A selective reaction of fructose bisphosphate aldolase with fluorescein isothiocyanate in chicken muscle extracts.
The present work describes the selective covalent modification of fructose bisphosphate aldolase in crude extracts of chicken breast muscle by fluorescein 5'-isothiocyanate (5'-FITC) at pH 7.0 and 35 degrees C. The modification was observed after 1 min while no other major soluble protein was labeled even after 30 min. We calculated that ca. one 5'-FITC molecule was incorporated into each aldolase tetramer after a 30 min reaction which resulted in a minimal loss of enzyme activity. The "native" structure of aldolase was required for the selective modification by 5'-FITC since high pH, high temperature, and ionic detergents either inhibited or prevented the reaction of 5'-FITC with aldolase. Certain metabolites (ATP, ADP, CTP, GTP, FBP) and erythrosin B also inhibited the 5'-FITC modification of aldolase. In contrast, F-6-P, AMP, NADH, and NAD(+) as well as free lysine and most importantly, the 6'-isomer of FITC exhibited no competition with 5'-FITC for the labeling of aldolase. Alone, the 6'-isomer of FITC did not exhibit preferential reaction when combined with aldolase. 5'-FITC-labeled and -unlabeled aldolases were not distinguished by their ability to bind to muscle myofibrils (MFs) or by their abilities to refold following reversible denaturation in urea. Structural analysis revealed that 5'-FITC-labeled a tryptic peptide corresponding to residues 112-134 in the primary structure of aldolase, a peptide that does not contain lysine, the amino acid believed to be the primary target of this reagent. Unlike chicken and rabbit muscle aldolases, chicken brain and liver aldolase isoforms along with several other aldolases derived from diverse biological sources did not exhibit this highly selective modification by 5'-FITC.
Gehring
Andrew G
AG
United States Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA 19038, USA. andrew.gehring@ars.usda.gov
Ezzell
John L
JL
Lebherz
Herbert G
HG
D000595:Amino Acid Sequence; D000818:Animals; D030161:Avian Proteins / Q000378:metabolism; D001665:Binding Sites; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000378:metabolism; D009132:Muscles / Q000187:drug effects / Q000201:enzymology*; D009711:Nucleotides / Q000494:pharmacology; D013194:Staining and Labeling; D013696:Temperature; D014020:Tissue Extracts / Q000378:metabolism*
D030161:Avian Proteins; D005456:Fluorescent Dyes; D007527:Isoenzymes; D046912:Multiprotein Complexes; D009711:Nucleotides; D014020:Tissue Extracts; D005634:Fructose-Bisphosphate Aldolase; D016650:Fluorescein-5-isothiocyanate