaperones and proteases suggests that cells distinguish between proteins that can be refolded and "hopeless" cases fated to enter the proteolytic pathway. The mechanisms controlling this key metabolic decision are not well understood. We show here that the widely conserved heat shock protein DegP (HtrA) has both general molecular chaperone and proteolytic activities. The chaperone function dominates at low temperatures, while the proteolytic activity is present at elevated temperatures. These results show that a single cellular factor can switch between two key pathways, controlling protein stability and turnover. Implications of this finding for intracellular protein metabolism are discussed. (+info)
Intestinal tumorigenesis in the Apc1638N mouse treated with aspirin and resistant starch for up to 5 months.
(13/1178)
The Apc1638N mouse model, which carries a targeted mutant allele within the adenomatous polyposis (Apc) gene and develops intestinal tumours spontaneously, predominantly in the small bowel, was used to investigate the effects of two potential chemopreventive agents, aspirin and alpha-amylase resistant starch (RS). Heterozygous Apc+/Apc1638N mice were fed semi-purified diets rich in animal fat, animal proteins and sucrose and low in dietary fibre (Western style diets) from approximately 6 weeks up to 6 months of age. Two of the diets contained aspirin (300 mg/kg diet) and two RS (1:1 mixture of raw potato starch: Hylon VII at 200 g/kg diet) in a 2 x 2 factorial design. A fifth treatment group were fed a conventional rodent chow diet. The mice fed the Western style diets became almost three times as fat as the chow-fed mice but this did not affect tumour yield. Treatment with RS resulted in significantly more intestinal tumours whereas aspirin alone had no effect. However, there was a significant aspirin x RS interaction, which suggests that aspirin could prevent the small intestine tumour-enhancing effects of RS in this Apc-driven tumorigenesis model. The possibility that large amounts of purified forms of resistant starch may have adverse effects within the small bowel is a novel observation that requires further investigation since greater intakes of starchy foods (and of RS) are being encouraged as a public health measure in compensation for reduced dietary fat intake. However, it remains possible that any increased risk is restricted to carriers of germline mutations in APC. (+info)
Study on genetic variation and DNA polymorphism for alpha-amylase gene of D. melanogaster in Korea.
(14/1178)
The restriction maps of the 20 kb Amy region of Drosophila melanogaster in Korean populations were surveyed from 27 isogenic second chromosome lines. A subset of these populations were also scored for isozyme type and adult enzyme activity of alpha-amylase. Among 34 kinds of restriction site variations that were observed in the 20 kb region surrounding the two transcriptional units of the duplicated Amy locus, nine were polymorphic (26.5%). The low frequencies at which each of the large insertions was found are consistent with earlier reports of variation in other loci. Restriction site variation in Korean populations provided an estimated heterozygosity per nucleotide pair of 0.007. Three pairwise comparisons showed relatively high levels of linkage disequilibria. The enzyme activity of the Chounan population showed no significant difference compared with that of the Pusan population, and the activity of heterozygotes was higher than that of Amy1. Lines bearing insertion/deletion exhibited relatively low enzyme activities. All lines examined showed the duplication of Amy transcriptional units that were highly conserved, and the manner of concerted evolution. (+info)
Coordinate transcriptional control in the hyperthermophilic archaeon Sulfolobus solfataricus.
(15/1178)
The existence of a global gene regulatory system in the hyperthermophilic archaeon Sulfolobus solfataricus is described. The system is responsive to carbon source quality and acts at the level of transcription to coordinate synthesis of three physically unlinked glycosyl hydrolases implicated in carbohydrate utilization. The specific activities of three enzymes, an alpha-glucosidase (malA), a beta-glycosidase (lacS), and an alpha-amylase, were reduced 4-, 20-, and 10-fold, respectively, in response to the addition of supplementary carbon sources to a minimal sucrose medium. Western blot analysis using anti-alpha-glucosidase and anti-beta-glycosidase antibodies indicated that reduced enzyme activities resulted exclusively from decreased enzyme levels. Northern blot analysis of malA and lacS mRNAs revealed that changes in enzyme abundance arose primarily from reductions in transcript concentrations. Culture conditions precipitating rapid changes in lacS gene expression were established to determine the response time of the regulatory system in vivo. Full induction occurred within a single generation whereas full repression occurred more slowly, requiring nearly 38 generations. Since lacS mRNA abundance changed much more rapidly in response to a nutrient down shift than to a nutrient up shift, transcript synthesis rather than degradation likely plays a role in the regulatory response. (+info)
Alcoholysis reactions from starch with alpha-amylases.
(16/1178)
The ability of alpha-amylases from different sources to carry out reactions of alcoholysis was studied using methanol as substrate. It was found that while the enzymes from Aspergillus niger and Aspergillus oryzae, two well-studied saccharifying amylases, are capable of alcoholysis reactions, the classical bacterial liquefying alpha-amylases from Bacillus licheniformis and Bacillus stearothermophilus are not. The effect of starch and methanol concentration, temperature and pH on the synthesis of glucosides with alpha-amylase from A. niger was studied. Although methanol may inactivate alpha-amylase, a 90% substrate relative conversion can be obtained in 20% methanol at a high starch concentration (15% w/v) due to a stabilizing effect of starch on the enzyme. As the products of alcoholysis are a series of methyl-oligosaccharides, from methyl-glucoside to methyl-hexomaltoside, alcoholysis was indirectly quantified by high performance liquid chromatography analysis of the total methyl-glucoside produced after the addition of glucoamylase to the alpha-amylase reaction products. More alcoholysis was obtained from intact soluble starch than with maltodextrins or pre-hydrolyzed starch. The biotechnological implications of using starch as substrate for the production of alkyl-glucosides is analyzed in the context of these results. (+info)