Involvement of matrix NADP turnover in the oxidation of NAD-linked substrates by pea leaf mitochondria

The involvement of the internal rotenone-insensitive NADPH dehydrogenase on the inner surface of the inner mitochondrial membrane [ND_in(NADPH)] in the oxidation of strictly NAD⁺-linked substrates by pea ( Pisum sativum L.) leaf mitochondria was measured. As estimated by the inhibition caused by 5 μ M diphenyleneiodonium (DPI) in the presence of rotenone to inhibit complex I, the activity of ND_in(NADPH) during glycine oxidation (measured both as O₂ uptake and as CO₂ release) was 40–50 nmol mg⁻¹ protein min⁻¹. No significant activity of ND_in(NADPH) could be detected during the oxidation of 2-oxoglutarate, another strictly NAD⁺-linked substrate; this was possibly due to its relatively low oxidation rate. Control experiments showed that, even at 125 μ M , DPI had no effect on the activity of glycine decarboxylase complex (GDC) and lipoamide dehydrogenase. The relative activity of complex I, ND_in(NADPH), and ND_in(NADH) during glycine oxidation, estimated using rotenone and DPI, differed depending on the pyridine nucleotide supply in the mitochondrial matrix. This was shown by loading the mitochondria with NAD⁺ and NADP⁺, both of which were taken up by the organelle. We conclude that the involvement of NADP turnover during glycine oxidation is not due to the direct production of NADPH by GDC but is an indirect result of this process. It probably occurs via the interconversion of NADH to NADPH by the two non-energy-linked transhydrogenase activities recently identified in plant mitochondria.     

l-Myo-inositol-1-phosphate synthase from lower plant groups: Partial purification and properties of the enzyme from Euglena gracilis

A survey for the enzyme L-myo-inositol-1-phosphate synthase (EC 5.5.1.4) has been conducted among various members of the lower plant groups, mainly algac, bryophytes and fungi; some properties of the partially purified enzyme from Euglena gracilis Z . are presented. The enzyme was detected in Chloropycean algae, Marchantiales and the Basidiomycetous fungi. The enzyme from Euglena had a pH optimum at 7.5. The Km for glucose-6-P was 2.1 m M and for NAD⁺ 80 μ M . When assayed in the absence of added NAD⁺, the enzyme showed a basal activity suggesting the presence of bund NAD⁺ in the system. NH₄Cl increased the enzyme activity two-fold, altough the enzyme was inactivated by (NH₄)₂SO₄.     

Nickel and rubidium uptake by whole oat plants in solution culture

Nickel and rubidium uptake by oat plants ( Avena sativa L. cv. Victory) were examined in relation to solution temperature, solution concentrations, metabolic inhibitors, anaerobic root conditions, transpiration and time. Over a 4-h period, uptake rates for both Ni²⁺ and Rb⁺ remained constant at 23°C. Decreasing temperatures to 2°C, 20 μ M concentrations of 2,4-dinitrophenol (DNP), or anaerobic root conditions decreased Ni²⁺ and Rb⁺ uptake rates by 97 to 86% in whole plants. Treatment of excised roots with 20 μ M DNP decreased Ni²⁺ uptake by 93%. Nickel and Rb⁺ uptake rates measured as a function of the external solution concentration followed a typical parabolic curve. K_m (0.012 m M ) and V_max [2.72 μmol (g dry weight)^-1 h^-1] values for Ni²⁺ were nearly 7 times lower than those for Rb⁺ [0.09 m M and 19.2 μmol (g dry weight)^-1 h^-1]. In all experiments, Ni²⁺ and Rb⁺ showed qualitatively similar uptake patterns, but Rb⁺ uptake was quantitatively more sensitive than Ni²⁺ to experimental manipulations.     

The regulation of Mn2+ and Cu2+ uptake in cells of the yeast Candida utilis grown in continuous culture

Abstract Transport of Mn²⁺ was repressed in Candida utilis cells grown in continuous culture in high-Mn²⁺ (100 μM Mn²⁺) medium as compared to cells grown in basic (0.45 μM Mn²⁺) and low-Mn²⁺ (< 0.05 μM Mn²⁺) media. In contrast, no repression of Cu²⁺ uptake occurred in high-Cu²⁺-grown (25 μM Cu²⁺) cells as compared to cells grown in basic medium (0.54 μM Cu²⁺). Cu²⁺-limited cells did not hyperaccumulate Cu²⁺ and there was not significant difference in initial uptake rates for all 3 Cu²⁺ conditions. Mn²⁺ uptake appears to be regulated by a mechanism sensitive to the external Mn²⁺ concentration, whereas Cu²⁺ transport is not governed in this way by the external Cu²⁺.     

Subcellular distribution of pyruvate-degrading enzymes in Chlamydomonas reinhardtii studied by an improved protoplast fractionation procedure

The subcellular distribution of pyruvate-degrading enzymes has been determined in Chlamydomonas reinhardtii (Dangeard) by protoplast induction with autolysine, dig-itonin lysis and further fractionation by differential centrifugation using a Percoll cushion. Mitochondrial and plastidic fractions contained intact and physiologically competent organelles - RC 1.7, ADP/O 2.7 and rate of malate oxidation 76 nmol O, (mg protein)^-1min^-1 for mitochondria, CO_2; fixation 46.8 μmol (mg Chi)^-1 h^-1 for chloroplasts.
Results from protoplast fractionation were further confirmed by the determination of enzyme activities within trypsin-treated organelles. Mitochondria (formate fermentation) and chloroplasts (chlorofermentation) were shown to possess the capacity for anaerobic pyruvate degradation. Pyruvate dehydrogenase (NAD⁺, EC 1.2.4.1), pyruvate formate-lyase (EC 2.3.1.54) and lactate dehydrogenase (NADH, EC 1.1.1.27) showed equal distribution between mitochondria and chloroplasts, whereas activities of phosphotransacetylase (EC 2.3.1.8) and acetate kinase (EC 2.7.2.1) were only detectable in the mitochondrial fraction. NADH- and NADPH-dependent activities of both alcohol dehydrogenase (EC 1.1.1.1) and aldehyde dehydrogenase (acylating, EC 1.2.1.10) were localized in the mitochondrial and cytoplasmic or the plastidic and cytoplasmic fractions, respectively, whereas pyruvate decarboxylase (EC 4.1.1.1) was only detected in the cytoplasmic fraction.     

Turgor- dependent membrane permeability in relation to calcium level

The relationship between the inhibiting effect of Ca²⁺ and of low turgor pressure on K⁺ release from fresh-cut discs of carrot ( Daucus carota var. Nantes) storage tissue was studied. A range of Ca²⁺ concentrations in the tissue was obtained by adding 0.5 m M EDTA or CaSO₄ at different concentrations to the medium. Calcium inhibited K⁺ release in fully turgid cells (2.5 μmol K⁺ g⁻¹ h⁻¹ in 0.5 m M EDTA vs 0.4 μmol K⁺ g⁻¹ h⁻¹ in 10 m M CaSO₄). Less turgid cells, obtained by equilibration with 0.2 M mannitol, released K⁺ at only 30% of the rate of the turgid cells, yet the pattern of K⁺ release as a function of Ca²⁺ level was similar in both turgid and non-turgid cells. Removal of calcium by EDTA occasionally injured cell membranes in the fully turgid discs but never in the less turgid ones. In view of the additive effect of Ca²⁺ and low turgor on K⁺ release regardless of the treatment order, it is suggested that the two factors exert their effect on membrane permeability independently of each other.     

4-Aminobutyraldehyde Dehydrogenase Activity in Rat Brain

Abstract: An enzyme with NAD⁺-dependent 4-aminobutyraldehyde dehydrogenase activity was purified about 360-fold from rat brain extract. AMP-Sepharose chromatography was effective in separating the enzyme from other NAD⁺-dependent aldehyde dehydrogenases included in the extract. The K _ms for the substrates NAD⁺ and 4-aminobutyraldehyde were 4.8 × 10⁻⁴ and 8.3 × 10⁻⁵ M , respectively. The pH optimum for the enzyme was about 8.0. The ratio of activities toward 4-aminobutyraldehyde, propionaldehyde, succinate semialdehyde, and benzaldehyde was 1.00:0.17:0.24:0.09:0.03 when the activity toward 4-aminobutyraldehyde was set equal to 1.00. The enzyme activity in subcellular fractions of rat brain was localized in cytosol.     

A method for long-term measurement of respiration in intertidal fishes during simulated intertidal conditions

Aquatic and aerial respiration of the amphibious fishes Lipophrys pholis and Periophthalmus barbarus were examined using a newly designed flow-through respirometer system. The system allowed long-term measurements of oxygen consumption and carbon dioxide release during periods of aquatic and aerial respiration. The M o ₂ of L. pholis , measured at 15° C, was 2·1 μmol O₂ g^–1 h^–1 during aquatic and 1·99 μmol O₂ g^–1 h^–1 during aerial exposure. The corresponding values of the M co₂ were 1.67 and 1.59 μmol O₂ g^–1 h^–1 respectively, giving an aquatic respiratory exchange ratio (RER) of 0·80 and an aerial RER of 0·79. The M o₂ of P. barbarus , measured at 28°C, was 4·05 μmol O₂ g^–1 h^–1 during aquatic and 3·44 μmol O₂ g^–1 h^–1 during aerial exposure. The corresponding values of the Mco₂ were 3·29 μmol CO₂ g^–1 h^–1 and 2·63 μmol CO₂ g^–1 h^–1 respectively, giving an aquatic RER of 0·81 and an aerial RER of 0·77. While exposed to air for at least 10 h, both species showed no decrease in metabolic rate or carbon dioxide release. The RER of these fishes equalled their respiratory quotient. After re-immersion an increased oxygen consumption, due to the payment of an oxygen debt, could not be detected.     

Light activates H2 15O flow in rice: Detailed monitoring using a positron-emitting tracer imaging system (PETIS)

Water (H₂ ¹⁵O) translocation from the roots to the top of rice plants ( Oryza saliva L. cv. Nipponbare) was visualized over time by a positron-emitting tracer imaging system (PETIS). H₂ ¹⁵O flow was activated 8 min after plants were exposed to bright light (1 500 μmol m⁻² s⁻¹). When the light was subsequently removed, the flow gradually slowed and completely stopped after 12 min. In plants exposed to low light (500 μmol m⁻² s⁻¹), H₂ ¹⁵O flow was activated more slowly, and a higher translocation rate of H₂ ¹⁵O was observed in the same low light at the end of the next dark period. NaCl (80 m M ) and methylmercury (1 m M ) directly suppressed absorption of H₂ ¹⁵O by the roots, while methionine sulfoximine (1 m M ), abscisic acid (10 μ M ) and carbonyl cyanide m -chlorophenylhydrazone (10 m M ) were transported to the leaves and enhanced stomatal closure, reducing H₂ ¹⁵O translocation.     

Effect of ultraviolet radiation on accumulation and leakage of 86Rb+ in guard cells of Vicia faba

The effects of UV-C (254 nm), UV-A (365 nm) and broad-band UV (280–380 nm) on guard cells of Vicia faba L. cv. Long Pod were investigated in the presence of white light (450 μmol m⁻² s⁻¹). UV-C (7 μmol m⁻² s⁻¹) was found to cause leakage of ⁸⁶Rb⁺ from guard cells, while UV-A (0.3 μmol m⁻² s⁻¹) stimulated increased uptake in these cells. A relatively small stimulatory effect was observed by broad-band UV (3 μmol m⁻² s⁻¹) during the first 30 min of irradiation with an apparent equilibration of influx and efflux thereafter. Leakage of ⁸⁶Rb⁺ from guard cells continued despite the removal of UV-C and an increase in the amount of white light from 450 to 1500 μmol m⁻² s⁻¹, suggesting that membranes were irreversibly damaged. Irradiation of guard cells with UV-C for 30, 45 and 90 min indicated that these cells began to be affected already by 30 min UV-C irradiation.     

Phloretin as an Antagonist of Prostaglandin F2α Receptor in Cultured Rat Astrocytes

Abstract: The effect of phloretin on prostaglandin (PG) F_2α-induced phosphoinositide hydrolysis and elevation of intracellular Ca²⁺ concentration was examined in cultured rat astrocytes. Phloretin inhibited PGF_2α (1 μ M )-induced phosphoinositide hydrolysis in a concentration-dependent manner with an IC₅₀ value of 16 μ M . The inhibitory action of phloretin was specific for PGs. The addition of increasing concentrations of phloretin caused progressive shifts of the dose-response curves of PGF_2α to the right. In digitoninpermeabilized astrocytes, phloretin (100 μ M ) inhibited the stimulation induced by PGF_2α (1 μ M ) plus GTPγS (50 μ M ) without affecting that induced by GTPγS alone. PGF_2α at 1 μ M transiently increased astrocytic intracellular Ca²⁺ concentration in 39% of the cells tested. The response was completely blocked by 100 μ M phloretin and the calcium response recovered again after washing out phloretin. These results suggest that phloretin is an antagonist of PGF_2α receptor linked to phospholipase C in astrocytes.     

Allosteric Properties of Ox Brain Nicotinamide–Adenine Dinucleotide Dependent Isocitrate Dehydrogenase

Abstract: The kinetic and regulatory properties of a partly purified preparation of ox brain NAD⁺-dependent isocitrate dehydrogenase have been studied at pH 7.5. The enzyme exhibits rate cooperativity with respect to isocitrate but shows normal hyperbolic kinetics with respect to NAD⁺. ADP activates the enzyme by decreasing the substrate concentrations that are necessary to give half-maximal velocity, but it has no effect on the Hill constant for isocitrate unless Mg²⁺ ions are replaced by Mn²⁺ ions in the reaction mixture. Citrate and tricarballylate activate the enzyme in a similar fashion to ADP. Higher concentrations of citrate cause inhibition but this could be overcome by raising the concentration of Mg²⁺ ions, suggesting that the inhibition by this compound might be due to its acting as a chelating agent. NADH and NADPH were competitive inhibitors with respect to NAD⁺ but the product, 2-oxoglutarate, was not inhibitory. γ-Aminobutyrate and a number of other compounds involved in the γ-aminobutyrate pathway had no significant effect on the activity of the enzyme.     

Evidence of a quinoprotein glucose dehydrogenase apoenzyme in several strains of Escherichia coli

Abstract When grown on glucose in K⁺-limited chemostat culture, or in batch culture with or without 2,4-dinitrophenol, several strains of Escherichia coli (including the type strain) were found to synthesize a quinoprotein glucose dehydrogenase apoenzyme. The pyridine nucleotides, NAD⁺ and NADP⁺, would not serve as cofactor, but activity could be demonstrated upon addition of 2,7,9-tricarboxy-1 H -pyrrolo(2,3- f )quinoline-4,5-dione (PQQ). Thus, in the presence of PQQ, but not in its absence, glucose was oxidized to gluconic acid. A mutant of E. coli PC 1000 was isolated that lacked Enzyme I of the phospho enol pyruvate phosphotransferase system (PTS) but still synthesized the glucose dehydrogenase apoenzyme. Whereas this mutant would not grow on glucose in the absence of PQQ, it would do so in the presence of low concentrations (1 μM) of this cofactor. On the basis of these observations, it is concluded that the protein (apoenzyme) formed is a genuine glucose dehydrogenase, but that it is not functional in growing cells due to their inability to synthesize the appropriate cofactor (PQQ), at least under these conditions.     

Photosynthesis and photoassimilation of glucose during photomixotrophic growth of Marchantia polymorpha cells in suspension culture

Even in the presence of glucose the growth of Marchantia polymorpha L. (cell line HYH-2F) requires light, and growth is more sensitive to 10⁻⁶ M 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea than to 10⁻⁴ Antimycin A. The inability of the cells to grow in the dark is due to the low level of respiration. The respiration rate under light increased to four times the dark value. The values of the compensation ratio (the photosyntehtic rate/the respiration rate) for the oxygen exchange were below 1.0 daring the growth period, although oxygen evolution was found. At the early exponential phase, oxygen evolution was 0.373 μmol (mg cell dry weight)⁻¹ h⁻¹ [61.7 μmol (mg chlorophyll)⁻¹ h⁻¹]. M. polymorpha cells are unable to grow anaerobically in the light without a supply of carbon dioxide. When 1% carbon dioxide in nitrogen is supplied, photochemically produced oxygen and energy are sufficient for sustained growth although at significantly reduced yields in both cell dry weight and chlorophyll. Photosyntehtic CO₂ assimilation rate was 0.13 μmol (mg cell dry weight)⁻¹ h⁻¹[11.3 μmol (mg chlorophyll)⁻¹ h⁻¹]. At least one-third of the carbon atoms in cellular constituents seem to be derived from atmospheric carbon dioxide, which indicates that M. polymorpha cells grow photomixotrophicaily.     

45Ca2+ Uptake into Rat Whole Brain Synaptosomes Unaltered by Dihydropyridine Calcium Antagonists

Abstract: Voltage-dependent ⁴⁵Ca²⁺ uptake into rat whole brain synaptosomes was measured after 3-s KCl-induced depolarization to investigate possible inhibitory effects of calcium antagonists, nitrendipine, nimodipine, and nisoldipine. At a Ca²⁺ concentration of 1.2 m M , nitrendipine, in concentrations ranging from 0.1 n M to 10 μ M , had no effect on ⁴⁵Ca²⁺ uptake. When the Ca²⁺ concentration was lowered to 0.06 and 0.12 m M , nitrendipine, 10 μ M , inhibited ⁴⁵Ca²⁺ uptake in response to 109 m M KCl depolarization. However, in a separate concentration response study, nitrendipine, nimodipine, and nisoldipine, 0.1 n M to 10 μ M , failed to alter the uptake of ⁴⁵Ca²⁺ (0.06 m M Ca²⁺) into 30 m M KCl-depolarized synaptosomes. The high concentrations of these agents required to depress ⁴⁵Ca²⁺ uptake indicate that the dihydropyridine calcium antagonists are considerably less potent in brain tissue than in peripheral tissue.     

The Effects of Electroshock Convulsions on Calcium Transport within Synaptic Terminals

Abstract: Calcium transport was assessed within synaptic terminals isolated from cerebral cortices of rats which experienced one maximal electroshock (ES) convulsion daily. No significant change in calcium content [(Ca₁)] of synaptosomes was present after 2 consecutive days of maximal convulsions. After 4 and 6 days of maximal seizures, (Ca₁) rose 20% and 37%, respectively. ¹⁵Ca²⁺ influx within synaptosomes in vitro increased after 6 days of ES convulsions (1.94 ± 0.4 μmol/g protein/min in ES convulsions versus 1.54 ± .03 μmol/g protein/min in controls). The higher rate of ⁴⁵Ca²⁺ influx in convulsed animals was accounted for by elevated internal sodium [(Na₁)] values. Maximal ⁴⁵Ca²⁺ efflux decreased after ES convulsions (0.48 μmol/g protei/min in ES convulsions versus 0.8 μmol/g protei/min in controls). The slower rate of ⁴⁵Ca²⁺ efflux after convulsions was also accounted for by elevated (Na₁). Our results suggest that (Ca₁) increased within synapses after in vivo ES convulsions secondary to a primary ionic event, namely, elevated (Na₁).     

Heavy metal inhibition of methanogenesis by Methanospirillum hungatei GP1

Abstract Washed whole cells of Methanospirillum hungatei incubated in TES buffer retained methanogenic activity in the absence of any reducing agents. Washed cells grown with 80% H₂-20% CO₂ and acetate produced methane from H₂/CO₂ and 50 mM formate at 1.1 to 1.8 and 15 μmol methane · h⁻¹· mg⁻¹ protein, respectively. Cadmium at a concentration of 15 μM and 50 μM mercury, copper or zinc completely inhibited methane production from H₂/CO₂ by M. hungatei . The chelating agent, EDTA, protected the cells from inhibition by cadmium but acetate and citrate did not. The activity of formate dehydrogenase and hydrogenase remaining in cells after incubation with copper, mercury, zinc or cadmium was reduced with formate dehydrogenase being the more sensitive.     

Purification and characterization of adenine phosphoribosyltransferase from Arabidopsis thaliana

Adenine phosphoribosyltransferase (APRT; EC 2. 4,2. 7) from Arabidopsis thaliana was purified approximately 3800-fold, to apparent homogeneity. The purification procedure involved subjecting a leaf extract to heat denaturation, (NH₄)₂SO₄ precipitation, Sephadex G-25 salt separation, ultracentrifugation and liquid chromatography on Diethylaminoethyl Sephacel, Phenyl Sepharose CL-4B, Blue Sepharose CL-6B and adenosine 5'-monophosphate-Agarose. The purified APRT was a homodimer of approximately 54 kDa and it had a specific activity of approximately 300 μmol (mg total protein)^-1 min^-1. Under standard assay conditions, the temperature optimum for APRT activity was 65°C and the pH optimum was temperature dependent. High enzyme activity was dependent upon the presence of divalent cations (Mn²⁺ or Mg²⁺). In the presence of MnCl₂₊ other divalent cations (Mg²⁺, Ca²⁺, Ba²⁺, Hg²⁺ and Cd²⁺) inhibited the APRT reaction. Kinetic studies indicated that 5-phosphoribose-1-pyrophosphate (PRPP) caused substrate inhibition whereas adenine did not. The K_m for adenine was 4.5±1.5 μ M , the K_m for PRPP was 0.29±0.06 m M and the K_i for PRPP was 1.96±0.45 m M . Assays using radiolabelled cytokinins showed that purified APRT can also catalyze the phosphoribosylation of isopentenyladenine and benzyladenine. The K_m for benzyladenine was approximately 0.73±0.06 m M     

Picomolar concentrations of tentoxin affect Mg2+- and Ca2+-ATPase activities of plasma membrane vesicles from roots of winter wheat seedlings

Purified plasmalemma vesicles were isolated in the presence of 250 m M sucrose from roots of 14-day-old seedlings of winter wheat ( Triticum aestivum L. Martonvásári-8) by phase partitioning of salt-washed microsomal fractions in a Dextran-polyethylene glycol two-phase system, and both Mg²⁺- and Ca²⁺-ATPase activities were detected. Orthovanadate-sensitive Mg²⁺-ATPase activity associated with the inside of right side-out plasmalemma (PM) vesicles (latency 98%) was inhibited 76% by 0.3 m M Ca²⁺, Ca²⁺-dependent ATPase activity located partly on the inside and partly on the outside of plasmalemma vesicles (latency 47%) was not affected by Mg²⁺.
Mg²⁺-ATPase activity was inhibited by 68% and inhibition of Mg²⁺ activation by 0.3 m M Ca²⁺ partly disappeared in the presence of 10 p M tentoxin, a fungal phytotoxin. Mg²⁺-ATPase activity remained inhibited up to 10 n M tentoxin while at 1 μ M tentoxin Mg²⁺ activation was as high as without tentoxin. K⁺-stimulation and vanadate inhibition was increased and decreased, respectively, by 100 p M -10 n M tentoxin. Ca²⁺-dependent ATPase activity was continuously increased by 1 p M -10 n M tentoxin, but at 1 μ M tentoxin the stimulation disappeared. The effects of p M tentoxin on plasma-lemma Mg²⁺-ATPase are discussed in relation to its influence on K⁺ transport in wheat seedlings.     

Detection of Intracellular Free Na+ Concentration of Synaptosomes by a Fluorescent Indicator, Na+-Binding Benzofuran Isophthalate: The Effect of Veratridine, Ouabain, and α-Latrotoxin

Abstract: A novel fluorescent Na⁺ indicator, Na⁺-binding benzofuran isophthalate (SBFI), was used to follow changes in the intracellular free Na⁺ concentration ([Na⁺]₁) of synaptosomes. The dye, when loaded into synapto- somes in the form of its acetoxymethyl ester, was responsive to changes of [Na⁺]₁. Calibration was made using the 340/380 nm excitation ratio when the cytoplasmic Na⁺ concentration was equilibrated with different concentrations of extracellular Na⁺ in the presence of 2 μ M gramicidin D. The basal value of [Na⁺]₁ in synaptosomes in the presence of 140 m M extracellular Na⁺ was found to be 10.9 ± 1.8 m M. Veratridine, which opens potential-dependent Na⁺ channels, caused a sudden increase in [Na⁺]₁ in a concentration-dependent manner (1 -20 μ M ), whereas the effect of ouabain (20 and 50 μ M ), the inhibitor of the plasma membrane Na⁺,K⁺-ATPase, was more gradual. The rise in the fluorescence intensity upon addition of veratridine was prevented completely by 2 μ M tetrodotoxin. α-Latrotoxin, the black widow spider toxin, caused an increase in the fluorescence intensity, which became evident 1 min after the addition of the toxin. The rate of increase was proportional to the concentration of the toxin (0.19–1.5 n M ). This report confirms our earlier finding demonstrating a Na⁺-dependent component in the action of α-Iatrotoxin, and shows that changes in [Na⁺]₁ in synaptosomes can be followed by SBFI.