Mode of interactions of human aldolase isozymes with cytoskeletons.
Three isoforms of fructose-1,6-bisphosphate aldolase were found to bind specifically to the actin-containing filament of the cytoskeleton and to show tissue-specific binding patterns. Aldolase A (muscle type) bound more tightly to the skeletal muscle cytoskeleton among the three isozymes, while aldolase B (liver type) preferred the liver cytoskeleton to those of other tissues. The specific binding of aldolase A to the skeletal muscle cytoskeleton was inhibited strongly by the substrates fructose 1,6-bisphosphate and fructose 1-phosphate. Several mutant aldolases A were examined to identify the amino acid residues or regions that play a role in specific binding. Among the mutant aldolases tested, A-E34D, A-K41N, and A-Y363S exhibited remarkably reduced binding activities. Experiments using FITC-labeled enzymes and Rh-labeled phalloidin disclosed that aldolase A associated with the cytoskeleton. Specifically, when aldolase A was incubated with human fibroblast MRC-5 permeabilized with Triton X-100, aldolase A bound to the actin filaments in the stress fibers within the cell. Aldolase A reversibly inhibited the contraction of MRC-5 cells which usually occurred in the presence of Mg2(+)-ATP and Ca2+. These results provide direct evidence that aldolase binds specifically to the actin-containing stress fibers and suggest that aldolase may regulate cell contraction through its reversible binding to the filaments in the permeabilized MRC-5 fibroblast.
Kusakabe
T
T
Department of Biochemistry, Saga Medical School, Nabeshima, Saga, Japan. kusakabe@bcmp.med.harvard.edu
Motoki
K
K
Hori
K
K
D008841:Actin Cytoskeleton / Q000502:physiology; D000199:Actins / Q000378:metabolism / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001921:Brain / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005634:Fructose-Bisphosphate Aldolase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism; D009154:Mutation; D009243:NAD / Q000494:pharmacology; D011485:Protein Binding; D051381:Rats
D000199:Actins; D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D009243:NAD; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D005634:Fructose-Bisphosphate Aldolase; D016650:Fluorescein-5-isothiocyanate