Upon binding to a high‐affinity plasma membrane (PM) protein (a member of the 14‐3‐3 family of regulatory proteins), the fungal phytotoxin fusicoccin (FC) activates the H+‐ ATPase by hindering the inhibitory interaction of the enzyme’s C‐terminus with its catalytic site. Protease protection experiments carried out with sealed PM vesicles of different orientation proved that the FC‐binding site faces the cytoplasmic surface of the membrane. The in vivo induced activation of the H+‐ATPase by FC was retained during solubilization of PM proteins. Two‐dimensional gel systems combining a native separation of membrane protein complexes with a denaturing dimension as well as high‐performance anion‐exchange chromatography proved the existence of a labile ATPase:14‐3‐3 complex in plasma membranes. Stabilization of this complex could be achieved by FC treatment in vivo or in vitro. Mild proteolytic removal of the C‐terminal auto‐inhibitory domain of the H+ATPase liberated apparent hydrophobic 14‐3‐3 isoforms from the membrane in soluble form. During size exclusion chromatography of the proteolytically released proteins, co‐elution of 14‐3‐3 dimers, protein‐bound FC and the C‐terminus of the H+ATPase was observed. Moreover, the data suggest that 14‐3‐3 dimers themselves are not able to bind FC. Based on these results, it is proposed that the ‘FC receptor’ is represented by a labile complex between a 14‐3‐3 dimer and the H+‐ATPase whose formation is part of a mechanism regulating ATPase‐activity under physiological conditions. In our working model, binding of FC stabilizes this labile complex, thus leading to a strong and persistent activation of the H+‐ATPase in vivo. The possibility that the C‐terminus of the enzyme represents the binding domain for 14‐3‐3 homologs is discussed.

Whether physical activity increases risk or promotes progression of motor neurone degeneration in amyotrophic lateral sclerosis (ALS) is still debated. Current pathophysiological hypotheses include excitotoxicity, oxidative stress and increased calcium loads as causes of selective degeneration of vulnerable motor neurones. Vigorous exercise might amplify these factors by increasing firing rates at motor neurones. To test this hypothesis, we constrained a transgenic mouse model of ALS overexpressing the mutant human form of the Cu/Zn superoxide dismutase-1 (SOD-1) to a lifetime exercise on motor-driven running wheels for 10 h daily (active group, n = 12). Onset and progression of disease were assessed by grip strength, stride length and tight rope test. Data were compared with SOD-1 mice placed in running wheels set to slow speed (sedentary group, n = 13). Untreated SOD-1 mice were an additional control group (n = 12). We found no differences in disease onset, which was determined by a change-point analysis using an iterative fitting of segmented linear regression models, or in disease progression. However, the running group showed a non-significant 6-day improvement in survival (133.7 +/- 3.2 days) compared with the sedentary group (127.2 +/- 3.2 days) and a 4-day improvement compared with the control group (129.1 +/- 2.5 days). We demonstrate that a lifetime of vigorous exercise does not promote onset or progression of motor degeneration in SOD-1-mediated ALS. Moreover, the results suggest that the level of excitatory input and calcium turnover at motor neurones, both of which should be increased by running activity, do not interfere with the pathophysiology of SOD-1-mediated ALS.

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A monoclonal antibody PA3‐2‐B3, IgG1 (Λ) is described which specifically recognizes phaseic acid and shows very little cross‐reactivity (0.14%) with abscisic acid or dihydrophaseic acid (0.88%). Based on this antibody, an enzyme immunoassay was developed which displays a linearity range from 15 pg to 3 ng of phaseic acid. Results obtained with this assay agree with those obtained by gas chromatography‐electron capture detection. Using the novel enzyme immunoassay, as well as an established immunoassay for abscisic acid, levels of these two compounds in leaves of Phaseolus vulgaris were determined as a function of plant age, water stress, recovery from stress, and feeding of abscisic acid through the transpiration stream. The production of phaseic acid in a microsomal system from bean leaves was demonstrated. The results show a regulation of the plant's capacity to metabolize abscisic acid to phaseic acid as a function of water stress.

Monoclonal antibodies were raised and selected against recombinant Plantago major PmSUC2 sucrose carrier protein. Epitopes of two monoclonal antibodies (PS2-1A2 and PS2-4D4) were mapped using N-terminally truncated PmSUC2 proteins and a lambda library displaying random PmSUC2 peptides. PS2-1A2 recognizes an octapeptide close to the N-terminus of PmSUC2, PS2-4D4 binds to a decapeptide at the very C-terminus. Analyses of antibody binding to yeast protoplasts with functionally active, tagged PmSUC2 protein revealed that both epitopes are located in cytoplasmic domains of PmSUC2. These results support a model for plant sucrose transporters containing 12 transmembrane helices with the N-terminus and the C-terminus on the cytoplasmic side of the plasma membrane.z 1999 Federation of European Biochemical Societies.