Effects of substrate branching characteristics on kinetics of enzymatic depolymerization of mixed linear and branched polysaccharides: II. Amylose/glycogen alpha-amylolysis

Enzymatic depolymerization of polysaccharides with alpha-amylase has been studied in mixed aqueous dimethylsulfoxide (DMSO)/water solvents. Polysaccharide substrate chemical compositions, configurational structures, and bonding pattersn are known to affect observed enzymatic reaction kinetics. The branching structures of polysaccharides and their effects on the kinetic mechanisms of depolymerization reactions via endo-acting hydrolyzing enzyme was studied via size exclusion chromatography coupled to low angle laser light scattering (SEC/LALLS). The glycogen branching structure is a heterogeneously distributed "cluster" structure rather than a homogeneously distributed "treelike" structure. The action pattern of alpha-amylase on glycogen, which is composed of highly branched clusters, as end-products, has a "pseudo-exo-attack" in contrast to an expected "endoattack" as seen in the hydrolysis of amylose or amylopectin substrates. These effects of branched substrates for mixed amylose/glycogen alpha-amylolysis have been predicted and demonstrated by both experimental and theoretical analysis using the kinetic model presented in this report. The "lumped" kinetic model employed, assumes that the enzyme simultaneously attacks both linear and branched substrates. In general, excellent agreement between the model predictions and the experimental observations, both qualitatively and quantitatively, was obtained. (c) 1995 John Wiley & Sons, Inc.     

Importance of spore mutants for fed-batch and continuous fermentation of Bacillus subtilis

To alleviate plasmid instability and to prolong the production phase of subtilisin, integrable plasmid and spore mutants are used. Compared with batch-type shake flask cultures, spore mutants' ability to produce subtilisin can be well pronounced in fed-batch and continuous cultures. Hence, the two culture methods make it possible to identify the peculiar characteristics of the spore mutants unobtainable in batch culture. Spore mutants can enhance subtilisin productivity and prolong subtilisin production time in fed-batch culture as well as enable us to use very low dilution rates (<0.1 h(-1)) without losing productivity in continuous culture, thereby improving the conversion yield of the nitrogen source. At 0.05 h(-1) the spollG mutant of Bacillus subtilis DB104 (Deltanpr Deltaapr) (Em(r)) spollG (Bim(r)):: pMK101 (Cm(r)) showed a subtilisin yield about ten times higher than that from wild-type DB104 (Deltanpr Deltaapr)::pMK101 (Cm(r)). (c) 1995 John Wiley & Sons, Inc.     

The centric region of the X chromosome rDNA functions in male meiotic pairing in Drosophila melanogaster

In Drosophila melanogaster males, sex chromosome pairing at meiosis is ensured by so-called pairing site(s) located discretely in the centric heterochromatin. The property of the pairing sites is not well understood. Recently, an hypothesis has been proposed that 240 bp repeats in the nontranscribed spacer region of rDNA function as the pairing sites in male meiosis. However, considerable cytogenetic evidence exists that is contrary to this hypothesis. Hence, the question is whether the chromosomal rDNA clusters, in which a high copy number of 240 bp repeats exists, are involved in the pairing. In order to resolve the problem we X-rayed Drosophila carrying the X chromosome inversion In(1)sc ^V2L sc ^8R and generated free, mini-X chromosomes carrying a substantial amount of rDNA. We defined cytogenetically the size of the mini-chromosomes and studied their meiotic behavior. Our results demonstrate that the heterochromatin at the distal end of the inversion, whose length is approximately 0.4 times that of the fourth chromosome, includes a meiotic pairing site in the male. We discuss the cytological location of the pairing site and the possible role of rDNA in meiotic pairing.     

Chaperone SecB: Conformational changes demonstrated by circular dichroism

The chaperone SecB, which is involved in protein export inEscherichia coli, is shown by circular dichroism measurements to contain a high content of-pleated sheets. Prediction of the secondary structure of SecB is in good agreement with the observed content of-sheet. In accordance with the previous studies in which changes in conformation were assessed indirectly [Randall (1992),Science 257, 241–245], here we show that the conformation of SecB changes with the concentration of salt in the milieu and also when SecB interacts with a peptide ligand.Abbreviations ANS 1-anilino-naphthalene-8-sulfonate - CD circular dichroism - NMR nuclear magnetic resonance - CCA convex constraint analysis     

Green fluorescent protein as a signal for protein-protein interactions.

Green fluorescent protein (GFP) is autofluorescent. This property has made GFP useful in monitoring in vivo activities such as gene expression and protein localization. We find that GFP can be used in vitro to reveal and characterize protein-protein interactions. The interaction between the S-peptide and S-protein fragments of ribonuclease A was chosen as a model system. GFP-tagged S-peptide was produced, and the interaction of this fusion protein with S-protein was analyzed by two distinct methods: fluorescence gel retardation and fluorescence polarization. The fluorescence gel retardation assay is a rapid method to demonstrate the existence of a protein-protein interaction and to estimate the dissociation constant (Kd) of the resulting complex. The fluorescence polarization assay is an accurate method to evaluate Kd in a specified homogeneous solution and can be adapted for the high-throughput screening of protein or peptide libraries. These two methods are powerful new tools to probe protein-protein interactions.     

Mutational analysis of the linker region of EnvZ, an osmosensor in Escherichia coli.

EnvZ, a transmembrane signal transducer, is composed of a periplasmic sensor domain, transmembrane domains, and a cytoplasmic signaling domain. Between the second transmembrane domain and the cytoplasmic signaling domain there is a linker domain consisting of approximately 50 residues. In this study, we investigated the functional role of the EnvZ linker domain with respect to signal transduction. Amino acid sequence alignment of linker regions among various bacterial signal transducer proteins does not show a high sequence identity but suggests a common helix 1-loop-helix 2 structure. Among several mutations introduced in the EnvZ linker region, it was found that hydrophobic-to-charged amino acid substitutions in helix 1 and helix 2 and deletions in helix 1, loop, and helix 2 (delta14, delta8, and delta7) resulted in constitutive OmpC expression. In the linker mutant EnvZ x delta7, both kinase and phosphatase activities were significantly reduced but the ratio of kinase to phosphatase activity increased, consistent with the constitutive OmpC expression. In contrast, the purified cytoplasmic fragment of EnvZ x delta7 possessed both kinase and phosphatase activities at levels similar to those of the cytoplasmic fragment of wild-type EnvZ. In addition, the linker mutations had no direct effect on EnvZ C-terminal dimerization. These results together with previous data suggest that the linker region is not directly involved in EnvZ enzymatic activities and that it may have a crucial role in propagating a conformational change to ensure correct positioning of two EnvZ molecules within a dimer during the transmembrane signaling.     

Optical characteristics of carboxyl group in relation to the circular dichroic properties and dissociation constants of glycosaminoglycans

Circular dichroism studies of glycosaminoglycan including chemically transformed heparins at various pH values reveal that carboxyl chromophore plays an important role in the dichroic behavior of the polymers. With decreasing pH, iduronic acid-containing glycosaminoglycans show increased negative ellipticity near 220 nm whereas the polymers containing glucuronic acid display enhanced negative dichroism near 230 nm and decreased negative dichroism around 210 nm. The pH-dependent optical properties have been utilized to determine the pK_a values of uronic acid moieties. The acid strengths of the iduronic acid-containing glycosaminoglycans are inherently smaller than those of corresponding glucuronic acid-containing polymers. Glycosaminoglycans in which the amino sugars are linked with iduronic acid display a very weak n → π^* amide transition, or none. The rotational strength at 210 nm of these polymers is largely due to iduronic acid moieties. The CD variations above 200 nm with change in pH do not indicate any major conformational transition of the molecules but the difference between dermatan sulfate and heparin can be attributed to difference either in iduronic acid conformation or in intersaccharide linkages.     

The role of glyoxylate in the regulation of biodegradative threonine dehydratase of Escherichia coli.

The activity of biodegradative threonine dehydratase of Escherichia coli K12 was reversibly inhibited by glyoxylate in the presence of AMP. Kinetic analysis showed that the inhibition was mixed with respect to L-threonine and competitive in terms of AMP; the inhibitory effect of glyoxylate was less pronounced at high protein concentrations. Incubation of dehydratase with L-threonine shifted the absorption maximum of the enzyme-bound pyridoxal phosphate from 413 to 425 nm; addition of glyoxylate completely prevented the threonine-mediated spectral shift. In addition to the inhibitory effect, incubation of purified enzyme with glyoxylate resulted in a progressive, irreversible inactivation of the enzyme and formation of inactive protein aggregates. The rates of inactivation were decreased with increasing concentrations of protein and AMP. During inactivation by glyoxylate, the 413-nm absorption maximum of the native enzyme was replaced by a new peak at 385 nm. Experiments with [14C]glyoxylate showed a rapid binding of 1 mol of glyoxylate per 147,000 g followed by a slow binding of 3 additional mol of glyoxylate; the glyoxylate-protein linkage was stable to acid precipitation and protein denaturants. Competition binding experiments revealed that pyruvate (which also inactivated the E. coli enzyme, Feldman, D.A., and Datta, P. (1975) Biochemistry 14, 1760-1767) did not interfere with the binding of glyoxylate or vice versa, suggesting that the two keto acids may occupy separate sites on the enzyme molecule. Nevertheless, experiments on enzyme inactivation using glyoxylate plus pyruvate reveal mutual interactions between these ligands in terms of lack of additive effect, retardation in the spectral shift due to glyoxylate, and stabilization of the enzyme in the presence and absence of AMP. We conclude from these results that the control of biodegradative threonine dehydratase is governed by a complex set of regulatory events resulting from reversible and irreversible association of these effectors with the enzyme molecule.     

Light effects on leaf development and photosynthetic capacity of Hydrocotyle bonariensis Lam.

Rates of net CO₂ uptake were examined in developing leaves of Hydrocotyle bonariensis. Leaves that developed under high photosynthetically active radiation (48 mol m^-2 day^-1 PAR) were smaller, thicker, and reached maximum size sooner than did leaves that developed under low PAR (4.8 mol m^-2 day^-1). Maximum net CO₂ uptake rates were reached after 5 to 6 days expansion for both the low and the high PAR leaves. Leaves grown at high PAR had higher maximum photosynthetic rates and a higher PAR required for light saturation but showed a more rapid decline in rate with age than did low PAR leaves. To assess the basis for the difference observed in photosynthetic rates, CO₂ diffusion conductances and the mesophyll surface available for CO₂ absorption were examined for mature leaves. Stomatal conductance was the largest conductance in all treatments and did not vary appreciably with growth PAR. Mesophyll conductance progressively increased with growth PAR (up to 48 mol m^-2 day^-1) as did the mesophyll surface area per unit leaf area, but the cellular conductance exhibited most of its increase at low PAR (up to 4.8 mol m^-2 day^-1).