betadex and maltohexaose

AMP-activated protein kinase (AMPK) coordinates cellular metabolism in response to energy demand as well as to a variety of stimuli. The AMPK beta subunit acts as a scaffold for the alpha catalytic and gamma regulatory subunits and targets the AMPK heterotrimer to glycogen. We have determined the structure of the AMPK beta glycogen binding domain in complex with beta-cyclodextrin. The structure reveals a carbohydrate binding pocket that consolidates all known aspects of carbohydrate binding observed in starch binding domains into one site, with extensive contact between several residues and five glucose units. beta-cyclodextrin is held in a pincer-like grasp with two tryptophan residues cradling two beta-cyclodextrin glucose units and a leucine residue piercing the beta-cyclodextrin ring. Mutation of key beta-cyclodextrin binding residues either partially or completely prevents the glycogen binding domain from binding glycogen. Modeling suggests that this binding pocket enables AMPK to interact with glycogen anywhere across the carbohydrate's helical surface.

Topics: Amino Acid Sequence; AMP-Activated Protein Kinases; Animals; beta-Cyclodextrins; Binding Sites; Binding, Competitive; Carbohydrate Conformation; Catalytic Domain; Crystallography, X-Ray; Glucans; Glucose; Glycogen; Leucine; Liver; Models, Molecular; Molecular Sequence Data; Multienzyme Complexes; Mutagenesis, Site-Directed; Mutation; Oligosaccharides; Protein Binding; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Protein Subunits; Rats; Sequence Homology, Amino Acid; Spectrum Analysis, Raman; Tryptophan; Water

The structures of Thermoactinomyces vulgaris R-47 alpha-amylase II mutant (d325nTVA II) complexed with substrate analogues, methyl beta-cyclodextrin (m beta-CD) and maltohexaose (G6), were solved by X-ray diffraction at 3.2 A and 3.3 A resolution, respectively. In d325nTVA II-m beta-CD complex, the orientation and binding-position of beta-CD in TVA II were identical to those in cyclodextin glucanotransferase (CGTase). The active site residues were essentialy conserved, while there are no residues corresponding to Tyr89, Phe183, and His233 of CGTase in TVA II. In d325nTVA II-G6 complex, the electron density maps of two glucosyl units at the non-reducing end were disordered and invisible. The four glucosyl units of G6 were bound to TVA II as in CGTase, while the others were not stacked and were probably flexible. The residues of TVA II corresponding to Tyr89, Lys232, and His233 of CGTase were completely lacking. These results suggest that the lack of the residues related to alpha-glucan and CD-stacking causes the functional distinctions between CGTase and TVA II.

Topics: alpha-Amylases; beta-Cyclodextrins; Cyclodextrins; Glucosyltransferases; Micromonosporaceae; Models, Molecular; Oligosaccharides; Protein Structure, Tertiary; Substrate Specificity

The separation of biological mixtures in open micro-channels using electrophoresis with rapid and simple coupling to mass spectrometry is introduced. Rapid open-access channel electrophoresis employs microchannels that are manufactured on microchips. Separation is performed in the open channels, and the chips are transferred to a matrix-assisted laser desorption/ionization (MALDI) source after the solvent is evaporated. The matrix (2,5-dihydroxybenzoic acid) is placed in the solution with the run buffer before the separation of the analyte components. After separation, the solvent is evaporated and the microchip is ready for MALDI-MS analysis. The microchip is placed directly into a specially designed ion source of an external source Fourier transform mass spectrometry instrument. Separation of simple mixtures containing oligosaccharides and peptides is shown.

Topics: Animals; beta-Cyclodextrins; Cyclodextrins; Electrophoresis, Capillary; Fourier Analysis; Gentisates; Hydroxybenzoates; Kallidin; Male; Oligosaccharides; Rats; Rats, Sprague-Dawley; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization