31P NUCLEAR MAGNETIC RESONANCE STUDY OF INTRACELLULAR PHOSPHATE POOLS AND POLYPHOSPHATE METABOLISM IN HETEROSIGMA AKASHIWO (HADA) HADA (RAPHIDOPHYCEAE)1

The effects of starvation and subsequent addition of phosphate-containing medium on the phosphate metabolic intermediates were studied by ³¹P-NMR spectroscope of perchloric acid extracts and intact cells of Heterosigma akashiwo (Hada) Hada. When orthophosphate in the medium was completely depleted the medium was enriched with orthophosphate (4.5 μM). In the phosphate starved condition, the P cell quota was 76 fmol·cell⁻¹ and the major components of phosphate intermediates were phosphodiester, sugar phosphate and orthophosphate (P_i). After addition of P_i, rapid uptake of P_i was observed and the P cell quota increased to 108 fmol·cell⁻¹ in 2 h, 134 fmol·cell⁻¹ in 5 h and 222 fmol·cell⁻¹ in 1 day after addition of phosphate. The ³¹P-NMR spectrum indicated that a major portion of P was stored as polyphosphate, in which the average chain length of polyphosphate increased from 10 to 20 phosphate residues in one day after addition of P_i.     

31P NUCLEAR MAGNETIC RESONANCE STUDY OF INTRACELLULAR PHOSPHATE POOLS AND POLYPHOSPHATE METABOLISM IN HETEROSIGMA AKASHIWO (HADA) HADA (RAPHIDOPHYCEAE)1

The effects of starvation and subsequent addition of phosphate-containing medium on the phosphate metabolic intermediates were studied by ³¹P-NMR spectroscopy of perchloric acid extracts and intact cells of Heterosigma akashiwo (Hada) hada. When orthophosphate in the medium was completely depleted the medium was enriched with orthophosphate (4.5 μM). In the phosphate starved condition, the P cell quota was 76 fmol-cell⁻¹ and the major components of phosphate intermediates were phosphodiester, sugar phosphate and orthophosphate (P_i) After addition of P_i' rapid uptake of P_i was observed and the P cell quota increased to 108 fmol. cell⁻¹ in 2 h, 134 fmol. cell⁻¹ in 5 h and 222 fmol. cell⁻¹ in 1 day after addition of phosphate. The ³¹P-NMR spectrum indicated that a major portion of P was stored as polyphosphate, in which the average chain length of polyphosphate increased from 10 to 20 phosphate residues in one day after addition of P_i-     

STORAGE OF PHOSPHORUS IN NITROGEN-FIXING ANABAENA FLOS-AQUAE (CYANOPHYCEAE)1

P accumulation and metabolic pathway in N₂-fixing Anabaena flos-aquae (Lyngb.) Bréb were investigated in P-sufficient (20 μMP) and P-limited (2 μMP) turbidostats in combined N-free medium. The cyanobacterium grew at its maximum rate (μ_max, 1.13 d^?1) at the high P concentration and at 65% of μ_max under P limitation, with total cell P concentrations (Q_P) at steady states of 12.0 and 5.2 fmol·cell^?1, respectively. At steady state, polyphosphates (PP_i) accounted for only 3% of Q_P (0.4 fmol·cell^?1) in P-rich cells. Its concentration in P-limited cells was 5.8% (0.3 fmol·cell^?1). On the other hand, sugar P was very high at 22% of Q_P in P-rich cells and was undetectable in P-limited cells. Pulse chase experiments with ³²P showed that P-rich cells initially incorporated the labeled P into the acid-soluble PP_i fraction within the first few minutes and to a lesser extent into nucleotide P. Radioactivity in the PP_i then declined rapidly with concomitant increases in sugar P and nucleotide P fractions. In contrast, in P-limited cells, no radiolabel was detected in acid-soluble PP_i, and ³²P was initially incorporated into nucleotide P, sugar P, and ortho P fractions. The latter two fractions then subsequently declined. Therefore, under N₂-fixing conditions the cyanobacteria appeared to store P as sugar P and also utilize P through different pathways under P-rich and -limited conditions. When nitrate was supplied as the N source under P-sufficient conditions, PP_i accounted for about 15% of steady-state Q_P, but no sugar P was detected. Therefore, the same organism stored P in different cell P fractions depending on its N sources.     

PHOSPHATE ACCUMULATION AND METABOLISM BY HETEROSIGMA AKASHIWO (RAPHIDOPHYCEAE) DURING DIEL VERTICAL MIGRATION IN A STRATIFIED MICROCOSM1

Diel vertical migration by Heterosigma akashiwo (Hada) Hada (Raphidophyceae) was monitored in a 1.5 in tall microcosm. Vertical stratification, with low salinity and low orthophosphate (P_i) concentration in the upper layer and high salinity and high P_i concentration in the lower layer, was simulated in the tank, analogous to summer stratification in the Seto Inland Sea. The phosphate metabolism of H. akashiwo during this vertical migration was studied using ³¹P-NMR spectroscopy. At night this species migrated to the lower phosphate-rich layer and took up inorganic phosphate (P_i) which then was accumulated as polyphosphate (PP_i) by an increase in the chain length of PP_i During the daytime this species migrated to the phosphate-depleted surface water and utilized the accumulated PP_i for photophosphorylation by decreasing the chain length of PP_i During the first night after the phosphorus was introduced to the previously impoverished waters, the cells took up inorganic phosphate, accumulating the new phosphorus nutrient internally as P_i But the cells did not convert P_i to PP_i presumably due to their lack of ATP. After the second day of the experiment, conversion of P_i to PP_i at night was much more rapid than on the first day, presumably due to increased ATP availability. Then the cycle continued, with uptake of P_i and conversion to PP_i at night at the bottom and its utilization during the day at the surface. These data suggest that the role of PP_i in the metabolism of this species appears to be as a phosphate pool which regulates the level of P_i and ATP in the cell. Diel vertical migration allows this red tide species to shuttle between the phosphate-rich lower layer and the photic upper layer in stratified waters. ³¹P-NMR is shown to be a valuable tool in studying the phosphorus metabolism in migrating organisms.     

Relationships between dynamics of red tide-causing raphidophycean flagellates and algicidal micro-organisms in the coastal sea of Japan

Temporal fluctuations of algicidal micro-organisms against the red tide causing raphidophycean flagellates Chattonella antiqua (Hada) Ono and Heterosigma akashiwo (Hada) Hada ex Hara et Chihara were investigated using the microplate most probable number (MPN) method in northern Hiroshima Bay and Harima-Nada, the Seto Inland Sea, in 1992 and 1993. In Har-ima-Nada, both flagellates appeared at low levels (< 1 cell mL^?1), and killer micro-organisms against the two flagellates (C-killer for C. antiqua and H-killer for H. akashiwo) also appeared at low densities (< 2 mL^?1). In northern Hiroshima Bay, C. antiqua cells were scarce (< 1 cell mL^?1), and C-killers occurred at a low level (≤ 3.4 mL^?1). Conversely, red tides of H. akashiwo occurred there in June of both years. The dynamics of H-killers revealed a close relationship with that of H. akashiwo populations. H-killers followed the increase of H. akashiwo cells, reached a maximum level after the beginning of decline of H. akashiwo, maintained a high level for at least 1 week after the crash of bloom, and then decreased. C-killers consistently remained at low densities during the period of H. akashiwo red tides in both 1992 and 1993. Hence, algicidal micro-organisms specifically associated with the occurrence and crash of H. akashiwo red tides, and presumably contributed to the rapid termination of the red tides in the coastal seas such as northern Hiroshima Bay.     

EFFECTS OF NH4+ ASSIMILATION ON DARK CARBON FIXATION AND β-1,3-GLUCAN METABOLISM IN THE MARINE DIATOM SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)

The effects of NH₄⁺ assimilation on dark carbon fixation and β-1,3-glucan metabolism in the N-limited marine diatom Skeletonema costatum (Grev.) Cleve (Bacillariophyceae) were investigated by chemical analysis of cell components and incorporation of ¹⁴C-bicarbonate. The diatom was grown in pH-regulated batch cultures with a 14:10 h LD cycle until N depletion. The cells were then incubated in the dark with ¹⁴C-bicarbonate, but without a source of N for 2 h, then in the dark with 63 μmol·L⁻¹ NH₄⁺ for 3 h. Without N, the cellular concentration of free amino acids was almost constant (∼4.5 fmol·cell⁻¹). Added NH₄⁺ was assimilated at a rate of 12 fmol·cell⁻¹·h⁻¹, and the cellular amino acid pool increased rapidly (doubled in <1 h, tripled in <3 h). The glutamine level increased steeply (45× within 3 h), and the Gln/ Glu ratio increased from 0.1 to 2.4 within 3 h. The rate of dark C fixation during N depletion was only 1.0 fmol·cell⁻¹·h⁻¹. The addition of NH₄⁺ strongly stimulated dark C fixation, leading to an assimilation rate of 4.0 fmol·cell⁻¹·h⁻¹, corresponding to a molar C/N uptake ratio of 0.33. Biochemical fractionation of organic ¹⁴C showed no significant ¹⁴C fixation into amino acids during N depletion, but during the first 1–2 h of NH₄⁺ assimilation, amino acids were rapidly radiolabeled, accounting for virtually all net ¹⁴C fixation. These results indicate that anaplerotic β-carboxylation is activated during NH₄⁺ assimilation to provide C₄ intermediates for amino acid biosynthesis. The level of cellular β-1,3-d-glucan was constant (16.5 pg·cell⁻¹) during N depletion, but NH₄⁺ assimilation activated a mobilization of 28% of the reserve glucan within 3 h. The results indicate that β-1,3-glucan in diatoms is the ultimate substrate for β-carboxylation, providing precursors for amino acid biosynthesis in addition to energy from respiration.     

INTERSTRAIN VARIABILITY IN PHYSIOLOGY AND GENETICS OF HETEROSIGMA AKASHIWO (RAPHIDOPHYCEAE) FROM THE WEST COAST OF NORTH AMERICA1

High levels of intraspecific variability are often associated with HAB species, and this variability is likely an important factor in their competitive success. Heterosigma akashiwo (Hada) Hada ex Y. Hara et M. Chihara is an ichthyotoxic raphidophyte capable of forming dense surface‐water blooms in temperate coastal regions throughout the world. We isolated four strains of H. akashiwo from fish‐killing northern Puget Sound blooms in 2006 and 2007. By assessing numerous aspects of biochemistry, physiology, and toxicity, we were able to describe distinct ecotypes that may be related to isolation location, source population, or bloom timing. Contrasting elements among strains were cell size, maximum growth and photosynthesis rates, tolerance of low salinities, amino acid use, and toxicity to the ciliate grazer Strombidinopsis acuminatum (Fauré‐Fremiet). In addition, the rDNA sequences and chloroplast genome of each isolate were examined, and while all rDNA sequences were identical, the chloroplast genome identified differences among the strains that tracked differences in ecotype. H. akashiwo strain 07A, which was isolated from an unusual spring bloom, had a significantly higher maximum potential photosynthesis rate (28.7 pg C · cell^?1 · h^?1) and consistently exhibited the highest growth rates. Strains 06A and 06B were not genetically distinct from one another and were able to grow on the amino acids glutamine and alanine, while the other two strains could not. Strain 07B, which is genetically distinct from the other three strains, exhibited the only nontoxic effect. Thus, molecular tools may support identification, tracking, and prediction of strains and/or ecotypes using distinctive chloroplast gene signatures.     

Growth and phosphate uptake kinetics of the toxic dinoflagellate Alexandrium tamarense from Hiroshima Bay in the Seto Inland Sea, Japan

Shellfish poisoning by the toxic dinoflagellate Alexandrium tamarense (Lebour) Balech occurred for the first time in Hiroshima Bay, Japan, in 1992. Oyster culture in the bay produces as much as 60% of the total production in Japan, and it suffered severe damage. In the present study, we experimentally investigated the growth rate and phosphate uptake kinetics of A. tamarense, Hiroshima Bay strain. A short-term phosphate uptake experiment revealed that the maximum uptake rate was 1.4 pmol P cell^-1 per h and the half-saturation constant was 2.6 umol L^-1. In semicontin-uous culture, the maximum specific growth rate and the minimum phosphorus cell quota were 0.54 day^-1 and 0.56 pmol P cell^-1, respectively. These uptake rates suggest that A. tamarense is a poor phosphorus competitor compared with other species. However, the large phosphorus storage capacity (Q_pmax/q_o= 36), the surge phosphorus uptake ability (V_s/V_i= 4.1) and the low growth rate would be advantageous for surviving brief periods of phosphorus limitation which frequently occur in Hiroshima Bay.     

TOXIN PROFILE,PIGMENT COMPOSITION,AND LARGE SUBUNIT RDNA PHYLOGENETIC ANALYSIS OF AN ALEXANDRIUM MINUTUM (DINOPHYCEAE) STRAIN ISOLATED FROM THE FLEET LAGOON,UNITED KINGDOM1

Paralytic shellfish toxins, pigment composition, and large subunit (LSU) rDNA sequence were analyzed for a clonal culture of Alexandrium minutum Halim isolated in 2000 from the coastal Fleet Lagoon, Dorset, United Kingdom. The HPLC pigment analysis revealed the presence of chl a, peridinin, and diadinoxanthin as major pigments and chl c₁+c₂ and c₃, diatoxanthin, and β‐carotene as minor components. The toxins responsible for paralytic shellfish poisoning were analyzed by HPLC with postcolumn derivatization and fluorescence detection. The paralytic shellfish poisoning toxin profile of the Fleet Lagoon strain of A. minutum in exponential growth phase was dominated by gonyautoxin‐3 up to 54%, whereas gonyautoxin‐2 made up 10% and saxitoxin (STX) 36%. The average toxicity of the culture was 3.8 pg STX Eq·cell^?1, and total toxin content varied from 5.6 fmol·cell^?1 on day 1 to a maximum of 16.8 fmol·cell^?1 during the early stationary phase. Sequence analysis of the LSU rDNA revealed the strain to be closely related to several European strains of A. minutum and one isolated from Australian waters, although most of these do not produce STX. The shallow Fleet Lagoon may provide a favorable environment for A. minutum to bloom, and the presence of highly potent saxitoxins in this strain indicates potential for future shellfish contamination.     

The regulation of liver phosphoprotein phosphatase by inorganic pyrophosphate and cobalt.

The preincubation of rat liver crude extracts with ATP caused a 60% inactivation of phosphoprotein phosphatase in 30 min at 30 °C. The presence of Mg²⁺, or cyclic AMP, along with ATP in the preincubation mixture had no effect on the inactivation of phosphatase caused by ATP. The crude liver phosphatase was also inactivated by ADP or PP_i; PP_i being the most potent inactivating metabolite. AMP, adenosine or P_i were without any effect. The effect of ATP or PP_i was completely reversed by cobalt. The cobalt effect was very specific and could not be replaced by several metal ions tested except by Mn²⁺ which was partly active. With the aid of sucrose density gradient studies, it was also shown that PP_icauses an apparent conversion of a 4.1 S form to a 7.8 S form of the enzyme in rat liver extracts. Cobalt, on the other hand, converts the higher 7.8 S form to a lower 4.1 S form of the enzyme. The preincubation of purified rabbit liver phosphoprotein phosphatase with PP_i also caused a complete inactivation of the enzyme in 40 min. The inactivation of the enzyme by PP_i was completely reversed by cobalt. Unlike the apparent interconversion between different molecular forms of the enzyme by PP_i and cobalt in rat liver crude extracts, no such interconversion of purified rabbit liver phosphoprotein phosphatase was observed in the presence of PP_i and cobalt.     

Nutrient uptake and alkaline phosphatase (ec 3:1:3:1) activity of emiliania huxleyi (PRYMNESIOPHYCEAE) during growth under n and p limitation in continuous cultures

Emiliania huxleyi (strain L) expressed an exceptional P assimilation capability. Under P limitation, the minimum cell P content was 2.6 fmol P·cell^?1, and cell N remained constant at all growth rates at 100 fmol N·cell^?1. Both, calcification of cells and the induction of the phosphate uptake system were inversely correlated with growth rate. The highest (cellular P based) maximum phosphate uptake rate (V_max^P) was 1400 times (i.e. 8.9 h^?1) higher than the actual uptake rate. The affinity of the P‐uptake system (dV/dS) was 19.8 L·μmol^?1·h^?1 at μ = 0.14 d^?1. This is the highest value ever reported for a phytoplankton species. V_max and dV/dS for phosphate uptake were 48% and 15% lower in the dark than in the light at the lowest growth rates. The half‐saturation constant for growth was 1.1 nM. The coefficient for luxury phosphate uptake (Q_maxt/Q_min) was 31. Under P limitation, E. huxleyi expressed two different types of alkaline phosphatase (APase) enzyme kinetics. One type was synthesized constitutively and possessed a V_max and half‐saturation constant of 43 fmol MFP·cell^?1·h^?1 and 1.9 μM, respectively. The other, inducible type of APase expressed its highest activity at the lowest growth rates, with a V_max and half‐saturation constant of 190 fmol MFP·cell^?1·h^?1 and 12.2 μM, respectively. Both APase systems were located in a lipid membrane close to the cell wall. Under N‐limiting growth conditions, the minimum N quotum was 43 fmol N·cell^?1. The highest value for the cell N‐specific maximum nitrate uptake rate (V_max^N) was 0.075 h^?1; for the affinity of nitrate uptake, 0.37 L·μmol^?1·h^?1. The uptake rate of nitrate in the dark was 70% lower than in the light. N‐limited cells were smaller than P‐limited cells and contained 50% less organic and inorganic carbon. In comparison with other algae, E. huxleyi is a poor competitor for nitrate under N limitation. As a consequence of its high affinity for inorganic phosphate, and the presence of two different types of APase in terms of kinetics, E. huxleyi is expected to perform well in P‐controlled ecosystems.     

Photosystem 2 activity of <Emphasis Type="Italic">Citrus volkameriana</Emphasis> (L.) leaves as affected by Mn nutrition and irradiance

Citrus volkameriana (L.) plants were grown for 43 d in nutrient solutions containing 0, 2, 14, 98, or 686 μM Mn (Mn₀, Mn₂, Mn₁₄, Mn₉₈, and Mn₆₈₆, respectively). To adequately investigate the combined effects of Mn nutrition and irradiance on photosystem 2 (PS2) activity, irradiance response curves for electron transport rate (ETR), nonphotochemical quenching (q_N), photochemical quenching (q_P), and real photochemical efficiency of PS2 (Φ_PS2) were recorded under 10 different irradiances (66, 96, 136, 226, 336, 536, 811, 1 211, 1 911, and 3 111 μmol m⁻² s⁻¹, I₆₆ to I₃₁₁₁, respectively) generated with the PAM-2000 fluorometer. Leaf chlorophyll content was significantly lower under Mn excess (Mn₆₈₆) compared to Mn₀; its highest values were recorded in the treatments Mn₂-Mn₉₈. However, ETR and Φ_PS2 values were significantly lower under Mn₀ compared to the other Mn treatments, when plants were exposed to irradiances ≥96 μmol m⁻² s⁻¹. Furthermore, Mn₀ plants had significantly higher values of q_N and lower values of q_P at irradiances ≤226 and ≥336 μmol m⁻² s⁻¹, respectively, than those grown under Mn₂-Mn₆₈₆. Irrespective of Mn treatment, the values of Φ_PS2 and q_N decreased, while those of q_P increased progressively by increasing irradiance from I₁₃₆ to I₃₁₁₁. Finally, Mn₂-Mn₉₈ plants were less sensitive to photoinhibition of photosynthesis (≥811 μmol m⁻² s⁻¹) than the Mn₆₈₆ (≥536 μmol m⁻² s⁻¹) and Mn₀ (≥336 μmol m⁻² s⁻¹) ones.     

Growth and uptake kinetics of phosphate by benthic microalga Nitzschia sp. isolated from Hiroshima Bay,Japan

In the present study, we experimentally investigated the phosphate uptake kinetics of benthic microalga Nitzschia sp. isolated from Hiroshima Bay, Japan. The maximum uptake rate (ρ_max) obtained by short‐term experiments was 6.84 pmol cell^?1 h^?1 for phosphate. The half‐saturation constant for uptake (K_S) was 61.2 µmol cell^?1 h^?1. Both the ρ_max and Ks of this species were extremely high, suggesting that Nitzschia sp. is adapted to benthic environments, where nutrient concentrations are much higher than in the water column. The specific maximum growth rate (µ'_max) and minimum cell quota (Q₀) for the P‐limited condition, obtained by a semi‐continuous growth experiment, were 0.48 day^?1 and 0.045 pmol cell^?1, respectively. It is concluded that Nitzschia sp. could be a ‘storage strategist’ species, meaning it adapts so as to minimize the influence of fluctuations in phosphate conditions resulting from the change in redox conditions of sediment due to bioturbation.     

A requirement for chelation in obtaining functional chloroplasts of sunflower and wheat.

In studying conditions for obtaining photosynthetically functional chloroplasts from mesophyll protoplasts of sunflower and wheat, a strong requirement for chelation was found. The concentration of chelator, either EDTA or pyrophosphate (PP_i), required for maximum activation depended on the pH, the concentration of orthophosphate (P_i) in the assay, and the chelator used. Studies with EDTA indicate that including the chelator in the isolation, resuspension, and assay media, in the absence of divalent cations, was most effective. Increased concentration of EDTA from 1 to 10 mm broadened the pH response curve for photosynthesis, inasmuch as a higher concentration of chelator was required for activation of photosynthesis at lower pH.Either EDTA, PP_i, or citrate could activate photosynthesis of sunflower chloroplasts isolated and assayed at pH 8.4. At pH 7.6, PP_i and EDTA were equally effective at low P_i concentrations but PP_i was particularly effective in shortening the induction period at high concentrations of P_i (2.5 mm) in the assay medium. Including 1 mm 3-phosphoglycerate in the assay medium with or without P_i could not replace the need for chelation. However, 3-phosphoglycerate + EDTA in the assay medium with 0.5 mm P_i, pH 7.6, gave a short induction period and rates of photosynthesis similar to those with 10 mm PP_i. The results suggest that PP_i can have a dual effect at the lower pH through chelation and inhibition of the phosphate transporter.Photosynthesis by sunflower chloroplasts isolated and assayed at pH 8.4 with 0.2 mm EDTA (+ 0.5 mm P_i in the assays) was severely inhibited by 2 mM CaCl₂, MgCl₂, or MnCl₂. Wheat chloroplasts isolated and assayed at pH 8.4 without chelation, and assayed with 0.2 mm P_i, had low rates of photosynthesis (25 μmol O₂ evolved mg^?1 chlorophyll h^?1) which were strongly inhibited by 2 to 4 mm MgCl₂, MnCl₂, or CaCl₂. With inclusion of EDTA and P_i at optimum levels, isolated chloroplasts of sunflower and wheat have high rates of photosynthesis and PP_i or divalent cations are not of benefit.     

Yeast inorganic pyrophosphatase substrate recognition

Monodentate Co(NH₃)₅PP_i was determined not to be a substrate for yeast inorganic pyrophosphatase while P¹,P²-bidentate Co(NH₃)₄PP_i was turned over by the enzyme at a rate of 7.5 min^?1. A kinetic analysis of the substrate activities of the P¹,P²-bidentate complexes, Co(en)₂PP_i, Cr(NH₃)₄PP_i, Cr(H₂O)(NH₃)₃PP_i, Cr(H₂O)₂(NH₃)₂PP_i, and Cr(H₂O)₄PP_i was carried out in order to access the potential role of the metal-water ligands in productive binding. While substitution of the H₂O ligands with NH₃ ligands had a minimal affect on the K_m for Mg²⁺, the binding affinity of the complexes decreased with an increasing $\text{NH}{}_3\text{H}{}_2\text{O}$ ligand ratio as did the turnover number of the corresponding central complexes. The Co(en)₂PP_i complex was hydrolyzed at a rate approximately 0.6% of that for the Co(NH₃)₄PP_i complex. The substrate activities of β,γ-bidentate Co(NH₃)₄PPP_i and α,β,γ-tridentate Co(NH₃)₃PPP with pyrophosphatase were also tested. While both complexes were shown to bind tightly to the Mg²⁺-activated enzyme neither was hydrolyzed. On the other hand, in the presence of the Zn²⁺-activated enzyme the tridentate complex was turned over at a rate of 0.17 min^?1 while the bidentate complex remained inert to hydrolysis.     

Adenosine triphosphate sulfurylase from Penicillium chrysogenum equilibrium binding, substrate hydrolysis, and isotope exchange studies.

ATP sulfurylase from Penicillium chrysogenum was purified to homogeneity. The enzyme binds 8 mol of free ATP (K_s = 0.53 mM) or AMP (K_s = 0.50 mM) per 440,000 g. The results are consistent with our earlier report that the enzyme is composed of eight identical subunits of M_r 55,000 (J. W. Tweedie and I. H. Segel, 1971, Prep. Biochem. 1, 91–117; J. Biol. Chem. 246, 2438–2446). In the absence of cosubstrates, the purified enzyme catalyzes the hydrolysis of MgATP (to AMP and MgPP_i) and adenosine 5′-phosphosulfate (APS) (to AMP and SO₄^2?). MgATP hydrolysis is inhibited by nonreactive sulfate analogs such as nitrate, chlorate, and formate (uncompetitive with MgATP). In spite of the hydrolytic reactions it is possible to observe the binding of MgATP and APS to the enzyme in a qualitative (nonequilibrium) manner. Neither inorganic sulfate (the cosubstrate of the forward reaction) nor formate or inorganic phosphate (inhibitors competitive with sulfate) will bind to the free enzyme in detectable amounts in the absence or in the presence of Mg²⁺, Ca²⁺, free ATP, or a nonreactive analog of MgATP such as Mg-α,β-methylene-ATP. Similarly, inorganic pyrophosphate (the cosubstrate of the reverse reaction) will not bind in the absence or in the presence of Mg²⁺ or Ca²⁺. The induced binding of ³²P_i (presumably to the sulfate site) can be observed in the presence of MgATP. The results are consistent with the obligately ordered binding sequence deduced from the steady-state kinetics (J. Farley et al., 1976, J. Biol. Chem. 251, 4389–4397) and suggest that the subsites for SO^2?₄ or MgPP_i appear only after nucleotide cleavage to form E~AMP · MgPP_i or E~AMP · SO₄ complexes. The suggestion is supported by the relative values of K_ia (ca. 1 mm for MgATP) and K_iq (ca. 1 αm for APS) and by the inconsistent value of k_?1 calculated from $\text{V}{}_{\mathrm{<mtext>f</mtext>}}\text{K}{}_{\mathrm{<mtext>i</mtext><mtext>a</mtext>}}\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>A</mtext>}}$ (The value is considerably less than V_r) Purified ATP sulfurylase will also catalyze a Mg³²PP_i-MgATP exchange in the absence of SO₄^2?. A ³⁵SO₄^2?-APS exchange could not be demonstrated in the absence or presence of MgPP_i. This result was not unexpected: The rate of APS hydrolysis (or conversion to MgATP) is extremely rapid compared to the expected exchange rate. Also, the pool of APS at equilibrium is extremely small compared to the sulfate pool. The V values for molybdolysis, APS hydrolysis (in the absence of PP_i), ATP synthesis (from APS + MgPP_i), and Mg³²PP_i-MgATP exchange at saturating sulfate are all about equal (12–19 μmol × min^?1 × mg of enzyme^?1). The rates of Mg³²PP_i-MgATP exchange in the absence of sulfate, APS synthesis (from MgATP + sulfate), and MgATP hydrolysis (in the absence of sulfate) are considerably slower (0.10 – 0.35 μmol × min^?1 × mg of enzyme^?1). These results and the fact that k₄ calculated from $\text{V}{}_{\mathrm{<mtext>r</mtext>}}\text{K}{}_{\mathrm{<mtext>i</mtext><mtext>q</mtext>}}\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>Q</mtext>}}$ is considerably larger than V_f suggest that the rate-limiting step in the overall forward reaction is the isomerization reaction E~AMP-SO^2?₄ → EAPS. In the reverse direction the rate-limiting step may be SO^2?₄ release or isomerization of the E~AMP · MgPP_i · SO₄^2? complex. (The reaction appears to be rapid equilibrium ordered.) Reactions involving the synthesis or cleavage of APS are specific for Mg²⁺. Reactions involving the synthesis or cleavage of ATP will proceed with Mg²⁺, with Mn²⁺, and, at a lower rate, with Co²⁺. The results suggest that the enzyme possesses a Mg²⁺-preferring divalent cation (activator) binding site that is involved in APS synthesis and cleavage and is distinct from the MeATP or MePP_i site. The equilibrium binding of about one atom of ⁴⁵Ca²⁺ per subunit (possibly to the activator site) could be demonstrated (K_s = 1.4 mM).     

Catalysis by isolated β-subunits of the ATP Synthase/ATPase from Thermophilic bacillus PS3. Hydrolysis of Pyrophosphate

Although the capacity of isolated β-subunits of the ATP synthase/ATPase to perform catalysis has been extensively studied, the results have not conclusively shown that the subunits are catalytically active. Since soluble F₁ of mitochondrial H⁺-ATPase can bind inorganic pyrophosphate (PP_i) and synthesize PP_i from medium phosphate, we examined if purified His-tagged β-subunits from Thermophilic bacillus PS3 can hydrolyze PP_i. The difference spectra in the near UV CD of β-subunits with and without PP_i show that PP_i binds to the subunits. Other studies show that β-subunits hydrolyze [³²P] PP_i through a Mg²⁺-dependent process with an optimal pH of 8.3. Free Mg²⁺ is required for maximal hydrolytic rates. The Km for PP_i is 75 μM and the Vmax is 800 pmol/min/mg. ATP is a weak inhibitor of the reaction, it diminishes the Vmax and increases the Km for PP_i. Thus, isolated β-subunits are catalytically competent with PP_i as substrate; apparently, the assembly of β-subunits into the ATPase complex changes substrate specificity, and leads to an increase in catalytic rates.     

PPiase-Activated ATP-Dependent H+ Transport at the Tonoplast of Mesophyll Cells of the CAM Plant Kalanchoë daigremontiana*

The co-ordinated action of the two proton-transporting enzymes at the tonoplast of the CAM plants. daigremontiana, viz. the ATPase and the PP_iase, was studied by measuring fluorescent dye quenching. The initial rates of ATP and PP_i-dependent H⁺ transport into tonoplast vesicles were additive, i.e. the sum of the rates obtained with each substrate alone was in the range obtained with both substrates added together at the same time. Conversely, the activities of the two H⁺ pumps were non-additive in establishing the steady-state level, indicating that the final steady state was under thermodynamic control of a maximal attainable proton gradient. The initial rates of ATP-dependent H⁺ transport were stimulated enormously if ATP was added a few minutes after pre-energization of the vesicles with PP_i. This stimulation was observed only when the PP_iase was active. A similar effect was not found for PP_i-dependent H⁺ transport after pre-energization with ATP. Hence, a PP_iase-activated ATP-dependent H⁺ transport can be distinguished from the basic ATP- and the basic PP_i-dependent H⁺ transport. In parallel a PP_i-dependent stimulation of ATP hydrolysis in the absence of ionophores was measured, which can only be attributed to the activity of the PP_iase. PP_iase-activated ATP-dependent H⁺ transport depends on the presence of permeant anions. It shows properties of both H⁺ transport activities, i.e. the chloride and malate stimulation and the DCCD inhibition of the ATP-dependent H⁺ transport activity, the nitrate stimulation and the KF inhibition of the PP_i-dependent H⁺ transport activity. Only MgPP_i and MgATP were effective as the respective substrates. The PP_iase-activated ATP-dependent H⁺ transport had a half life of about 5–9 minutes. It is concluded that the PP_iase may play an important role in kinetic regulation of the ATPase, and implications for CAM metabolism are discussed.     

Photosynthetic formation of inorganic pyrophosphate in phototrophic bacteria

In this paper we report studies on photosynthetic formation of inorganic pyrophosphate (PP_i) in three phototrophic bacteria. Formation of PP_i was found in chromatophores from Rhodopseudomonas viridis but not in chromatophores from Rhodopseudomonas blastica and Rhodobacter capsulatus. The maximal rate of PP_i synthesis in Rps. viridis was 0.15 mol PP_i formed/(min*mol Bacteriochlorophyll) at 23°C. The synthesis of PP_i was inhibited by electron transport inhibitors, uncouplers and fluoride, but was insensitive to oligomycin and venturicidin. The steady state rate of PP_i synthesis under continuous illumination was about 15% of the steady-state rate of ATP synthesis. The synthesis of PP_i after short light flashes was also studied. The yield of PP_i after a single 1 ms flash was equivalent to approximately 1 mol PP_i/500 mol Bacteriochlorophyll. In Rps. viridis chromatophores, PP_i was also found to induce a membrane potential, which was sensitive to carbonyl cyanide p-trifluoromethoxyphenylhydrazone and NaF.Abbreviations BChl Bacteriochlorophyll - F₀F₁-ATPase Membrane bound proton translocating ATP synthase - FCCP Carbonyl cyanide p-trifluoromethoxyphenylhydrazone - H⁺-PPase Membrane bound proton translocating PP_i synthase - TPP⁺ Tetraphenyl phosphonium ion - TPB^- Tetraphenyl boron ion - Transmembrane electrical potential difference     

INFLUENCE OF SALINITY AND TEMPERATURE ON GROWTH AND PHOTOSYNTHESIS IN THE EXTREMOPHILIC CHLOROPHYTE,NANNOCHLORIS SP.

Major, K. M. & Henley, W. J. Department of Botany, Oklahoma State University, Stillwater, OK 74078-3013 USA Preliminary data suggest Nannochloris sp., isolated from the Great Salt Plains National Wildlife Refuge, is a true extremophile. This alga is able to withstand salinities ranging from 0 to 150 ç and temperatures up to 45°C. To test the hypothesis that acclimation to high salinity confers tolerance to high temperature, experimental cultures were acclimated to salinities of 25 and 100 ç and/or temperatures of 23 and 38°C; irradiance (500 mol photons m^-2 s^-1) was saturating for both growth and photosynthesis. Cells acclimated to low salt and low temperature exhibited high photosynthetic performance in terms of both light-saturated photosynthesis (P_max; 45.0 fmol O₂ cell^-1 h^-1) and light-harvesting efficiency (0.103 fmol O₂ cell^-1 h^-1/mol photons m^-2 s^-1). However, high-salinity cells exhibited values for net P_max (18.1 fmol O₂ cell^-1 h^-1), (0.107 fmol O₂ cell^-1 h^-1/mol photons m^-2 s^-1) and growth rates (ca. 0.4 d^-1) that were equal to, or higher than, those of low-salinity cells when acclimated to high temperature. Both the amount of light required to achieve net photosynthesis (I_c) and that required to achieve light-saturated photosynthesis (I_k) were lower in high-salinity cells than those exhibited by low-salinity cells grown at high temperature; reductions in I_c and I_k were primarily due to increases in light-harvesting efficiency. We propose that an increase in growth temperature might release Nannochloris sp. from energy constraints associated with osmolyte production and low-temperature effects on enzyme activity. These data are consistent with effects of short-term temperature stress on Chl a fluorescence kinetics in this alga.