Evidence for a sequestered solvent space in the chloroplast ATPase

Isolated coupling factor of photophosphorylation (CF₁) covalently labeled with eosin isothiocyanate was studied by polarized laser spectroscopy. Judged by the access of oxygen bound to eosin isothiocyanate and by the librational mobility of eosin isothiocyanate we conclude that activated CF₁ encloses a volume with solvent character. In the membrane-bound enzyme the sequestered volume becomes exposed when the membrane is energized.     

Enhanced glutamate,IP3 and cAMP activity in the cerebral cortex of Unilateral 6-hydroxydopamine induced Parkinson's rats: Effect of 5-HT,GABA and bone marrow cell supplementation

Parkinson's disease is characterized by progressive cell death in the substantia nigra pars compacta, which leads to dopamine depletion in the striatum and indirectly to cortical dysfunction. Increased glutamatergic transmission in the basal ganglia is implicated in the pathophysiology of Parkinson's disease and glutamate receptor mediated excitotoxicity has been suggested to be one of the possible causes of the neuronal degeneration. In the present study, the effects of serotonin, gamma-aminobutyric acid and bone marrow cells infused intranigrally to substantia nigra individually and in combination on unilateral 6-hydroxydopamine induced Parkinson's rat model was analyzed. Scatchard analysis of total glutamate and NMDA receptor binding parameters showed a significant increase in B_max (P < 0.001) in the cerebral cortex of 6-hydroxydopamine infused rat compared to control. Real Time PCR amplification of NMDA2B, mGluR5, bax, and ubiquitin carboxy-terminal hydrolase were up regulated in cerebral cortex of 6-hydroxydopamine infused rats compared to control. Gene expression studies of GLAST, ά-Synuclien and Cyclic AMP response element-binding protein showed a significant (P < 0.001) down regulation in 6-OHDA infused rats compared to control. Behavioural studies were carried out to confirm the biochemical and molecular studies. Serotonin and GABA along with bone marrow cells in combination showed reversal of glutamate receptors and behaviour abnormality shown in the Parkinson's rat model. The therapeutic significance in Parkinson's disease is of prominence.     

Quaternary structure of chloroplast F1-ATPase in solution. Conformational changes in spatial arrangement of subunits upon activation

The hydrodynamic properties of isolated ATPases were studied via their rotational diffusion in buffer solution. Chloroplast F1-ATPase (CF1) and Escherichia coli F1-ATPase (EF1) were covalently labeled with eosinisothiocyanate and then investigated by polarized laser spectroscopy. The rotational correlation time in aqueous buffer of latent (five-subunit) CF1 was 390 ns. Four-subunit (delta-deficient) CF1 showed the same correlation time, however, for three-subunit (delta, epsilon-deficient) CF1 the rotational correlation time was more than eight times larger (3200 ns). The rotational correlation time of activated CF1 was three times larger than the one of latent CF1. These large changes in the rotational correlation times are directly related to changes in the quaternary structure of CF1 upon activation. EF1 was found to behave essentially as activated CF1. Based on the observed rotational correlation times we concluded that the mass distributions of latent CF1 and of delta-deficient CF1 resemble a dimeric arrangement. The structure of delta, epsilon-deficient CF1 more likely resembles a hexagon, the mass centers of the six main subunits lie in one plane. The structure of the activated forms of CF1 can be described best as an intermediate between the dimeric arrangement of latent CF1 and an octahedron. The large changes in the quaternary structure of isolated CF1 are reversed when the activation of the enzyme is reversed.     

Proximity between fluorescent probes attached to four essential lysyl residues in phosphoenolpyruvate carboxylase. A resonance energy transfer study

Phosphoenolpyruvate carboxylase, purified from maize leaves, is rapidly inactivated by the fluorescence probe dansyl chloride. The loss of activity can be ascribed to the covalent modification of an R-NH2 group, presumably the epsilon-NH2 group of lysine. Analysis of the data by the statistical method of Tsou [Sci. Sin. 11, 1535-1558 (1962)] provides clear evidence that a pH 8 eight R-NH2 groups can be modified in the tetrameric form of the enzyme, four of which are essential for catalytic activity. Essential groups are modified about five times more rapidly than the non-essential ones. The enzyme was completely protected against inactivation by Mg2+ plus phosphoenolpyruvate and consequently binding of the modifier to the essential groups is completely abolished. Hence the four essential groups seemed to be located at or near the active site(s). One of the four essential groups was modified with dansyl chloride and the other three progressively with eosin isothiocyanate. In the doubly labeled protein non-radiative single-singlet energy transfer between dansyl chloride (donor) and eosin isothiocyanate (acceptor) was observed. The low variance (+/- 5%) in the efficiency of energy transfer obtained at a particular acceptor stoichiometry (0.8-1.1, 1.9-2.1, 2.9-3.1) in triplicate samples provided confidence that the measured transfer efficiency may be interpreted as transfer between specific sites. The distances calculated from the efficiency of resonance energy transfer revealed two acceptor sites, equally separated, 4.8-5.1 nm from the donor site and third site being 6.4 nm apart from the donor. Under conditions where the tetrameric enzyme dissociates into the monomers, no transfer of resonance energy between the protein-bound dansyl chloride and eosin isothiocyanate was observed. Most likely the four essential lysyl residues in the tetrameric enzyme are located in different subunits of the enzyme, hence each of the subunits would contain a substrate-binding site with one lysyl residue crucial for activity.