Enhancement of chloroplast energization in Chlorella by treatments inactivating the nitrate-reducing system

Inactivation of the nitrate-reducing system in whole cells of Chlorella vulgaris Bejerinck by darkening, nitrogen starvation, ammonium, or cycloheximide brings cells into a state with a high yield of the millisecond-delayed fluorescence of chlorophyll. Activation of this system by illumination, by adding glucose to dark-adapted cells or nitrate to nitrogen-starved cells brings the cells into a low-yield state. The transitions between the lowand high-yield state induced by alternating light and dark periods are suppressed by tungstate and restored by subsequent molybdate addition. The drop in the delayed-fluorescence yield upon activation of the nitrate-reducing system is associated with the decrease of the amplitude of the electrochemical proton gradient across the thylakoid membrane of the chloroplast, as evidenced by the kinetics of the light-induced adsorption changes at 520 nm. The decrease of the proton gradient may be caused by the electron flow diverting from the cyclic path in photosystem I as a result of the activation of the electron transfer from ferredoxin to nitrite.Abbreviation DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Electron microscopic localization of calelectrin, a Mr 36 000 calcium-regulated protein, at the cholinergic electromotor synapse of Torpedo

Calelectrin is a calcium-binding protein of Mr 36 000 which has previously been shown to be associated with membranes of the cholinergic synapse in a calcium-dependent manner. We report here that calelectrin was solubilized from the electric organ of Torpedo marmorata in the absence of calcium together with proteins of Mr 54 000 and Mr 15 000. In cholinergic nerve endings isolated from the electric organ only calelectrin was solubilized in a calcium-dependent manner. A specific antiserum to calelectrin was used to localize the antigen by immunofluorescence microscopy on sections of electric organ and showed that calelectrin is distributed throughout the postsynaptic cell. Calelectrin was also detected in axons and in the cell bodies of the cholinergic neurones where it was concentrated in discrete patches throughout the cells. Electric organ tissue was processed to localize calelectrin with the electron microscope using an immunoperoxidase method. The most intense staining was observed on the cytoplasmic face of the acetylcholine receptor-containing postsynaptic membrane and also associated with the intracellular filaments of the electrocyte. The intensity of staining associated with these structures could be greatly reduced by preincubating the tissue with calcium chelators. In nerve terminals calelectrin was associated with synaptic vesicles in a polarized fashion. Calelectrin was also found on the cytoplasmic face of the synaptosomal plasma membrane and associated with neurofilaments. No extracellular staining was ever observed. Our results strongly support our original hypothesis that calelectrin is a calcium-regulated component of intracellular structure associated both with membranes and filaments.     

The localization and rate of disappearance of a synaptic vesicle antigen following denervation

Summary A proteoglycan-specific antiserum has been used to monitor the effects of denervation in the electric organ of Torpedo marmorata. The antiserum was produced by injecting a highly purified synaptic vesicle fraction prepared from the electric organs of Torpedo marmorata. Following absorption the serum appears to be specific towards synaptic vesicles. The ultrastructural localization of the antigen determined by immuno-electron microscopy confirmed the specificity of the antiserum and showed that it did not crossreact with the proteoglycans of the basal lamina. The rate of disappearance of the vesicle proteoglycans following denervation was evaluated by means of the antiserum and was compared to the rate of disappearance of other vesicular and nerve terminal-associated markers. The results suggest that degeneration affects the vesicular constituents at varying rates resulting in a progressive disappearance of the entire functional capacity of the synaptic vesicles.     

Uptake of Magnesium by Chromaffin Granules In Vitro: Role of the Proton Electrochemical Gradient

Abstract: The chromaffin granule membrane in vitro is impermeable to protons as well as to Mg²⁺; however, when granules are incubated in the presence of the proton ionophore carbonyl cyanide p -trifluoromethoxy-phenylhydrazone or an inhibitor of the granule membrane Mg²⁺-dependent ATPase, the metal ion is accumulated inside the granules. This accumulation is dependent upon the granule transmembrane potential. The simultaneous presence of the ATPase inhibitor and the proton ionophore markedly increases metal ion incorporation. Mg²⁺ incorporation is also promoted by nigericin in the presence of potassium or sodium ions, indicating that Mg²⁺ accumulation is also dependent upon the transmembrane pH gradient. Concomitant with the Mg²⁺ accumulation, there is a significant loss of endogenous catecholamines. It is concluded that Mg²⁺ accumulation is determined by the electrochemical gradient maintained across the membrane. Once the metal ion has accumulated into the granules it displaces catecholamines from their storage sites.     

An immunohistochemical study of synaptogenesis in the electric organ of Torpedo marmorata by use of antisera to vesicular and presynaptic plasma membrane components

Summary Synaptogenesis has been studied in the electric organ of embryonic Torpedo marmorata by use of two antisera directed against components of synaptic vesicles (anti-SV) and presynaptic plasma membranes (ap-anti-TSM), respectively. The anti-SV serum was previously shown to recognize a proteoglycan specific for synaptic vesicles. The ap-anti-TSM serum was raised to plasma membranes of synaptosomes derived from the electromotor nerve terminals and affinity-purified on electric-organ gangliosides. The vesicular antigen was first detectable at the 81-mm stage of development, which is 1–2 weeks earlier than the formation of morphologically mature presynaptic terminals, but is coincident with a rise in choline acetyltransferase levels and the ability of the electric organ to generate discharges. The gangliosidic antigen recognized by the ap-anti-TSM was first detectable on the ventral electrocyte surface at the 93-mm stage of development. This indicates that specific carbohydrate epitopes, not present on the growth cones, are expressed during maturation of the nerve terminal. The nerve terminal components recognized by these sera arose pari passu with neurite coverage of the ventral surface of the electrocyte, reaching a maximum in the adult. In contrast, postsynaptic aggregates of acetylcholine receptor, rendered visible with rhodamine-labeled -bungarotoxin, arose previous to the presynaptic antigens, reaching a maximum surface density at 110 mm and then declining in the adult.     

Teichoicase from Bacillus subtilis Marburg.

The properties of a teichoic acid degrading enzyme (teichoicase) isolated from Bacillus subtilis Marburg are described. The purified enzyme showed phosphodiesterase activity but not phosphomonoesterase activity, and it had an absolute substrate specificity for alpha-glucosylated glycerol teichoic acid, the endogenous cell wall teichoic acid of the enzyme-producing cell. The substrate was degraded by an exo-mechanism yielding the monomer alpha-D-glucose 1 leads to 2 (sn)glycero-3-phosphate. When B. subtilis Marburg was grown in a rich medium, enzyme activity was detected in extracts from sporulating cells. Teichoicase activity was present in a mutant blocked in stage II of the sporulation process but was absent in a mutant blocked in stage O. It was concluded that teichoicase is active on enzyme-producing cells since the reaction product could be detected in their culture supernatant. Attempts to demonstrate analogous enzyme activity in other Bacillus strains failed. The enzyme could be used for the rapid detection of alpha-glucosylated glycerol teichoic acid and for the controlled alteration of native bacterial cell surfaces exhibiting the appropriate structure.     

The mechanisms of fatty-acid inhibition of electron transport in chloroplasts

Unsaturated fatty acids at concentrations of 1–2 μmol mg^-1 chlorophyll decrease the intensity of long-lived delayed fluorescence and inhibit the Hill reaction in Pisum sativum L. chloroplasts in a pH-dependent and reversible manner. A charged form of the fatty acids is two times more effective than an undissociated form. Fatty acids, anionic and cationic detergents and urea inhibit activity and decrease the temperature of heat inactivation of the water-spilitting system. Sucrose at a concentration of 2.5 M protects chloroplasts against the effects of these compounds. It is concluded that their action can be explained by the denaturation of the water-splitting protein.     

Effect of 3-phenylindole on lipophilic ion and carrier-mediated ion transport across bilayer lipid membranes

The physical effects of 3-phenylindole, an antimicrobial compound which interacts with phospholipids, on ion transport across phosphatidylcholine-cholesterol bilayers have been investigated using three lipophilic ions and one ion-carrier complex. It was found that 3-phenylindole increased membrane electrical conductance of positively charged membrane probes and decreased electrical conductance of negatively charged probes. The enhancement of conductance detected by nonactin-K+ complex and tetraphenylarsonium+ was several orders of magnitude, whereas the suppression of conductance due to tetraphenylborate- and dipicrylamine- was less than a factor of ten. Presence of 3-phenylindole in aqueous phase slightly decreased adsorption of tetraphenylborate- and dipicrylamine- at the membrane surface. From the voltage dependence of the steady-state conductance it was shown that 3-phenylindole induced kinetic limitation of membrane transport of potassium mediated by nonactin. No such limitation was found in the case of tetraphenylarsonium+ transport. These results are shown to be consistent with the present concept of ion diffusion in membranes and the assumption that 3-phenylindole decreases the electric potential in the membrane interior. The asymmetry of the effect of 3-phenylindole on the magnitude of conductance changes for positively and negatively charged membrane permeable ions is also discussed as a reflection of the discreteness of both the absorbed 3-phenylindole and lipid dipoles.