ABSTRACT
When a derivatized oligosaccharide isolated from ovalbumin and containing 6 mannose residues was incubated with yeast membranes and GDP-mannose, two sets of products were obtained, a high molecular weight one containing about 25 mannose residues and a low molecular weight one consisting of compounds with 7, 8, and 9 mannose residues, respectively. When the low molecular weight products were reincubated with the yeast membranes and GDP-mannose, no further mannose incorporation was observed, showing that these compounds must be of the wrong structure as substrates for yeast glycan processing enzymes. The structures were investigated by 1H NMR spectroscopy. The high molecular weight products contained an outer chain of an average length of 18 1----6-linked mannose residues attached to a core structure made up of the original 6 mannose residues with one additional 1----2-linked mannose added. The low molecular weight product with 8 mannose residues was deduced to contain a terminal 1----6-linked mannose (on the 1----6 arm) substituted by mannose at the 2-position, and the ones with 7 and 9 mannose residues were identified as having an additional 1----3-linked mannose on the starting Man6 substrate and on the Man8 product, respectively. The results lend further support to the picture that the processing steps must occur in proper sequence for specific products to form.
Subject(s)
Polysaccharides/metabolism , Saccharomyces cerevisiae/metabolism , Carbohydrate Conformation , Cell Membrane/metabolism , Chromatography, Ion Exchange , Magnetic Resonance Spectroscopy , Mass SpectrometryABSTRACT
Two compounds have been designed to serve as photoaffinity reagents for use with carboxyl proteases. 1,2-Epoxy-3-(4'-azido-2'-nitrophenoxy)propane has been synthesized and shown to react with porcine pepsin in the same fashion as the traditional inhibitor 1,2-epoxy-3-(p-nitrophenoxy)propane, while p-azidophenacyl bromide is similar to other phenacyl bromides in its reaction with pepsin. In combination with p-azido-alpha-diazoacetophenone, previously shown to resemble alpha-diazo carbonyl reagents in its reaction with pepsin, photoaffinity analogs are now available for all three of the widely-used carboxyl protease inhibitors.
Subject(s)
Affinity Labels/metabolism , Endopeptidases/metabolism , Pepsin A/metabolism , Animals , Aspartic Acid Endopeptidases , Azides/metabolism , Photochemistry , SwineSubject(s)
Azides , Enzymes/metabolism , Acetophenones , Alcohol Oxidoreductases/metabolism , Animals , Binding Sites , Carbonic Anhydrases/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Iodoacetates , NAD/analogs & derivatives , Pepsin A/metabolism , Rabbits , Saccharomyces cerevisiae/enzymology , Sulfanilamides , SwineSubject(s)
Alcohol Oxidoreductases , Azides , Saccharomyces cerevisiae/enzymology , Binding Sites , Cysteine , Iodoacetates , Kinetics , Photolysis , Protein BindingSubject(s)
Affinity Labels/chemical synthesis , RNA, Transfer, Amino Acyl/pharmacology , Ribosomes/metabolism , Binding Sites , Methods , Molecular Conformation , N-Formylmethionine , Nucleic Acid Conformation , Peptide Initiation Factors , Phenylalanine , Photochemistry , RNA, Transfer/metabolism , Ribosomes/drug effects , Structure-Activity Relationship , Thiouridine/pharmacology , ValineABSTRACT
The synthesis of the photochemically labile bifunctional reagent p-azidophenacyl bromide (1) is described. This compound may be covalently attached to a known site of an enzyme or other macromolecule by nucleophilic displacement at the alpha-bromo ketone moiety. Subsequent irradiation of the bound reagent gives a nitrene which may insert into a second portion of the enzyme forming a cross-link. Regeant 1 proved to be an excellent inhibitor of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase.
