Purification and characterization of cytosolic aldolase from carrot storage root.
A single fructose-1,6-bisphosphate (FBP) aldolase has been detected in extracts from carrot storage roots (Daucus carota L.). The enzyme was purified 850-fold to electrophoretic homogeneity and a final specific activity of 26.3 mumols of FBP utilized/min per mg of protein. SDS/PAGE of the final preparation revealed a single protein-staining band of 40 kDa. The native molecular mass was determined by analytical gel filtration to be 159 kDa, indicating that the enzyme is a homotetramer. Denaturing isoelectric focusing revealed two predominant protein-staining bands, with pI values of 5.6 and 5.7. The enzyme is a class I aldolase, since EDTA or metal ions had no effect on its activity. The enzyme was relatively heat-stable, had an activation energy (Ea) of 68.3 kJ.mol-1, and had an absorption coefficient of 8.08 x 10(4) M-1.cm-1 at 280 nm. Km values for FBP and sedoheptulose 1,7-bisphosphate (SBP) were both determined to be 6 microM (pH optima 7.4). The specificity constant with FBP was 2.6 times that obtained with SBP. Ribose 5-phosphate, 6-phosphogluconate, MgAMP, glucose 1-phosphate and phosphoenolpyruvate (PEP) were inhibitors. PEP was a mixed-type inhibitor with respect to FBP (Ki = 3.2 mM, K'i = 5.1 mM). No activators were found. Rabbit anti-(carrot aldolase) polyclonal antibodies immunoprecipitated the activity of both carrot root aldolase and spinach leaf cytosolic aldolase, but not that of spinach leaf plastid aldolase. Western-blot analysis also revealed cross-reactivity with cytosolic, but not plastid, spinach leaf aldolase, indicating that the single carrot root aldolase is cytosolic.
Department of Biology, Queen's University, Kingston, Ontario, Canada.
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Moorhead
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Plaxton
D000596:Amino Acids / Q000032:analysis; D015153:Blotting, Western; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002845:Chromatography; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005634:Fructose-Bisphosphate Aldolase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007163:Immunosorbent Techniques; D007525:Isoelectric Focusing; D046911:Macromolecular Substances; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology*; D011092:Polyethylene Glycols; D013379:Substrate Specificity; D013403:Sugar Phosphates / Q000378:metabolism; D013816:Thermodynamics
D000596:Amino Acids; D046911:Macromolecular Substances; D013403:Sugar Phosphates; D011092:Polyethylene Glycols; C022079:sedoheptulose 1,7-bisphosphate; D005634:Fructose-Bisphosphate Aldolase