N-Linked glycosylation and sialylation of the acid-labile subunit. Role in complex formation with insulin-like growth factor (IGF)-binding protein-3 and the IGFs.
Over 75% of the circulating insulin-like growth factors (IGF-I and -II) are bound in 140-kDa ternary complexes with IGF-binding protein-3 (IGFBP-3) and the 84-86-kDa acid-labile subunit (ALS), a glycoprotein containing 20 kDa of carbohydrate. The ternary complexes regulate IGF availability to the tissues. Since interactions of glycoproteins can be influenced by their glycan moieties, this study aimed to determine the role of ALS glycosylation in ternary complex formation. Complete deglycosylation abolished the ability of ALS to associate with IGFBP-3. To examine this further, seven recombinant ALS mutants each lacking one of the seven glycan attachment sites were expressed in CHO cells. All the mutants bound IGFBP-3, demonstrating that this interaction is not dependent on any single glycan chain. Enzymatic desialylation of ALS caused a shift in isoelectric point from 4.5 toward 7, demonstrating a substantial contribution of anionic charge by sialic acid. Ionic interactions are known to be involved in the association between ALS and IGFBP-3. Desialylation reduced the affinity of ALS for IGFBP-3. IGF complexes by 50-80%. Since serum protein glycosylation is often modified in disease states, the dependence of IGF ternary complex formation on the glycosylation state of ALS suggests a novel mechanism for regulation of IGF bioavailability. (+info)
The structure of a glycopeptide (GP-II) isolated from Rhizopus saccharogenic amylase.
Mild alkaline treatment of glycopeptide (GP-II) resulted in the loss of 1 mole of serine and 5 moles of threonine per mole of GP-II, suggesting the presence of O-glycosyl bonds between 1 serine and 5 threonine residues and carbohydrate chains. Treatment of GP-II with alkaline borohydride released only disaccharide. Methylation studies of the carbohydrate moiety gave 2,3,4,6-tetra-O-methyl and 2,4,6-tri-O-methyl derivatives of mannose in a ratio of approximately 1:1. In addition, one step of Smith degradation resulted in the loss of about 6 residues of mannose per mole of GP-II. Moreover, alpha-mannosidase [EC 22.214.171.124] liberated about 6 residles of mannose per mole of GP-II. On the basis of these data, the structure of the carbohydrate moiety of GP-II was confirmed to be 3-O-alpha-mannosylmannose. The amino- and carboxyl-terminal amino acids of GP-II were determined to be threonine and serine, respectively. On reductive cleavage of N-proline bonds with metallic sodium in liquid ammonia, 2 moles of alanine per mole of GP-II were lost. From the compositions of three fragments isolated from the reductive cleavage products, the amino acid sequence of the peptide portion of GP-II was determined. Based on these data, a probable structure was proposed for GP-II. (+info)
Relationship between glycosyl hydrolase inventory and growth physiology of the hyperthermophile Pyrococcus furiosus on carbohydrate-based media.
Utilization of a range of carbohydrates for growth by the hyperthermophile Pyrococcus furiosus was investigated by examining the spectrum of glycosyl hydrolases produced by this microorganism and the thermal labilities of various saccharides. Previously, P. furiosus had been found to grow in batch cultures on several alpha-linked carbohydrates and cellobiose but not on glucose or other beta-linked sugars. Although P. furiosus was not able to grow on any nonglucan carbohydrate or any form of cellulose in this study (growth on oat spelt arabinoxylan was attributed to glucan contamination of this substrate), significant growth at 98 degrees C occurred on beta-1,3- and beta-1,3-beta-1,4-linked glucans. Oligosaccharides generated by digestion with a recombinant laminarinase derived from P. furiosus were the compounds that were most effective in stimulating growth of the microorganism. In several cases, periodic addition of beta-glucan substrates to fed-batch cultures limited adverse thermochemical modifications of the carbohydrates (i.e., Maillard reactions and caramelization) and led to significant increases (as much as two- to threefold) in the cell yields. While glucose had only a marginally positive effect on growth in batch culture, the final cell densities nearly tripled when glucose was added by the fed-batch procedure. Nonenzymatic browning reactions were found to be significant at 98 degrees C for saccharides with degrees of polymerization (DP) ranging from 1 to 6; glucose was the most labile compound on a mass basis and the least labile compound on a molar basis. This suggests that for DP of 2 or greater protection of the nonreducing monosaccharide component may be a factor in substrate availability. For P. furiosus, carbohydrate utilization patterns were found to reflect the distribution of the glycosyl hydrolases which are known to be produced by this microorganism. (+info)
Rapid identification of Actinomycetaceae and related bacteria.
Identification of new isolates belonging to the family Actinomycetaceae requires extensive numbers of biochemical tests, supplemented with gas-liquid chromatography determination of fermentation end products and, often, analysis of cell wall composition. This paper describes the results of the testing of 162 strains of Actinomycetaceae and related taxa for 20 different enzymatic activities including phosphatases, esterases, aminopeptidases, and glycosidases. The results of all tests were read after 4 h of incubation. The results obtained in the study provide significant new information on the biochemical properties of these groups of bacteria. An identification scheme based upon 13 selected tests, which allow the identification of these groups of bacteria within 4 h, is proposed. (+info)
Purification and characterization of Aspergillus ficuum endoinulinase.
Endoinulinase from Aspergillus ficuum, which catalyzes the hydrolysis of inulin via an endo-cleavage mode, was purified by chromatography from Novozym 230 as a starting commercial enzyme mixture on CM-Sephadex and DEAE-Sepharose, and by preparative electrophoresis under native conditions. The enzyme was estimated to be pure on the basis of its I/S ratio, whose value was infinite in our assay conditions. Two forms separated by using this method. SDS gel electrophoresis showed the two purified forms to respectively exhibit molecular weights of 64,000 +/- 500 and 66,000 +/- 1,000. The results of deglycosylation indicated that the two forms were originally the same protein but with different sugar contents. A molecular weight of 54,800 +/- 1,500 was found by gel filtration of the native enzyme, indicating the native functional protein to be a monomer. The enzyme showed nearly absolute substrate specificity towards inulin and inulooligosaccharides, and acted via an endo-attack to produce mainly inulotriose during the late stage of the reaction. The apparent Km and Vmax values for inulin hydrolysis were 8.1 +/- 1.0 mM and 773 +/- 60 U/mg, respectively. The internal peptides of the enzyme showed sequence homology to the endoinulinase of Penicillium purpurogenum. (+info)
Insertion analysis of putative functional elements in the promoter region of the Aspergillus oryzae Taka-amylase A gene (amyB) using a heterologous Aspergillus nidulans amdS-lacZ fusion gene system.
Expression of the Taka-amylase A gene (amyB) of Aspergillus oryzae is induced by starch or maltose. The A. oryzae amyB gene promoter contains three highly conserved sequences, designated Regions I, II, and III, compared with promoter regions of the A. oryzae glaA encoding glucoamylase and the agdA encoding alpha-glucosidase. To identify the function of these sequences within the amyB promoter, various fragments containing conserved sequences in the amyB promoter were introduced into the upstream region of the heterologous A. nidulans amdS gene (encoding acetamidase) fused to the Escherichia coli lacZ gene as a reporter. Introduction of the sequence between -290 to -233 (the number indicates the distance in base pairs from the translation initiation point (+1)) containing Region III significantly increased the expression of the lacZ reporter gene in the presence of maltose. The sequence between -377 to -290 containing Region I also increased the lacZ activity, but its maltose inducibility was less than that of Region III. The sequence between -233 to -181 containing Region II had no effect on the expression. These results indicated that Region III is most likely involved in the maltose induction of the amyB gene expression. (+info)
A single limit dextrinase gene is expressed both in the developing endosperm and in germinated grains of barley.
The single gene encoding limit dextrinase (pullulan 6-glucanohydrolase; EC 126.96.36.199) in barley (Hordeum vulgare) has 26 introns that range in size from 93 to 822 base pairs. The mature polypeptide encoded by the gene has 884 amino acid residues and a calculated molecular mass of 97,417 D. Limit dextrinase mRNA is abundant in gibberellic acid-treated aleurone layers and in germinated grain. Gibberellic acid response elements were found in the promoter region of the gene. These observations suggest that the enzyme participates in starch hydrolysis during endosperm mobilization in germinated grain. The mRNA encoding the enzyme is present at lower levels in the developing endosperm of immature grain, a location consistent with a role for limit dextrinase in starch synthesis. Enzyme activity was also detected in developing grain. The limit dextrinase has a presequence typical of transit peptides that target nascent polypeptides to amyloplasts, but this would not be expected to direct secretion of the mature enzyme from aleurone cells in germinated grain. It remains to be discovered how the enzyme is released from the aleurone and whether another enzyme, possibly of the isoamylase group, might be equally important for starch hydrolysis in germinated grain. (+info)
Characterization of the divergent sacBK and sacAR operons, involved in sucrose utilization by Lactococcus lactis.
The divergently transcribed sacBK and sacAR operons, which are involved in the utilization of sucrose by Lactococcus lactis NZ9800, were examined by transcriptional and gene inactivation studies. Northern analyses of RNA isolated from cells grown at the expense of different carbon sources revealed three sucrose-inducible transcripts: one of 3.2 kb containing sacB and sacK, a second of 3.4 kb containing sacA and sacR, and a third of 1.8 kb containing only sacR. The inactivation of the sacR gene by replacement recombination resulted in the constitutive transcription of the sacBK and sacAR operons in the presence of different carbon sources, indicating that SacR acts as a repressor of transcription. (+info)