ABSTRACT
Glycogen phosphorylases catalyze the breakdown of glycogen to glucose-1-phosphate, which enters glycolysis to fulfill the energetic requirements of the organism. Maintaining control of blood glucose levels is critical in minimizing the debilitating effects of diabetes, making liver glycogen phosphorylase a potential therapeutic target. To support inhibitor design, we determined the crystal structures of the active and inactive forms of human liver glycogen phosphorylase a. During activation, forty residues of the catalytic site undergo order/disorder transitions, changes in secondary structure, or packing to reorganize the catalytic site for substrate binding and catalysis. Knowing the inactive and active conformations of the liver enzyme and how each differs from its counterpart in muscle phosphorylase provides the basis for designing inhibitors that bind preferentially to the inactive conformation of the liver isozyme.
Subject(s)
Liver/enzymology , Phosphorylases/chemistry , Phosphorylases/metabolism , Adenosine Monophosphate/metabolism , Animals , Binding Sites , Catalytic Domain , Crystallography, X-Ray , Diabetes Mellitus/drug therapy , Diabetes Mellitus/metabolism , Drug Design , Enzyme Activation , Enzyme Inhibitors/pharmacology , Humans , In Vitro Techniques , Models, Molecular , Muscles/enzymology , Phosphorylases/genetics , Protein Conformation , Protein Structure, Secondary , RabbitsABSTRACT
Bisulfite or sulfite was found to be inhibitory to Helicobacter pylori growth. A modified version of Brucella broth (BB), bisulfite-less BB (BLBB), supported rapid, robust, and consistent growth of H. pylori. We suggest that BLBB simply be called "Pylori broth" for distinction from Brucella broth.
Subject(s)
Culture Media , Helicobacter pylori/drug effects , Helicobacter pylori/growth & development , Sulfites/pharmacology , Bacteriological Techniques , Culture Media/chemistry , Evaluation Studies as Topic , Helicobacter pylori/isolation & purification , Humans , Oxidation-ReductionABSTRACT
The use of vortex flow filtration for harvesting cells or conditioned medium from large scale bioreactors has proven to be an efficient, low shear method of cell concentration and conditioned medium clarification. Several 8-10 L batches of the human histiocytic lymphoma U-937 cell line (ATCC CRL 1593) were concentrated to less than 1 L by vortex flow filtration through a 3.0 microns membrane. An aggressive filtration regimen caused a 17% loss of cell viability and a 32% loss of IL-4 receptor binding capacity when compared to a batch centrifuged control. A reduction of the rotor speed from 1500 to 500 RPM and reduction of system back pressure from 10 to 0 PSIG resulted in cell viability and IL-4 binding capacity comparable to the control. Several 10 L batches of baculovirus infected Sf-9 cells were also concentrated to less than 1 L by vortex flow filtration through a 3.0 microns membrane. SDS-PAGE analysis of filtrate samples showed that aggressive filtration caused cell damage which led to contamination of the process stream by cellular lysate. When rotor speed was reduced to 500 RPM and system back pressure was reduced to 0 PSIG, the amount of contaminating lysate proteins in filtrate samples was comparable to a batch centrifuged control.
