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.
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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)
Paracellular glucose transport plays a minor role in the unanesthetized dog.
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Traditionally, intestinal glucose absorption was thought to occur through active, carrier-mediated transport. However, proponents of paracellular transport have argued that previous experiments neglected effects of solvent drag coming from high local concentrations of glucose at the brush-border membrane. The purpose of this study was to evaluate glucose absorption in the awake dog under conditions that would maximize any contribution of paracellular transport. Jejunal Thiry-Vella loops were constructed in six female mongrel dogs. After surgical recovery, isotonic buffers containing L-glucose as the probe for paracellular permeability were given over 2-h periods by constant infusion pump. At physiological concentrations of D-glucose (1-50 mM), the fractional absorption of L-glucose was only 4-7% of total glucose absorption. Infusion of supraphysiological concentrations (150 mM) of D-glucose, D-maltose, or D-mannitol yielded low-fractional absorptions of L-glucose (2-5%), so too did complex or nonabsorbable carbohydrates. In all experiments, there was significant fractional water absorption (5-19%), a prerequisite for solvent drag. Therefore, with even up to high concentrations of luminal carbohydrates in the presence of significant water absorption, the relative contribution of paracellular glucose absorption remained low. (+info)
AlphaB-crystallin selectively targets intermediate filament proteins during thermal stress.
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PURPOSE: AlphaB-Crystallin is a small heat shock protein (sHsp) expressed at high levels in the lens of the eye, where its molecular chaperone functions may protect against cataract formation in vivo. The purpose of this study was to identify protein targets for the sHsp alphaB-crystallin in lens cell homogenates during conditions of mild thermal stress. METHODS: The authors report the use of a fusion protein, maltose-binding protein alphaB-crystallin (MBP-alphaB), immobilized on amylose resin as a novel method for isolating endogenous alphaB-crystallin-binding proteins from lens cell homogenates after mild thermal stress. RESULTS: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western immunoblot analyses showed selective interactions in lens cell homogenates between MBP-alphaB and endogenous alphaA- and alphaB-crystallins, the lens-specific intermediate filament proteins phakinin (CP49) and filensin (CP115), and vimentin during a mild 20-minute heat shock at 45 degrees C. No interactions were observed with the beta- or gamma-crystallins, or the cytoskeletal proteins actin, alpha-tubulin, and spectrin, although these proteins were present in lens cell homogenates. In contrast, gamma-crystallin and actin interacted with MBP-alphaB at 45 degrees C only in their purified states. The results obtained with MBP-alphaB were confirmed by immunoprecipitation reactions in which immunoprecipitation of native bovine alphaB-crystallin from heat-shocked lens cell homogenates resulted in the coprecipitation of phakinin and filensin. CONCLUSIONS: In the lens the sHsp alphaB-crystallin may selectively target intermediate filaments for protection against unfolding during conditions of stress. (+info)
Latency of some glycosidases of rat liver lysosomes.
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The latency of the alpha-glucosidase activity of intact rat liver lysosomes was studied by using four substrates (glycogen, maltose, p-nitrophenyl, alpha-glucoside, alpha-fluoroglucoside) at a range of substrate concentrations. The results indicate that the entire lysosome population is impermeable to glycogen and maltose, but a proportion of lysosomes are permeable to alpha-fluoroglucoside and a still higher proportion permeable to p-nitrophenyl alpha-glucoside. Incubation at 37 degrees C in an osmotically protected buffer of of pH 5.0 caused lysosomes to become permeable to previously impermeant substrates and ultimately to release their alpha-glucosidase into the medium. The latencies of lysosomal beta-glucosidase and beta-galactosidase were examined by using p-nitrophenyl beta-glucoside and beta-galactoside as substrates. The results indicate permeability properties to these substrates similar to that to p-nitrophenyl alpha-glucoside. On incubation in an osmotically protected buffer of pH 5, lysosomes progressively released their beta-galactosidase in soluble form, but beta-glucosidase remained attached to sedimentable material. Lysosomal beta-glucosidase was inhibited by 0.1% Triton X-100; alpha-glucosidase and beta-galactosidase were not inhibited. (+info)
Taste qualities of solutions preferred by hamsters.
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Molecules of diverse chemical structure are sweet to humans and several lines of evidence (genetic, physiological, behavioral) suggest that there may be distinct sweet perceptual qualities. To address how many perceptual categories these molecules elicit in hamsters (Mesocricetus auratus), we studied patterns of generalization of conditioned taste aversions for seven sweeteners: 100 mM sucrose, 320 mM maltose, 32 mM D-phenylalanine, 3.2 mM sodium saccharin, 16 mM calcium cyclamate, 10 mM dulcin and 32 mM sodium m-nitrobenzene sulfonate. Each stimulus was preferred versus water in two-bottle intake tests and stimulated the chorda tympani nerve. For each of seven experimental groups the conditional stimulus (CS) was a sweetener and for the control group the CS was water. Apomorphine.HCl was injected i.p. after a CS was sampled and, after recovery, test stimuli (TS) were presented for 1 h daily. The intake (ml) of each TS consumed by experimental animals was compared with mean TS intake by the control group. Learned aversions for 18/21 stimulus pairs cross-generalized, resulting in a single cluster of generalization patterns for the seven stimuli. Cross-generalization failures (maltose-cyclamate, maltose-sucrose, cyclamate-NaNBS) may be the consequence of particular stimulus features (e.g. salience, cation taste), rather than the absence of a 'sucrose-like' quality. The results are consistent with a single hamster perceptual quality for a diverse set of chemical structures that are sweet to humans. (+info)
Acarbose, a pseudooligosaccharide, is transported but not metabolized by the maltose-maltodextrin system of Escherichia coli.
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The pseudooligosaccharide acarbose is a potent inhibitor of amylases, glucosidases, and cyclodextrin glycosyltransferase and is clinically used for the treatment of so-called type II or insulin-independent diabetes. The compound consists of an unsaturated aminocyclitol, a deoxyhexose, and a maltose. The unsaturated aminocyclitol moiety (also called valienamine) is primarily responsible for the inhibition of glucosidases. Due to its structural similarity to maltotetraose, we have investigated whether acarbose is recognized as a substrate by the maltose/maltodextrin system of Escherichia coli. Acarbose at millimolar concentrations specifically affected the growth of E. coli K-12 on maltose as the sole source of carbon and energy. Uptake of radiolabeled maltose was competitively inhibited by acarbose, with a Ki of 1.1 microM. Maltose-grown cells transported radiolabeled acarbose, indicating that the compound is recognized as a substrate. Studying the interaction of acarbose with purified maltoporin in black lipid membranes revealed that the kinetics of acarbose binding to LamB is asymmetric. The on-rate of acarbose is approximately 30 times lower when the molecule enters the pore from the extracellular side than when it enters from the periplasmic side. Acarbose could not be utilized as a carbon source since the compound alone was not a substrate of amylomaltase (MalQ) and was only poorly attacked by maltodextrin glucosidase (MalZ). (+info)
A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation.
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The Escherichia coli biotin holoenzyme synthetase, BirA, catalyzes transfer of biotin to the epsilon amino group of a specific lysine residue of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. Sequences of naturally biotinylated substrates are highly conserved across evolutionary boundaries, and cross-species biotinylation has been demonstrated in several systems. To define the minimal substrate requirements in BirA-catalyzed biotinylation, we have measured the kinetics of modification of a 23-residue peptide previously identified by combinatorial methods. Although the sequence of the peptide bears little resemblance to the biotinylated sequence in BCCP, it is enzymatically biotinylated in vivo. Rates of biotin transfer to the 23-residue peptide are similar to those determined for BCCP. To further elucidate the sequence requirements for biotinylation, transient kinetic measurements were performed on a series of amino- and carboxy-terminal truncations of the 23-mer. The results, determined by stopped-flow fluorescence, allowed identification of a 14-residue peptide as the minimum required sequence. Additional support was obtained using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometric analysis of peptides that had been incubated with an excess of biotinyl-5'-adenylate intermediate and catalytic amounts of BirA. Results of these measurements indicate that while kinetically inactive truncations showed no significant shift in molecular mass to the values expected for biotinylated species, kinetically active truncations exhibited 100% biotinylation. The specificity constant (k(cat)/Km) governing BirA-catalyzed biotinylation of the 14-mer minimal substrate is similar to that determined for the natural substrate, BCCP. We conclude that the 14-mer peptide efficiently mimics the biotin acceptor function of the much larger protein domain normally recognized by BirA. (+info)
A Cys-less variant of the bacterial ATP binding cassette protein MalK is functional in maltose transport and regulation.
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The cysteine residues of the ABC protein MalK from Salmonella typhimurium maltose transport system (C40, C350, C360) were consecutively replaced by serines. Cys-less MalK was fully functional in maltose transport in vivo. Moreover, the activity of MalK as a repressor of other maltose-regulated genes was also retained. The absence of cysteine residues in the purified protein was verified by its failure to react with fluorescein-5-maleimide. In contrast to purified wild-type MalK, the ATPase activity of the C40S variant was insensitive to inhibition by N-ethylmaleimide. (+info)