Equilibration of methionine into the Met-tRNA pool of rabbit reticulocyte lysates: Additional evidence that deacylation of ribosomal subunit-bound Met-tRNAf does occur in heme-deficiency

We have measured the rate of equilibration of [³⁵S]methionine into the Met-tRNA pool of rabbit reticulocyte lysate as in [FEBS Lett. (1982) 143, 301–305]. Our results indicate that hemin-deficiency inhibits the equilibration of methionine into the tRNA pool much less than protein synthesis or the equilibration of alanine into the tRNA pool, whereas cycloheximide inhibits these processes similarly. This finding is consistent with our previous data and supports the hypothesis that with hemin-deficiency much of the Met-tRNA_f that becomes bound to 40 S subunits subsequently undergoes enzymatic deacylation.     

Role of voltage-dependent ionic currents in coupling glucose stimulation to insulin secretion in canine pancreatic islet B-Cells

Summary Glucose-induced electrical activity in canine pancreatic islet B cells is distinct from that in rodent islets, though both display Ca²⁺-dependent insulin secretion. Rodent islet B cells undergo regular bursts of Ca²⁺-dependent action potentials, while canine islet B cells generate isolated Na⁺-dependent action potentials which often give way to a plateau depolarization. Here we present evidence to reconcile the species difference in electrical activity with the similarity of Ca²⁺ dependence of secretion. (i) In canine B cells increasing glucose concentrations produce membrane depolarization and increasing frequency of Na_o-dependent action potentials until a background membrane potential (-40mV) is reached where Na⁺ currents are inactivated. (ii) Voltage-dependent Ca²⁺ currents are present which are activated over the voltage excursion of the action potential (–50 to +20 mV) and inactivate slowly, (over seconds) in the range of the plateau depolarization (–40 to –25 mV). Hence, they are available to contribute to both phases of depolarization. (iii) Tetrodotoxin (TTX) reduces by half an early transient phase of glucosestimulated insulin secretion but not a subsequent prolonged plateau phase. The transient phase of secretion often corresponds well in time to the period of initial high frequency action potential activity. These latter results suggest that in canine B cells voltagedependent Na⁺ and Ca²⁺ currents mediate biphasic glucose-induced insulin secretion. The early train of Na⁺-dependent action potentials, by transiently activating Ca²⁺ channels and allowing pulsatile Ca²⁺ entry, may promote an early transient phase of insulin secretion. The subsequent sustained plateau depolarization, by allowing sustained Ca²⁺ entry, may permit steady insulin release.     

Chronic Electroconvulsive Treatment Augments Coupling of the GTP-Binding Protein Gs to the Catalytic Moiety of Adenylyl Cyclase in a Manner Similar to That Seen with Chronic Antidepressant Drugs

A significant increase of guanylylimidodiphosphate (GppNHp)-, fluoride-, and forskolin-stimulated adenylyl cyclase was observed in synaptic membrane preparations from rat cerebral cortex subsequent to chronic electroconvulsive shock (ECS) treatment. This effect required at least five treatments over a course of 10 days. The inhibition of adenylyl cyclase induced by GppNHp was not affected by these treatments. The dissociation constant (KD) and maximal binding for the photoaffinity GTP analog, [32P]P3-(4-azidoanilido)-P1-5'-GTP [( 32P]AAGTP), to each of the synaptic membrane G proteins also were unchanged after ECS treatment. Nonetheless, the transfer of [32P]AAGTP from Gi to Gs, which we suggest is indicative of the coupling between Gs and the adenylyl cyclase catalytic moiety, was accelerated by chronic ECS treatment but not by acute or sham treatment. Furthermore, chemical uncoupling of Gs from adenylyl cyclase rendered membranes from treated animals indistinguishable from controls. Finally, in all cases tested, membranes prepared from animals subjected to chronic treatment with amitriptyline or iprindole showed similar changes in the Gs-mediated activation of adenylyl cyclase. Acute treatments produced effects similar to controls, and liver and kidney membranes from animals receiving chronic treatment showed no changes in adenylyl cyclase despite the marked changes seen in brain. These results suggest that chronic administration of ECS enhances coupling between Gs and adenylyl cyclase enzyme and modifies interactions between Gs and Gi.     

The proton-translocating nicotinamide adenine dinucleotide transhydrogenase

H⁺-transhydrogenase couples the reversible transfer of hydride ion equivalents between NAD(H) and NADP(H) to the translocation of protons across a membrane. There are separate sites on the enzyme for the binding of NAD(H) and of NADP(H). There are some indications of the position of the binding sites in the primary sequence of the enzymes from mitochondria andEscherichia coli. Transfer of hydride ion equivalents only proceeds when a reduced and an oxidized nucleotide are simultaneously bound to the enzyme. When p=0 the rate of interconversion of the ternary complexes of enzyme and nucleotide substrates is probably limiting. An increase in p accelerates the rate of interconversion in the direction of NADH NADP⁺ until another kinetic component, possibly product release, becomes limiting. The available data are consistent with either direct or indirect mechanisms of energy coupling.Abbreviations DCCD N N¹-dicyclohexylcarbodiimide - FSBA 5¹-[p-(fluorosulfonyl)benzoyl] adenosine - FCCP carbonylcyanide-p-fluoromethoxyphenylhydrazone - H⁺-Thase H⁺-transhydrogenase - thio-NADP⁺ thionicotinamide adenine dinucleotide phosphate - AcPdAd⁺ 3-acetylpyridine adenine dinucleotide - p proton electrochemical gradient - membane potential - pH pH difference across the membrane     

The mechanism of cumulus cell-oocyte uncoupling: Evidence for the participation of both cumulus cells and oocytes

Cumulus cells are metabolically coupled to oocytes via heterologous gap junctions. This coupling terminates near the time of ovulation, and the termination appears to be correlated with the mucification of the cumulus cells lying immediately adjacent to the oocytes. The first objective of this project was to determine whether follicle stimulating hormone (FSH) induction of cumulus cell-oocyte uncoupling could occur independently of FSH-stimulated cumulus mucification (expansion). Intercellular coupling was measured as a percentage of radiolabeled choline (or its metabolites) that was incorporated into the oocyte relative to the total amount of radiolabel incorporated into the entire cumulus cell-oocyte complex. It was found that the complete suppression of FSH-stimulated cumulus expansion with chondroitin sulfate B had no suppressive effect on FSH-stimulated cumulus cell-oocyte uncoupling. This finding showed that FSH-stimulated cumulus expansion was not required for cumulus cell-oocyte uncoupling. Since 17β-estradiol, testosterone, or progesterone could not induce maximal cumulus cell uncoupling, it was concluded that the uncoupling-promoting action of FSH was probably not mediated by steroid hormones. A partial uncoupling of cumulus cells and oocytes was found when spontaneous oocyte maturation had occurred in the absence of FSH. This partial uncoupling was prevented by incubation of cumulus cell-oocyte complexes in concentrations of dibutyryl cyclic adenosine monophosphate (dbcAMP) or 3-isobutyl-1-methyl xanthine (IBMX) (0.25 and 0.10 mM respectively) that suppressed spontaneous oocyte maturation without inducing cumulus expansion. These inhibitors also prevented the maximal induction of uncoupling that would have been provoked by biological grade preparations of either FSH or luteinizing hormone (LH). It was concluded that two factors were required to bring about maximal cumulus cell-oocyte uncoupling: one factor was dependent upon the action of gonadotropins on cumulus cell function, the other factor appeared to be a function of the oocytes, since maximal uncoupling could occur only after the germinal vesicles had broken down.     

Increase in the translatable mRNA for acetylcholine receptor during embryonic development of Torpedo ocellata electric organ

Single-turnover flash-induced ATP synthesis in chloroplasts was measured in situ with the luciferin luminescence method. In dark-adapted chloroplasts the first flashes only induce ATP hydrolysis. Once the reversible ATPase is fully activated, ATP hydrolysis persists for extended periods of darkness and flash-induced ATP-synthesis is optimal even at flash frequencies lower than 0.1 Hz. About one molecule of ATP is formed per 1000 chlorophyll and flash. In a low frequency flashing regime under steady state conditions, the newly formed ATP is stable. There is no threshold light intensity for flash-induced ATP synthesis. The data are in agreement with models involving short-range interaction between electron transport and the coupling factor.     

The reconstitution of a Photosystem II protein complex, P-700-chlorophyll a-protein complex and light-harvesting chlorophyll ab-protein

A Photosystem II reaction centre protein complex was extracted from spinach chloroplasts using digitonin. This complex showed (i) high rates of dichloroindophenol and ferricyanide reduction in the presence of suitable donors, (ii) low-temperature fluorescence at 685 nm with a variable shoulder at 695 nm which increased as the complex aggregated due to depletion of digitonin and (iii) four major polypeptides of 47, 39, 31 and 6 kDa on dissociating polyacrylamide gels. The Photosystem II protein complex, together woth the P-700-chlorophylla protein complex and light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$ complex (LHCP) also isolated using digitonin, were reconstituted with lipids from spinach chloroplasts to form proteoliposomes. The low-temperature (77 K) fluorescence properties of the various proteoliposomes were analysed. The $\text{F}{}_{685}\text{F}{}_{695}$ ratios of the Photosystem II reaction centre protein complex-liposomes decreased as the lipid to protein ratios were increased. The $\text{F}{}_{681}\text{F}{}_{697}$ ratios of LHCP-liposomes were found to behave similarly. Light excitation of chlorophyll b at 475 nm stimulated emission from both the Photosystem II protein complex (F₆₈₅ and F₆₉₅) and the P-700-chlorophyll a-protein complex (F₇₃₅) when LHCP was reconstituted with either of these complexes, demonstrating energy transfer between LHCP and PS I or II complexes in liposomes. No evidence was found for energy transfer from the PS II complex to the P-700-chlorophyll a-protein complex reconstituted in the same proteoliposome preparation. Proteoliposome preparations containing all three chlorophyll-protein complexes showed fluorescence emission at 685, 700 and 735 nm.     

Modulation of the chloroplast ATPase by tight binding of nucleotides

Inactivation of the chloroplast ATPase upon tight nucleotide binding was studied with several adenine nucleotide analogs. Compared with ADP, the other nucleoside diphosphates were less effective in the follwing order: IDP >?-ADP > 1-oxido-ADP > GDP. The nucleotide analogs compete with ADP for binding to the tight nucleotide-binding site(s) on the ATPase and also prevent further inactivation by ADP. AdoPP[NH]P also causes inactivation but has a lower affinity than ADP. [³H]GDP binds tightly to the ATPase, but the resulting enzyme-GDP complex is more readily dissociable than the enzyme-ADP complex. Although both nucleotides appear to bind to the same site, the catalytic and binding properties of the coresponding nucletide-enzyme complexes differ. Binding of GDP also decreases the rate and extent of the sontaneous decay of the activated enzyme. PP_i decreases the rate of inacivation caused by ADP and also the level of tigthly buond ADP. Based on these results, we suggest that two different confomations of the ATPase exist which contain tigthly bound ADP. The active conformation is conveted to the inactive conformation in the absence of a continued supply of energy by illumination or ATP hydrolysis.     

The induction kinetics of bacterial photophosphorylation. Threshold effects by the phosphate potential and correlation with the amplitude of the carotenoid absorption band shift

1. ATP synthesis (monitored by luciferin-luciferase) can be elicited by a single turnover flash of saturating intensity in chromatophores from Rhodopseudomonas capsulata, Kb1. The ATP yield from the first to the fourth turnover is strongly influenced by the phosphate potential: at high phosphate potential (?11.5 kcal/mol) no ATP is formed in the first three turnovers while at lower phosphate potential (?8.2 kcal/mol) the yield in the first flash is already one half of the maximum, which is reached after 2–3 turnovers.2. The response to ionophores indicates that the driving force for ATP synthesis in the first 20 turnovers is mainly given by a membrane potential. The amplitude of the carotenoid band shift shows that during a train of flashes an increasing ΔΨ is built up, which reaches a stationary level after a few turnovers; at high phosphate potential, therefore, more turnovers of the same photosynthetic unit are required to overcome an energetic threshold.3. After several (six to seven) flashes the ATP yield becomes constant, independently from the phosphate potential; the yield varies, however, as a function of dark time (t_d) between flashes, with an optimum for t_d = 160–320 ms.4. The decay kinetics of the high energy state generated by a long (125 ms) flash have been studied directly measuring the ATP yield produced in post-illumination by one single turnover flash, under conditions of phosphate potential (?10 kcal/mol), which will not allow ATP formation by one single turnover. The high energy state decays within 20 s after the illumination. The decay rate is strongly accelerated by 10^?8 M valinomycin.5. Under all the experimental conditions described, the amplitude of the carotenoid signal correlates univocally with the ATP yield per flash, demonstrating that this signal monitores accurately an energetic state of the membrane directly involved in ATP synthesis.6. Although values of the carotenoid signal much larger than the minimal threshold are present, relax slowly, and contribute to the energy input for phosphorylation, no ATP is formed unless electron flow is induced by a single turnover flash.7. The conclusions drawn are independent from the assumption that a ΔΨ between bulk phases is evaluable from the carotenoid signal.