Changes in phosphometabolites and intracellular pH in the tail muscle of the prawnPalaemon serratus as shown by in vivo31P-NMR

Summary ³¹P NMR spectra were recorded from tail muscles of the prawnPalaemon serratus, at rest, after exhaustive work and during subsequent recovery. At rest, the spectra indicated concentrations of phosphoarginine and ATP in good agreement with those obtained from resting fast skeletal muscles in mammals, which are characterized by a high phosphocreatine/P_i ratio. Following exhaustive work, phosphoarginine dropped by ca. 60% and ATP by 20%, while inorganic phosphate increased by 160%. The increase in inorganic phosphate immediately after contractions and in the first minutes of recovery corresponded partially to the changes in phosphoarginine and ATP. During recovery, the decrease of inorganic phosphate balanced the resynthesized phosphoarginine which was fully replenished within 30–40 min. The position of the inorganic phosphate resonance peak was used to monitor changes in intracellular pH (pH_i). The average pH_i in resting tail muscles was 7.20. After stimulation it was observed to decrease by 0.22 units. The return to pre-stimulation value was not achieved within 45 min. A NMR index (ATP+Arg-P)/(ATP+Arg-P+P_i) was calculated to characterize the extent of energetic changes caused by exercise.     

Induction of a non-specific permeability transition in mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts

In this study we used tightly-coupled mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts, possessing a respiratory chain with the usual three points of energy conservation. High-amplitude swelling and collapse of the membrane potential were used as parameters for demonstrating induction of the mitochondrial permeability transition due to opening of a pore (mPTP). Mitochondria from Y. lipolytica, lacking a natural mitochondrial Ca²⁺ uptake pathway, and from D. magnusii, harboring a high-capacitive, regulated mitochondrial Ca²⁺ transport system (Bazhenova et al. J Biol Chem 273:4372–4377, 1998a; Bazhenova et al. Biochim Biophys Acta 1371:96–100, 1998b; Deryabina and Zvyagilskaya Biochemistry (Moscow) 65:1352–1356, 2000; Deryabina et al. J Biol Chem 276:47801–47806, 2001) were very resistant to Ca²⁺ overload. However, exposure of yeast mitochondria to 50–100 μM Ca²⁺ in the presence of the Ca²⁺ ionophore ETH129 induced collapse of the membrane potential, possibly due to activation of the fatty acid-dependent Ca²⁺/nH⁺-antiporter, with no classical mPTP induction. The absence of response in yeast mitochondria was not simply due to structural limitations, since large-amplitude swelling occurred in the presence of alamethicin, a hydrophobic, helical peptide, forming voltage-sensitive ion channels in lipid membranes. Ca²⁺- ETH129-induced activation of the Ca²⁺/H⁺-antiport system was inhibited and prevented by bovine serum albumin, and partially by inorganic phosphate and ATP. We subjected yeast mitochondria to other conditions known to induce the permeability transition in animal mitochondria, i.e., Ca²⁺ overload (in the presence of ETH129) combined with palmitic acid (Mironova et al. J Bioenerg Biomembr 33:319–331, 2001; Sultan and Sokolove Arch Biochem Biophys 386:37–51, 2001), SH-reagents, carboxyatractyloside (an inhibitor of the ADP/ATP translocator), depletion of intramitochondrial adenine nucleotide pools, deenergization of mitochondria, and shifting to acidic pH values in the presence of high phosphate concentrations. None of the above-mentioned substances or conditions induced a mPTP-like pore. It is thus evident that the permeability transition in yeast mitochondria is not coupled with Ca²⁺ uptake and is differently regulated compared to the mPTP of animal mitochondria.     

The use of Escherichia coli bearing a phoN gene for the removal of uranium and nickel from aqueous flows

A Citrobacter sp. originally isolated from metal-polluted soil accumulates heavy metals via metal-phosphate deposition utilizing inorganic phosphate liberated via PhoN phosphatase activity. Further strain development was limited by the non-transformability of this environmental isolate. Recombinant Escherichia coli DH5α bearing cloned phoN or the related phoC acquired metal-accumulating ability, which was compared with that of the Citrobacter sp. with respect to removal of uranyl ion (UO₂ ²⁺) from dilute aqueous flows and its deposition in the form of polycrystalline hydrogen uranyl phosphate (HUO₂PO₄). Subsequently, HUO₂PO₄-laden cells removed Ni²⁺ from dilute aqueous flows via intercalation of Ni²⁺ into the HUO₂PO₄ lattice. Despite comparable acid phosphatase activity in all three strains, the E. coli DH5α (phoN) construct was superior to Citrobacter N14 in both uranyl and nickel accumulation, while the E. coli DH5α (phoC) construct was greatly inferior in both respects. Expression of phosphatase activity alone is not the only factor that permits efficient and prolonged metal phosphate accumulation, and the data highlight possible differences in the PhoN and PhoC phosphatases, which are otherwise considered to be related in many respects. Received: 30 December 1997 / Received revision: 25 March 1998 / Accepted: 26 March 1998     

In vivo nuclear magnetic resonance studies on the lugworm Arenicola marina. II Seasonal changes of metabolism

In vivo ³¹P- and ¹³C-NMR spectra of the lugworm Arenicola marina (Polychaeta, Annelida) gathered between 1988 and 1994 at different times of the year were evaluated for seasonally dependent metabolic changes. Beside the typical ³¹P-NMR signals of ATP and (phospho)taurocyamine, other seasonally dependent signals were observed: from January to March an intensive signal at 1.4–1.8 ppm was identified as inorganic phosphate compartmented in an acidic intestinal lumen. Between April and September signals at 1.2–1.4 ppm were assigned to phosphodiester. Starting in July males showed a second phosphagen signal [(phospho)creatine of spermatozoa, cf. Kamp and Juretschke (1989a)] whose intensity increased until spawning in September. The (phospho)taurocyamine/ATP ratio was also dependent on the season. In January or February the ratio reached 11, while in summer and autumn the ratio was between 4 and 5. As verified by biochemical assays this effect was caused by a significant decrease of ATP in the lugworm body wall during winter (December–February). The phosphagen (phospho)taurocyamine and the respective unphosphorylated guanidine taurocyamine remained constant throughout the year. Levels of free inorganic phosphate incurred similar changes to ATP. ¹³C-NMR spectra of lugworms showed a dramatic change in lipid stores. They were below the detection limit between January and March but developed into the most intensive signals during summer. The most abundant amino acids, glycine and alanine, were observed throughout the year while glycogen could not be detected in the ¹³C-NMR spectra. After treating tissue extracts with amyloglucosidase, the signals of the hydrolytic product glucose were recorded indicative of NMR-invisible glycogen stores.Abbreviations AN adenine nucleotides - NMR nuclear magnetic resonance - P _i inorganic phosphate - (P)Cr (phospho)creatine - PDE phosphodiester - PME phosphomonoester - PPA phenylphosphonic acid - (P)Tc (phospho)taurocyamine     

Fatty acid synthesis by isolated leucoplasts from developingBrassica seeds: Role of nucleoside triphosphates and DHAP-shuttle as the source of energy

Fatty acid synthesis in leucoplasts isolated from developing seeds ofBrassica campestris was absolutely dependent on external source of ATP. None of the other nucleoside triphosphates could replace ATP in the reaction mixture. Use of ADP alone also resulted in reduced rates of fatty acid synthesis. However, in combination with inorganic phosphate or inorganic pyrophosphate, it improved the rate of fatty acid synthesis, giving up to 50% of the ATP-control activity. Inorganic phosphate or inorganic pyrophosphate alone again did not serve as an energy source for fatty acid synthesis. AMP, alongwith inorganic pyrophosphate could promote fatty acid synthesis to up to 42% of the activity obtained with ATP. The three components dihydroxy acetone phosphate, oxaloacetic acid, inorganic phosphate of dihydroxy acetone phosphate-shuttle together could restore 50% of the activity obtained with ATP. Omission of any one of the components of this shuttle drastically reduced the rate of fatty acid synthesis to 15–24% of the ATP-control activity. Inclusion of ATP in reaction mixtures containing shuttle components enhanced the rate of synthesis over control. The optimum ratio of shuttle components dihydroxy acetone phosphate, oxaloacetic acid, inorganic phosphate determined was 1:1:2. Maximum rates of fatty acid synthesis were obtained when dihydroxy acetate phosphate was used as the shuttle triose. Glyceraldehyde-3-P, 3-phosphoglycerate, 2-phosphoglycerate and phosphoenolpyruvate as shuttle trioses were around 35–60% as effective as dihydroxy acetone phosphate in promoting fatty acid synthesis. The results presented here indicate that although the isolated leucoplasts readily utilize exogenously supplied ATP for fatty acid synthesis, intraplastidic ATP could also arise from dihydroxy acetone phosphate shuttle components or other appropriate metabolites     

Activation of the Cardiac Ryanodine Receptor by Sulfhydryl Oxidation is Modified by Mg2+ and ATP

The reactive disulfide 4,4′-dithiodipyridine (4,4′DTDP) was added to single cardiac ryanodine receptors (RyRs) in lipid bilayers. The activity of native RyRs, with cytoplasmic (cis) [Ca²⁺] of 10⁻⁷ m (in the absence of Mg²⁺ and ATP), increased within ∼1 min of addition of 1 mm 4,4′-DTDP, and then irreversibly ceased 5 to 6 min after the addition. Channels, inhibited by either 1 mm cis Mg²⁺ (10⁻⁷ m cis Ca²⁺) or by 10 mm cis Mg²⁺ (10⁻³ m cis Ca²⁺), or activated by 4 mm ATP (10⁻⁷ m cis Ca²⁺), also responded to 1 mm cis 4,4′-DTDP with activation and then loss of activity. P _o and mean open time (T _o ) of the maximally activated channels were lower in the presence of Mg²⁺ than in its absence, and the number of openings within the long time constant components of the open time distribution was reduced. In contrast to the reduced activation by 1 mm 4,4′-DTDP in channels inhibited by Mg²⁺, and the previously reported enhanced activation by 4,4′-DTDP in channels activated by Ca²⁺ or caffeine (Eager et al., 1997), the activation produced by 1 mm cis 4,4′-DTDP was the same in the presence and absence of ATP. These results suggest that there is a physical interaction between the ATP binding domain of the cardiac RyR and the SH groups whose oxidation leads to channel activation. Received: 8 September 1997/Revised: 20 January 1998     

Characterization of ATP-dependent proteolysis in embryos of the brine shrimp, Artemia franciscana

Under anoxia, embryos of Artemia franciscana enter a state of quiescence. During this time protein synthesis is depressed, and continued degradation of proteins could jeopardize the ability to recover from quiescence upon return to favorable conditions. In this study, we developed an assay for monitoring ATP/ubiquitin-dependent proteolysis in order to establish the presence of this degradation mechanism in A. franciscana embryos, and to describe some characteristics that may regulate its function during anoxia-induced quiescence. For lysates experimentally depleted of adenylates, supplementation with ATP and ubiquitin stimulated protein degradation rates by 92 ± 17% (mean ± SE) compared to control rates. The stimulation by ATP was maximal at concentrations ≥11 μmol · l⁻¹. In the presence of ATP and ubiquitin, ubiquitin-conjugated proteins were produced by lysates during the course of the 4-h assays, as detected by Western blotting. Acute acidification of lysates to values approximating the intracellular pH observed under anoxia completely inhibited ATP/ubiquitin-dependent proteolysis. Depressed degradation was also observed under conditions where net ATP hydrolysis occurred. These results suggest that ATP/ubiquitin-dependent proteolysis is markedly inhibited under cellular conditions promoted by anoxia. Inhibition of proteolysis during quiescence may be one critical factor that increases macromolecular stability, which may ultimately govern the duration of embryo survival under anoxia. Accepted: 2 November 1999     

Effects of Mechano-Gated Cation Channel Blockers on Xenopus Oocyte Growth and Development

The putative role(s) of a mechanically gated (MG) cation channel in Xenopus oocyte growth, maturation, fertilization and embryogenesis has been examined. Using a pharmacological approach, we have tested the effects of the MG channel blockers, gadolinium, gentamicin and amiloride on the above developmental events. Our results indicate that oocyte maturation, fertilization and early embryogenesis (up to the free-swimming stage 45) can proceed normally in the presence of concentrations of agents that either completely abolish (i.e., ≥10 μm Gd³⁺) or partially block (i.e., 1 mm gentamicin) single MG channel activity as measured by patch-clamp recording. However, we also find that higher concentrations of Gd³⁺ (≥50 μm) can lead to an increased percentage (>20%) of axis-perturbed embryos compared with control (<1%) and that amiloride (0.5 mm) reduces the success of fertilization (from 100% to <50%) and increases mortality (by ∼75%) in developing embryos. Furthermore, we find that all three agents inhibit oocyte growth in vitro. However, their order of effectiveness (amiloride > gentamicin > Gd³⁺) is opposite to their order for blocking MG channels (Gd³⁺≫ gentamicin > amiloride). These discrepancies indicated that the drugs effects occur by mechanisms other than, or in addition to, MG channel block. Our results provide no compelling evidence for the idea that MG channel activity is critical for development in Xenopus. This could mean that there are other mechanisms in the oocyte that can compensate when MG channel activity is blocked or that the protein that forms the channel can undergo additional interactions that result in a function insensitive to MG channel blockers. Received: 27 March 1998/Revised: 10 June 1998     

The role of suspended matter in the distribution of dissolved inorganic phosphate,iron and aluminium in the Ems estuary

In winter 1992/1993, a persistent local maximum in fluorescence, dissolved iron, dissolved aluminium and dissolved inorganic phosphate was found, upstream of the turbidity maximum in the freshwater zone of the Ems estuary (The Netherlands — Federal Republic Germany; western Europe). Upstream of this local maximum values ranged from 6 to 9 rel. units fluorescence, 0.9 to 2.4 μmol dm⁻³ iron, 0.5 to 0.7 μmol dm⁻³ aluminium and 0.6 to 2.3 μmol dm⁻³ dissolved inorganic phosphate. Within the maximum peak values of 24 rel. units fluorescence, 5.8 μmol dm⁻³ iron, 1.4 μmol dm⁻³ aluminium and 8.3 μmol dm⁻³ dissolved inorganic phosphate were observed. Downstream, fluorescence (indicator of dissolved organic carbon) showed conservative mixing with sea water, whereas dissolved iron, aluminium and dissolved inorganic phosphate did not. Dissolved aluminium and iron were quickly removed from solution to reach values of ∼100 nmol dm⁻³ aluminium and ∼0.3 μmol·dm⁻³ Fe at salinities of approximately 7 PSU. Further seaward iron concentrations gradually decreased to levels below 0.04 μmol dm⁻³. Dissolved aluminium first decreased to ∼20 nmol dm⁻³ at 29 PSU and increased again to concentrations of 30–44 nmol dm⁻³ at higher salinities. Dissolved inorganic phosphate, however, first decreased to upstream concentrations before reaching a secondary peak in the mid-estuarine reaches. At salinities >25 PSU dissolved inorganic phosphate mixed conservatively with sea water. It is hypothesized that adsorption-desorption equilibria are responsible for the local maximum values of fluorescence (DOC), iron, aluminium and dissolved inorganic phosphate. The similarity between the observed curves suggests a common underlying process, possibly related to the adjustment of new equilibria between suspended matter of marine and riverine origin.     

The SR Ca2+ ATPase of the Antarctic scallop Adamussium colbecki: cold adaptation and heavy metal effects

Cell calcium is accumulated in intracellular stores by sarco-endoplasmic reticulum Ca²⁺ ATPases functionally interacting with the membrane lipid environment. Cold adaptations of membrane lipids in Antarctic Sea organisms suggest possible adaptive effects also on sarco-endoplasmic reticulum Ca²⁺ ATPases. We investigated the SR Ca²⁺ ATPase of an Antarctic scallop, Adamussium colbecki, by characterising the enzyme activity and studying temperature effects. Ca²⁺ ATPase, assayed by following ATP hydrolysis, was thapsigargin- and vanadate-sensitive, showed maximum activity under 2 μM Ca²⁺, 200 mM KCl and pH 7.2, and had a K _M for ATP of 22 ± 7 μM. Temperature effects showed an Arrhenius inversion between −1.8 and 0°C, indicating cold adaptation, an Arrhenius break at 10°C, and a collapse above 20°C. A. colbecki accumulates high amounts of cadmium in the digestive gland; heavy metal effects on sarco-endoplasmic reticulum Ca²⁺ ATPases were therefore tested, finding an IC₅₀ = 0.9 μM for Hg²⁺ and 3 μM for Cd²⁺. Finally, SDS-PAGE analysis showed a main band at about 100 kDa, which was identified as sarco-endoplasmic reticulum Ca²⁺ ATPase after trypsin digestion, and accounted for 60% total protein. Accepted: 10 December 1998     

<Emphasis Type="Italic">Paracoccus denitrificans</Emphasis> proton-translocating ATPase: Kinetics of oxidative phosphorylation

The initial rates of ATP synthesis catalyzed by tightly coupled Paracoccus denitrificans plasma membrane were measured. The reaction rate was hyperbolically dependent on the substrates, ADP and inorganic phosphate (P_i). Apparent K _m values for ADP and P_i were 7–11 and 60–120 μM, respectively, at saturating concentration of the second substrate (pH 8.0, saturating Mg²⁺). These values were dependent on coupling efficiency. The substrate binding in the ATP synthesis reaction proceeds randomly: K _m value for a given substrate was independent of the concentration of the other one. A decrease of electrochemical proton gradient by the addition of malonate (when succinate served as the respiratory substrate) or by a decrease of steady-state level of NADH (when NADH served as the respiratory substrate) resulted in a proportional decrease of the maximal rates and apparent K _m values for ADP and P_i (double substitution, ping-pong mechanism). The kinetic scheme for ATP synthesis was compared with that described previously for the proton-translocating ATP hydrolysis catalyzed by the same enzyme preparation (T. V. Zharova and A. D. Vinogradov (2006) Biochemistry, 45, 14552–14558).     

The root surface phosphatases ofEriophorum vaginatum: Effects of temperature,pH, substrate concentration and inorganic phosphorus

Eriophorum vaginatum L. subsp.spissum (Fern.) Hult., a dominant plant in arctic tundra ecosystems, has acid phosphatase activity evenly distributed along its root surface from the root tip to a distance at least 16 cm from the tip. These root surface phosphatases have optimal activity from pH 3.5 to 4.0; mean soil pH for soil samples collected with roots was 3.69. Apparent energy of activation and Q₁₀ values (14.0 kcal mol⁻¹ and 2.2, respectively) do not provide evidence for temperature acclimation, but substantial phosphatase activity was measured at 1°C. Kinetic parameters determined for this root surface phosphatase were as follows: Km=9.23 mM, Vmax=1.61×10⁻³ μmoles mm⁻²h⁻¹. The presence of inorganic phosphorus in the assay medium did not inhibit root surface phosphatase activity except at very high concentrations (100 mM); even then, only slight inhibition was detected (7 to 19%). A comparison of hydrolysis rates with inorganic phosphate assimilation rates measured forE. vaginatum indicates that organic phosphate hydrolysis may occur at approximately one third the rate of inorganic phosphate absorption. Calculations show that inorganic phosphate produced by root surface phosphatase activity may satisfy 65% of the annual phosphate demand ofE. vaginatum. Since arctic tundra soils are typically higher in dissolved organic phosphorus compounds than in inorganic phosphate, root surface phosphatase activity may make a considerable contribution to the phosphate nutrition of this widespread and abundant arctic plant.     

Tonoplast inorganic pyrophosphatase in Vicia faba guard cells

Lysed guard-cell protoplasts of Vicia faba L. exhibited hydrolytic activity characteristic of tonoplast inorganic pyrophosphatase (V-PPase; EC 3.6.1.1). Activity was inhibited by the specific V-PPase inhibitor aminomethylenediphosphonate, stimulated by K⁺ (K _m = 51 mM) and inhibited by Ca²⁺ (80 nM free Ca²⁺ was required for 50% inhibition at 0.27 mM free Mg²⁺). Patch-clamp measurements of electrogenic activity confirmed enzyme localisation at the tonoplast. This is the first report of V-PPase activity in guard cells; its possible involvement in stomatal opening is discussed. Received: 12 February 1998 / Accepted: 24 April 1998     

Role of microorganisms in corrosion inhibition of metals in aquatic habitats

In acetate-limited chemostat cultures of Acinetobacter johnsonii 210A at a dilution rate of 0.1 h⁻¹ the polyphosphate content of the cells increased from 13% to 24% of the biomass dry weight by glucose (100 mM), which was only oxidized to gluconic acid. At this dilution rate, only about 17% of the energy from glucose oxidation was calculated to be used for polyphosphate synthesis, the remaining 83% being used for biomass formation. Suspensions of non-growing, phosphate-deficient cells had a six- to tenfold increased uptake rate of phosphate and accumulated polyphosphate aerobically up to 53% of the biomass dry weight when supplied with only orthophosphate and Mg²⁺. The initial polyphosphate synthesis rate was 98 ± 17 nmol phosphate min⁻¹ mg protein⁻¹. Intracellular poly-β-hydroxybutyrate and lipids served as energy sources for the active uptake of phosphate and its subsequent sequestration to polyphosphate. The H⁺-ATPase inhibitor N,N′-dicyclohexylcarbodiimide caused low ATP levels and a severe inhibition of polyphosphate formation, suggesting the involvement of polyphosphate kinase in polyphosphate synthesis. It is concluded that, in A. johnsonii 210A, (i) polyphosphate is accumulated as the energy supply is in excess of that required for biosynthesis, (ii) not only intracellular poly-β-hydroxybutyrate but also neutral lipids can serve as an energy source for polyphosphate-kinase-mediated polyphosphate formation, (iii) phosphate-deficient cells may accumulate as much polyphosphate as activated sludges and recombinants of Escherichia coli designed for polyphosphate accumulation. Received: 23 October 1998 / Received revision: 18 January 1999 / Accepted: 22 January 1999     

Propionyl-CoA carboxylase of Myxococcus xanthus: catalytic properties and function in developing cells

An acyl-coenzyme A carboxylase that carboxylates acetyl-CoA, butyryl-CoA, propionyl-CoA, and succinyl-CoA was purified from Myxococcus xanthus. Since the enzyme showed maximal rates of carboxylation with propionyl-CoA, the enzyme is thought to be propionyl-CoA carboxylase. The apparent K _m values for acetyl-CoA, butyryl-CoA, propionyl-CoA, and succinyl-CoA were found to be 0.2, 0.2, 0.03, and 1.0 mM, respectively. The native enzyme has a molecular mass of 605–615 kDa and is composed of nonidentical subunits (α and β) with molecular masses of 53 and 56 kDa, respectively. The enzyme showed maximal activity at pH 7.0–7.5 and at 25–30°C, and was affected by variation in concentrations of ATP and Mg²⁺. During development of M. xanthus, the propionyl-CoA carboxylase activity increased gradually, with maximum activity observed during the sporulation stage. Previous work has shown that a propionyl-CoA-carboxylase-deficient mutant of M. xanthus reduces levels of long-chain fatty acids. These results suggest that the propionyl-CoA carboxylase is also responsible for the carboxylation of acetyl-CoA to malonyl-CoA used for the synthesis of long-chain fatty acids during development. Received: 24 February 1998 / Accepted: 25 May 1998     

Inhibitory Effects of Adenine Nucleotides on Brain Mitochondrial Permeability Transition

The adenine nucleotides ADP and ATP are probably the most important endogenous inhibitors of the mitochondrial permeability transition (MPT). We studied the inhibitory effects of adenine nucleotides on brain MPT by measuring mitochondrial swelling and Ca²⁺ and cytochrome c release. We observed that in the presence of either ADP or ATP, at 250 μM, brain mitochondria accumulated more than 1 μmol Ca²⁺ × mg protein⁻¹. ADP or ATP also prevented Ca²⁺-induced mitochondrial swelling and cytochrome c release. Interestingly, ATP lost most of its inhibitory effects on MPT when the experiments were carried out in the presence of ATP-regenerating systems. These results indicate that MPT inhibition observed in the presence of added ATP could be mainly due to hydrolysis of ATP to ADP. From mitochondrial swelling measurements, half-maximal inhibitory values (K _i) of 4.5 and 98 μM were obtained for ADP and ATP, respectively. In addition, a delayed mitochondrial swelling sensitive to higher ADP concentrations was observed. Mitochondrial anoxia/reoxygenation did not interfere with the inhibitory effect of ADP on Ca²⁺-induced MPT, but oxidative phosphorylation markedly decreased this effect. We conclude that ADP is a potent inhibitor of brain MPT whereas ATP is a weaker inhibitor of this phenomenon. Our results suggest that ADP can have an important protective role against MPT-mediated tissue damage under conditions of brain ischemia and hypoglycemia.     

Environmental and physiological factors affecting the uptake of phosphate by Chlorobium limicola

The uptake of soluble phosphate by the green sulfur bacterium Chlorobium limicola UdG6040 was studied in batch culture and in continuous cultures operating at dilution rates of 0.042 or 0.064 h^–1. At higher dilution rates, washout occurred at phosphate concentrations below 7.1 μM. This concentration was reduced to 5.1 μM when lower dilution rates were used. The saturation constant for growth on phosphate (K _μ) was between 2.8 and 3.7 μM. The specific rates of phosphate uptake in continuous culture were fitted to a hyperbolic saturation model and yielded a maximum rate (Va _max) of 66 nmol P (mg protein)^–1 h^–1 and a saturation constant for transport (K _t) of 1.6 μM. In batch cultures specific rates of phosphate uptake up to 144 nmol P (mg protein)^–1 h^–1 were measured. This indicates a difference between the potential transport of cells and the utilization of soluble phosphate for growth, which results in a significant change in the specific phosphorus content. The phosphorus accumulated within the cells ranged from 0.4 to 1.1 μmol P (mg protein)^–1 depending on the growth conditions and the availability of external phosphate. Transport rates of phosphate increased in response to sudden increases in soluble phosphate, even in exponentially growing cultures. This is interpreted as an advantage that enables Chl. limicola to thrive in changing environments. Received: 9 February 1998 / Accepted: June 1998     

Regulation of ribulose-1,5-bisphosphate carboxylase activity by the activase system in lysed spinach chloroplasts

Ribulose-1,5-bisphosphate (RuBP) carboxylase in lysed spinach (Spinacia oleracea L. cv virtuosa) chloroplasts that had been partly inactivated at low CO₂ and Mg²⁺ by incubating in darkness with 4 millimolar partially purified RuBP was reactivated by light. If purified RuBP was used to inhibit dark activation of the enzyme, reactivation by light was not observed unless fructose-1,6-bisphosphate, ATP, or ADP plus inorganic phosphate were also added. Presumably, ADP plus inorganic phosphate acted as an ATP-generating system with a requirement for the generation of ΔpH across the thylakoid membrane. When the RuBP obtained from Sigma Chemical Co. was used, light did not reactivate the enzyme. There was no direct correlation between ΔpH and activation. Therefore, thylakoids are required in the ribulose-1,5-bisphosphate carboxylase activase system largely to synthesize ATP. Inactivation of RuBP carboxylase in isolated chloroplasts or in the lysed chloroplast system was not promoted simply by a transition from light to dark conditions but was caused by low CO₂ and Mg²⁺.     

Regulation of ANP-stimulated guanylate cyclase in the presence of Mn2+ in rat lung membranes

The catalytic activity of guanylate cyclase (GCase) coupled to atrial natriuretic peptide (ANP) receptor depends on the metal co-factor, Mn²⁺ or Mg²⁺. ATP synergistically stimulates the ANP-stimulated GCase in the presence of Mg²⁺. We have now shown the ATP regulation of the ANP-stimulated GCase in the presence of Mn²⁺ in rat lung membranes. ANP stimulated the GCase 2.1-fold compared to the control. ATP enhanced both the basal (basal-GCase) and the ANP-stimulated GCase maximally 1.7- and 2.3- fold compared to the control, respectively, at a concentration of 0.1 mM. The stimulation by ATP was smaller in the presence of Mn²⁺ than in the presence of Mg²⁺. The addition of inorganic phosphate to the reaction mixture altered the GCase activities in the presence of Mn²⁺ with or without ANP and/or ATP. In the presence of 10 mM phosphate, ATP dose-dependently stimulated the basal GCase 5-fold compared to the control at a concentration of 1 mM and augmented the ANP-stimulated GCase, which was 4.2-fold compared to the basal-GCase, 5.5-fold compared to the control at a concentration of 0.5 mM. Protein phosphatase inhibitors, okadaic acid (100 nM), H8 (1 M) and staurosporin (1 M), did not alter the activity. Orthovanadate (1 mM), an inorganic phosphate analogue, significantly stimulated both the basal-GCase and the ANP-stimulated GCase, which were inhibited by ATP. It was assumed that phosphate and orthovanadate might interact with the GCase to regulate the activity in the opposite manner. This was the first report that inorganic phosphate and orthovanadate affected the ATP-regulation of the ANP-stimulated GCase in the presence of Mn²⁺.     

Isolation, culture and regeneration of avocado (Persea americana Mill.) protoplasts

Protoplasts were isolated from embryogenic suspension cultures derived from avocado (Persea americana Mill.) zygotic embryos and nucellus in an enzyme digestion solution consisting of 1% cellulase Onozuka RS, 1% Macerase R10, 0.2% Pectolyase Y-23, 0.7 M mannitol. 24.5 mM CaCl₂, 0.92 mM NaH₂PO₄ and 6.25 2-[N-morpholino]ethanesulfonic acid (1.5 ml) mixed with 0.7 M MS^–8P (2.5 ml). MS^-8P medium consisted of Murashige and Skoog salts without NH₄NO₃, 1 mg l^–1 thiamine HCl, 100 mg l^–1 myo-inositol, 3.1 g l^–1 glutamine and 8P organic addenda. Medium osmolarity was adjusted with 0.15 M sucrose and 0–0.55 M mannitol. Protoplast yields of 3.5×10⁶ protoplasts g^–1 were obtained. Growth and development of the protoplasts were significantly affected by osmolarity, nitrogen source, plating density and culture medium dilution. Under optimum conditions, proembryos developed directly from embryogenic protoplasts and subsequently into somatic embryos. Optimum conditions for somatic embryo development included the culture of protoplasts at a density of 0.8–1.6×10⁵ ml^–1 in 0.4 M MS^–8P for 2–3 weeks, followed by subculture in 0.15 M MS^–8P at a diluted density of 20–40× for 1 month in darkness to obtain somatic embryos. Mature somatic embryos were recovered on semisolid medium; however, a low frequency of plantlet recovery (≤1%) from protoplast-derived somatic embryos was observed. Received: 9 February 1998 / Revision received: 4 May 1998 / Accepted: 15 May 1998