Purification and properties of isocitrate lyase from flax seedlings

Khan, Farah; Saleemuddin, M.; Siddiqi, Majid; McFadden, Bruce A.

doi:10.1016/0003-9861(77)90413-1

Cited by 52 publications

(28 citation statements)

References 33 publications

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supporting

confidence: 77%

“…This compares favorably with the 14% recovery reported for flax seedlings (18), the only other angiosperm source from which the enzyme has to date been isolated. Based on a final specific activity of 5.8 units/mg and an over-all purification of 180-fold, cucumber ICL represents about 0.56% of extractable (18,19). The mol wt of 325,000 determined for the native cucumber enzyme is substantially higher than most literature values, including 170,000 for Chlorella (15), 222,000 for Pseudomonas (26), 265,000 for Neurospora (14), and 264,000 for flax (18), although a mol wt of 480,000 has been reported for ICL from the vinegar eelworm, Turbatrix aceti (28).…”

supporting

confidence: 77%

“…Catalase appears to consist of four subunits each with a mol wt of 54,500. ICL appears from these data to consist of five similar subunits, though this seems unlikely in light of the tetrameric nature of the enzyme from other sources (14,15,18,26).…”

mentioning

confidence: 74%

“…The formation of glyoxylate phenylhydrazone was monitored as AA32nm (extinction coefficient: 1.7 x 104 M-1 cm-'). One unit of ICL activity is that amount of enzyme required to degrade I ,umol of threo-D.-isocitrate to glyoxylate and succinate per min at 30 C. For kinetic assays, the incubation mixture (14) contained 18.4 mm MOPS (pH 6.8), 1.4 mm MgC12, 1.62 mm phenylhydrazine hydrochloride, 1.1 mm DTT, variable concentrations (0.02-0.15 mM) of DL-isocitrate, and 10 to 20,Al of enzyme in a final volume of 2.5 ml.…”

mentioning

confidence: 99%

“…Based on a final specific activity of 5.8 units/mg and an over-all purification of 180-fold, cucumber ICL represents about 0.56% of extractable (18,19). The mol wt of 325,000 determined for the native cucumber enzyme is substantially higher than most literature values, including 170,000 for Chlorella (15), 222,000 for Pseudomonas (26), 265,000 for Neurospora (14), and 264,000 for flax (18), although a mol wt of 480,000 has been reported for ICL from the vinegar eelworm, Turbatrix aceti (28). The value of 325,000 appears reliable, since it was determined simultaneously with that for catalase, using an appropriate range of mol wt markers.…”

mentioning

confidence: 99%

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Regulation of Glyoxysomal Enzymes during Germination of Cucumber

Lamb

Riezman²,

Becker³

et al. 1978

Plant Physiol.

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The glyoxysomal enzymes isocitrate lyase and catalase have been isolated from etiolated cucumber (Cucumis satdvus) cotyledons. The enzymes co-purified through polyethyleneimine precipitation and (NH4)2SO4 precipitation, and were resolved by gel filtration on Sepharose 6B foUlowed by chromatography on diethylaminoethyl-cefulose (isocitrate lyase) or hydroxylapatite (catalase). Purity of the isolated enzymes was assessed by sodium dodecyl sulfate-polyacrylamide electrophoresis, isoelectric focusing, and immunoelectrophoresis. Antibodies raised to both enzymes in rabbits and in tumor-bearing mice were shown to be monospecific by immunoelectrophoresis against total homogenate protein. Isocitrate lyase and catalase represent about 0.56% and 0.1%, respectively, of total extractable cotyledonary protein. Both enzymes appear to be present in a single form. Molecular weights of the native enzymes and its subunits are 225,000 and 54,500 for catalase, and 325,000 and 63,500 for isocitrate lyase. The pH optimum for isocitrate lyase is about 6.75 in morpholinopropane sulfonic acid buffer, but varies significantly with buffer used. The Km for D-isocitrate is 39 micromolar. A double antibody technique (rabbit antiisocitrate lyase followed by 1251-labeled goat anti-rabbit immunoglobulin G) has been used to visualize isocitrate lyase subunit protein on sodium dodecyl sulfate-polyacrylamide with high specificity and sensitivity.Seed germination in fat-storing species requires a functional glyoxylate cycle to effect net gluconeogenesis from the acetyl-CoA derived by fl-oxidation of storage triglycerides (4). During early germination, glyoxylate cycle enzymes such as isocitrate lyase (threo-D-isocitrate glyoxylate lyase, EC 4.1.3.1) undergo a well characterized increase and subsequent decline in activity, with peak activity corresponding to the period of maximum fat metabolism (3,4,7,16,32,35). Much is already known about the developmental physiology of the glyoxylate cycle enzymes and the glyoxysomal compartment in which they are localized, but little is presently understood about the regulatory mechanisms which underlie their appearance and subcellular compartmentation in the germinating seed. 'To whom reprint requests should be addressed.enzyme with a characteristic increase and subsequent decline in activity during germination (35). We are interested both in the level(s) at which the activities of glyoxysomal enzymes are regulated during cucumber germination (3) and in the relationship between appearance of enzyme activity and organelle biogenesis.Critical to such studies is the availability of purified glyoxysomal enzymes and of monospecific antibodies to them. Few reports have appeared to date concerning the isolation of glyoxysomal enzymes from plant sources. Specifically, ICL4 has been isolated from bacterial (25), fungal (14), algal (15), and nematode (9, 28) sources, but its purification from angiosperms has thus far been described only for flax by Khan et al. (18) and for cucumber in our own preliminary communica...

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supporting

confidence: 77%