Article Figures & Data
Figures
- Fig. 1.
Western blot of tomato and tobacco membrane fractions. Supernatant (S) and microsomal membranes (M) were separated by centrifugation at 203,000g. The membrane pellet was then fractionated by the aqueous two-phase partitioning method into a lower phase (L) enriched for intracellular membranes and the upper phase (U) enriched with plasma membranes. Proteins (25 μg per lane) from each fraction (S, M, L, and U) were fractionated by denaturing SDS-PAGE, and immunoblotted. Blots were probed with antibodies raised against plasma membrane H-ATPase (P-ATPase), plasma membrane tobacco calmodulin-binding protein (NtCBP4), and the 60-kD subunit of vacuolar H-ATPase from oat root (V-ATPase). The relative density was established by scanning the gel as described in “Materials and Methods.” In the case of the P-ATPase the lower band was scanned.
- Fig. 2.
NADPH oxidase activity gels of tomato and tobacco leaf upper phase plasma membranes fractionated by native and denaturing SDS-PAGE. A, Tomato and tobacco membranes (50 μg) were fractionated by native PAGE and assayed for NADPH oxidase activity with or without the addition of DPI or O2 −dismutase (SOD). B, Tomato and tobacco membranes (100 μg) were fractionated by denaturing SDS-PAGE and assayed for NADPH oxidase activity with or without the addition of DPI or SOD. C, Activity gel of refractionated NADPH oxidase activity bands. Membrane proteins of the upper phase were fractionated by native PAGE as in A. The formazan stained bands were excised, subsequently refractionated by denaturating SDS-PAGE, and an in-gel activity assay was carried out.
- Fig. 3.
Activity gel assay and immunodetection of NADPH oxidase in tomato plasma membranes. Membranes (M) were fractionated by the aqueous two-phase partitioning method into a lower phase (L) enriched with intracellular membranes and an upper phase (U) enriched with plasma membranes. Proteins from each fraction (60 μg per lane) were fractionated by denaturating SDS-PAGE and immunoblotted with antisera against the C-terminal portion of the tomato Rboh (western blot), or stained for NADPH activity (SDS activity) or Coomassie Blue (Coomassie) as described in “Materials and Methods.”
- Fig. 4.
NADPH oxidase activity in isolated tomato and tobacco membranes. NADPH activity was assayed in membranes of the upper phase of aqueous two-phase partitioned membranes. XTT reduction by O2 − is shown corrected for reduction in the presence of SOD (50 units mL−1). Where indicated 10 mm EGTA was added. Data are mean ± se of four repeats and represent one of three different experiments that yielded essentially identical results.
- Fig. 5.
In-gel assay for NADPH oxidase activity in tomato membranes as affected by Ca+2 in the reaction medium. Plasma membrane proteins (60 μg per lane) were fractionated by denaturating SDS-PAGE or native PAGE and stained for activity in the presence or absence of Ca+2 in the reaction buffer.
- Fig. 6.
Plasma membrane NADPH oxidase activities and O2 − production in leaf discs and membranes isolated from TMV-inoculated and control tobacco leaves. A, NADPH oxidase in isolated membranes (left) and extracellular O2 − generating activity of leaf discs (right). In each measurement, amounts measured are based on equal fresh weight as described in “Results.” B, In-gel assay for NADPH oxidase activity in plasma membranes isolated from control and TMV-infected leaves. In each measurement, amounts loaded on the gels are based on equal fresh weight as described in “Results.”
Tables
- Table I.
Effect of the detergents SDS, CHAPS, and Triton X-100 on tomato plasma membrane NADPH oxidase activity1-a
Treatment Concentration Relative Activity μM and % % ± se No detergent 0 100 ± 6 SDS 40 (0.0012%) 85 ± 5 100 (0.0029%) 54 ± 4 200 (0.0058%) 36 ± 4 3,467 (0.1%) 0.8 ± 2 34,674 (1%) 0 CHAPS 1,626 (0.1%) 75 ± 7 16,263 (1%) 25 ± 5 32,526 (2%) 21 ± 7 Triton X-100 15,480 (1%) 33 ± 5 -
↵F1-a Membrane fractions were obtained from the upper phase of aqueous two-phase partitioning. The superoxide production (reduction of 2, 3-bis [2-methoxy-4-nitro-5-sulfophenyl 2H-tetrazolium-5-carboxanilicle [XTT]]) was quantified as described in “Materials and Methods.” The activity is expressed as percentage of control values without detergents (100% = 0.395 ± 0.027 of XTT formazan absorbance at 470 0.6 h−1 3 μg−1 plasma membrane protein). Means and seare calculated for two to three independent plasma membrane preparations.
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- Table II.
Relative activity of NADPH oxidase activity bands (103–106 and 75–80 kD) in the fractionated supernatant and membrane proteins in the SDS in-gel assay
Fraction Molecular Soluble Protein Relative Activity Total Relative Activity (Soluble Protein × Relative Activity) kD μ fraction−1 Supernatant (203,000 g) 103–106 813,000 0 0 77–80 0 0 Pellet (203,000 g) 103–106 8,950 4 35,800 77–80 4 35,800 Lower phase 103–106 350 1 350 77–80 3 1,050 Upper phase 103–106 370 100 37,000 77–80 15 5,550 -
F2-a Extracts from 150 g of tomato leaves were centrifuged at 10,000g. The supernatant was subjected to centrifugation at 203,000g to yield the microsomal fraction. Lower and upper phases were partitioned from the microsomal fraction by the two-phase system and fractionated by SDS-PAGE as described in “Materials and Methods.” The activity gels were scanned and the results presented in arbitrary relative units.
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