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  • 1
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Physiologia plantarum 99 (1997), S. 0 
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: In our search for an endogenous ethylene trigger from tomato (Lycopersicon esculentum Mill. ev. Rutgers) fruit cell wall alkaline soluble pectin (ASP), we purified an active component using DEAE-Sepharose chromatography followed by elution on Bio-Gel P-100 or Superose 12. The purified active fraction produced a single band on silver-stained SDS-PAGE of approximately Mr 20000. Using two-dimensional proton-proton and proton-carbon correlation spectroscopy, we identified the repeating sub-unit as trans-1,2-diamino-cyclohexane- N,N,N′,N′-tetraacetic acid (CDTA), a chelator used to extract ASP. Although the ASP undergoes extensive dialysis during its extraction which should remove CDTA, the CDTA apparently forms a large molecular weight polymer which does not diffuse out of the dialysis tubing. Infiltration of commercially prepared CDTA into mature green tomato fruit stimulated ethylene production. The ethylene stimulatory effect of CDTA was not affected by the presence of equimolar amounts of CaCl2, or nmol g-1 amounts of the calcium channel blockers, nifedipine or verapamil. EDTA, EGTA, and diethylenetriaminepentaacetic acid, other divalent cation chelators, also stimulated ethylene production when they were infiltrated into tomato fruit. Neither the purified material nor commercial CDTA stimulated ethylene production when they were infiltrated into leaf tissue.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Copenhagen : Munksgaard International Publishers
    Physiologia plantarum 112 (2001), S. 0 
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Multi-subunit acetyl-coenzyme A carboxylase (MS-ACCase; EC 6.4.1.2) isolated from soybean chloroplasts is a labile enzyme that loses activity during purification. We found that incubating the chloroplast stromal fraction under anaerobic conditions or in the presence of 5 mM FeSO4 stimulated ACCase (acetyl-CoA→malonyl-CoA) and carboxyltransferase (malonyl-CoA→acetyl-CoA) activity. Fe-stimulation of activity was associated with 59Fe binding to a stromal protein fraction. ACCase and carboxyltransferase activities measured in the stromal protein fraction containing bound 59Fe were 2-fold and 6-fold greater, respectively, than the control (stromal fraction not pretreated with FeSO4). Superose 6 gel filtration chromatography indicated 59Fe comigrated with stromal protein of approximately 180 kDa that exhibited carboxyltransferase activity, but lacked ACCase activity. Anion exchange (Mono-Q) chromatography of the Superose 6 fraction yielded a protein peak that was enriched in carboxyltransferase activity and contained protein-bound 59Fe. Denaturing gels of the Mono-Q fraction indicated that the 180-kDa protein was composed of a 56-kDa subunit that was bound by an antibody raised against a synthetic β-carboxyltransferase (β-CTase) peptide. Incubation of the Mono-Q carboxyltransferase fraction with increasing concentrations of iron at a fixed substrate concentration resulted in increased initial velocities that fit well to a single rectangular three parameter hyperbola (v=vo+Vmax[FeSO4]/Km+[FeSO4]) consistent with iron functioning as a bound activator of catalysis. UV/Vis spectroscopy of the partially purified fraction before and after iron incubation yielded spectra consistent with a protein-bound metal cluster. These results suggest that the β-CTase subunit of MS-ACCase in soybean chloroplasts is an iron-containing enzyme, which may in part explain its labile nature.
    Type of Medium: Electronic Resource
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