Two pathways of electron transfer in quinol-mediated cyclic phosphorylation in spinach chloroplasts

Low potential quinones are mediators of cyclic phosphorylation in washed spinach thylakoid membranes if they are prereduced to provide the proper redox poise. Cyclic phosphorylation catalyzed by different quinols varies in its sensitivity to the electron transfer inhibitor 2-iodo-6-isopropyl-3-methyl-2′,4,4′-trinitrodiphenyl ether (DNPINT), which is thought to inhibit electron flux from the bound plastoquinone (B) to the plastoquinone pool (Trebst, A., Wietoska, H., Draber, W. and Knops, H.J. (1978) Z. Naturforsch. 33c, 919–927). Cyclic phosphorylation catalyzed by uncharged quinols is extremely sensitive to DNPINT, whereas cyclic phosphorylation catalyzed by negatively charged quinols is approximately two orders of magnitude less sensitive. Many quinols have pK₁ values in the physiological range (pH 7–9). Increasing the concentration of the deprotonated quinol either by raising the assay pH, increasing the mediator concentration, or increasing the fractional reduction of the quinone results in a decrease in the sensitivity of cyclic phosphorylation to DNPINT. At very high DNPINT concentrations, cyclic phosphorylation catalyzed by all quinols (and ferredoxin) is inhibited, but not phenazine methosulfate catalyzed cyclic phosphorylation.These data suggest that the deprotonated form of the quinol can donate electrons directly to the plastoquinone pool, whereas the uncharged quinol most obligately transfer electrons through the bound plastoquinone ‘B’. A second site of DNPINT action after the plastoquinone pool is also observed, which requires much higher DNPINT concentrations for inhibition of phosphorylation.     

Hormonal stimulation of cAMP-dependent protein kinase in rat pancreas

The phosphorylation of myelin (basic protein) purified from rabbit brain was markedly stimulated by exogenously added calmodulin in the presence of calcium and inhibited by W-7(N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), a calmodulin interacting agent, in a dose-dependent fashion. However, exogenously added myelin basic protein free from protein kinase activity could not serve as a substrate of this calmodulin dependent protein kinase, suggesting that this kinase catalyzes the phosphorylation of the enzyme-substrate complex. These results suggest that a calmodulin-dependent protein kinase complex with the substrate (basic protein) is located in the myelin membrane of the central nervous system.     

Inhibition by polyamines of lipid peroxide formation in rat liver microsomes.

Both NADPH- and ascorbic acid-dependent lipid peroxidations were inhibited by spermine, the degree of inhibition being greater with the former peroxidation. The effective concentration of spermine required for inhibition was higher when larger amounts of microsomes were used. However, the activities of NADPH-cytochrome c reductase and NADPH-peroxidase were not influenced by spermine. These results suggest that spermine inhibits lipid peroxidation by binding to phospholipids in the microsomes.     

Activation by a calcium-binding protein of guanylate cyclase in Tetrahymena pyriformis.

A Ca²⁺-binding protein (TCBP), which was isolated from $\text{Tetrahymena pyriformis}$, enhanced about 20-fold particulate-bound guanylate cyclase activity in $\text{Tetrahymena}$ cells in the presence of a low concentration of Ca²⁺, while the adenylate cyclase activity was not increased. The enhancement was eliminated by ethylene glycol-bis (β-aminoethyl ether)-N,N′-tetraacetic acid. The enzyme activity was not stimulated by rabbit skeletal muscle troponin-C, the Ca²⁺-binding component of troponin, or other some proteins. In the presence of TCBP, stimulating effect of calcium ion on the enzyme activity was observed within the range of pCa 6.0 to 4.6, and was immediate and reversible.     

Specificity of nucleotide binding and coupled reactions utilising the mitochondrial ATPase

1. 1. Tightly bound ATP and ADP, found on the isolated mitochondrial ATPase, exchange only slowly at pH 8, but the exchange is increased as the pH is reduced. At pH 5.5, more than 60% of the bound nucleotide exchanges within 2.5 min.

2. 2. Preincubation of the isolated ATPase with ADP leads to about 50% inhibition of ATP hydrolysis when the enzyme is subsequently assayed in the absence of free ADP. This effect, which is reversed by preincubation with ATP, is absent on the membrane-bound ATPase. This inhibition seems to involve the replacement of tightly bound ATP by ADP.

3. 3. Using these two findings, the binding specificity of the tight nucleotide binding sites was determined. iso-Guanosine, 2′-deoxyadenosine and formycin nucleotides displaced ATP from the tight binding sites, while all other nucleotides tested did not. The specificities of the tight sites of the isolated and membrane-bound ATPase were similar, and higher than that of the hydrolytic site.

4. 4. The nucleotide specificities of ‘coupled processes’ nucleoside triphosphate-driven reversal of electron transfer, nucleoside triphosphate-³²P_i exchange and phosphorylation were higher than that of the hydrolytic site of the ATPase and similar to that of the tight nucleotide binding sites.

5. 5. The different nucleotide specificities of uncoupled ATP hydrolysis and coupled processes can be explained even if both processes involve a single common site on the ATPase molecule. This model requires that energy can be ‘coupled’ only when it is released/utilised in the nucleotide binding steps of the mechanism.

6. 6. Adenosine β,γ-imidotriphosphate (AMP-PNP) is not a simple reversible inhibitor of the ATPase, since incubation requires preincubation and is not reversed when the compound is diluted out, or by addition of ATP. This compound inhibits the isolated and membrane-bound ATPase equally well. Its guanosine analogue does not act in this way.

7. 7. In submitochondrial particles, ADP inhibited uncoupled hydrolysis of ATP much more effectively than coupled hydrolysis, the latter being measured both directly (from ATP hydrolysis in the absence of uncoupler) or indirectly, by monitoring ATP-driven reduction of NAD⁺ by succinate.

8. 8. The effects of ADP and AMP-PNP were interpreted as providing evidence for two of the intermediates in the proposed scheme for coupled triphosphate hydrolysis.

Factors required for calcium dependent acetylcholine release from isolated torpedo synaptic vesicles.

The release of acetylcholine (Ach) from Torpedo synaptic vesicles has been investigated. Factors have been found which induce Ca⁺² dependent Ach release from the synaptic vesicles. In the absence of these factors, the vesicles are not affected by Ca⁺². Addition of a soluble factor to the vesicles induces a Ca⁺²-dependent release of their Ach. This secretion is enhanced by a non-vesicular membranous component which, by itself, does not induce the Ca⁺²-dependent release. These results demonstrate that vesicular Ach release may be studied $\text{in vitro}$ and thus will enable the study, at the molecular level, of the biochemical events underlying neurotransmission.     

Radioimmune assay of tubulin applied to oligomers, synaptic membranes, and plants

A radioimmune assay for microtubule protein, tubulin, is described, in which unknown amounts of native or denatured tubulin can be quantitated by the ability to compete with pure [¹²⁵I]tubulin for rabbit antibodies produced against purified bovine brain tubulin. The assay is used to demonstrate that crude extracts of mouse brain contain negligible amounts of 30–36S tubulin oligomers under conditions where purified tubulin forms substantial amounts of such structures. Also, the particulate fraction of osmotically shocked and sonicated brain synaptosomes contains negligible tubulin antigenic activity. By contrast, soluble extracts of soybean, especially rapidly dividing regions of the plant, were found to contain significant amounts of cross-reacting material, providing further evidence for the conservative evolutionary nature of this ubiquitous and important protein.     

Influence of systems biology response and environmental exposure level on between-subject variability in breath and blood biomarkers

To explain the underlying causes of apparently stochastic disease, current research is focusing on systems biology approaches wherein individual genetic makeup and specific ‘gene–environment’ interactions are considered. This is an extraordinarily complex task because both the environmental exposure profiles and the specific genetic susceptibilities presumably have large variance components. In this article, the focus is on the initial steps along the path to disease outcome namely environmental uptake, biologically available dose, and preclinical effect. The general approach is to articulate a conceptual model and identify biomarker measurements that could populate the model with hard data. Between-subject variance components from different exposure studies are used to estimate the source and magnitude of the variability of biomarker measurements. The intent is to determine the relative effects of different biological media (breath or blood), environmental compounds and their metabolites, different concentration levels, and levels of environmental exposure control. Examples are drawn from three distinct exposure biomarker studies performed by the US Environmental Protection Agency that studied aliphatic and aromatic hydrocarbons, trichloroethylene and methyl tertiary butyl ether. All results are based on empirical biomarker measurements of breath and blood from human subjects; biological specimens were collected under appropriate Institutional Review Board protocols with informed consent of the subjects. The ultimate goal of this work is to develop a framework for eventually assessing the total susceptibility ranges along the toxicological pathway from exposure to effect. The investigation showed that exposures are a greater contributor to biomarker variance than are internal biological parameters.     

Hyaluronic acid salt — a mechanoelectrical transducer

Hyaluronic acid transduces a very gentle pressure into an electrical potential. Such pressure, depending on its direction, changes the optical rotary dispersion properties of the salt, either increasing the rotation in the direction already shown by the unpressured salt or changing and increasing the rotation in the opposite direction. These findings have implications for understanding the funtion of the cochlear and vestibular fluids, renal function, and the approximation to frictionless motion of normal joints.