Vanadate inhibits blue light-stimulated swelling of vicia guard cell protoplasts

When supplied under low chloride concentrations, vanadate inhibits the blue light-stimulated swelling of Vicia faba L. guard cell protoplasts in a dose-dependent fashion. The volume of guard cell protoplasts incubated in 10 mm K-imino-diacetic acid, 0.4 m mannitol, and 1 mm CaCl₂ remained essentially constant under 1000 μmol m⁻² s⁻¹ red light, but increased an average of 27% after 8 min of the addition of 50 μmol m⁻² s⁻¹ blue light to the background red light. At 500 μm, vanadate completely inhibits the response to blue light. Vanadate also inhibits the swelling of guard cell protoplasts stimulated by the H⁺-ATPase agonist fusicoccin. The vanadate sensitivity of the blue light-stimulated swelling implicates a proton-pumping ATPase as a component of the sensory transduction of blue light in guard cells.     

Characterization of a NO(3)-Sensitive H-ATPase from Corn Roots

When assayed in the presence of azide, NO₃⁻ was shown to be a specific inhibitor of a proton-translocating ATPase present in corn (Zea mays L. cv WF9 × M017) root microsomal membranes. The distribution of the NO₃⁻-sensitive ATPase on sucrose gradients and its general characteristics are similar to those previously reported for the anion-stimulated H⁺-ATPase of corn roots believed to be of tonoplast origin. An ATPase inhibited by 20 μm vanadate and insensitive to molybdate was also identified in corn root microsomal membranes which could be largely separated from the NO₃⁻-sensitive ATPase on sucrose gradients and is believed to be of plasma membrane origin. Inasmuch as both ATPase most likely catalyze the efflux of H⁺ from the cytoplasm, our objective was to characterize and compare the properties of both ATPases under identical experimental conditions. The vanadate-sensitive ATPase was stimulated by cations (K⁺ > NH₄⁺ > Rb⁺ > Cs⁺ > Li⁺ > Na⁺ > choline⁺) whereas the NO₃⁻-sensitive ATPase was stimulated by anions (Cl⁻ > Br⁻ > C₂H₃O₂⁻ > SO₄²⁻ > I⁻ > HCO₃⁻ > SCN⁻). Both ATPases required divalent cations. However, the order of preference for the NO₃⁻-sensitive ATPase (Mn²⁺ > Mg²⁺ > Co²⁺ > Ca²⁺ > Zn²⁺) differed from that of the vanadate-sensitive ATPase (Co²⁺ > Mg²⁺ > Mn²⁺ > Zn²⁺ > Ca²⁺). The vanadate-sensitive ATPase required higher concentrations of Mg:ATP for full activity than did the NO₃⁻-sensitive ATPase. The kinetics for Mg:ATP were complex for the vanadate-sensitive ATPase, indicating positive cooperativity, but were simple for the NO₃⁻-sensitive ATPase. Both ATPases exhibited similar temperature and pH optima (pH 6.5). The NO₃⁻-sensitive ATPase was stimulated by gramicidin and was associated with NO₃⁻-inhibitable H⁺ transport measured as quenching of quinacrine fluorescence. It was insensitive to molybdate, azide, and vanadate, but exhibited slight sensitivity to ethyl-3-(3-dimethylaminopropyl carbodiimide) and mersalyl. Overall, these results indicate several properties which distinguish these two ATPases and suggest that under defined conditions NO₃⁻-sensitive ATPase activity may be used as a quantitative marker for those membranes identified tentatively as tonoplast in mixed or nonpurified membrane fractions. We feel that NO₃⁻ sensitivity is a better criterion by which to identify this ATPase than either Cl⁻ stimulation or H⁺ transport because it is less ambiguous. It is also useful in identifying the enzyme following solubilization.     

Kinesin-2 KIF3AB Exhibits Novel ATPase Characteristics

KIF3AB is an N-terminal processive kinesin-2 family member best known for its role in intraflagellar transport. There has been significant interest in KIF3AB in defining the key principles that underlie the processivity of KIF3AB in comparison with homodimeric processive kinesins. To define the ATPase mechanism and coordination of KIF3A and KIF3B stepping, a presteady-state kinetic analysis was pursued. For these studies, a truncated murine KIF3AB was generated. The results presented show that microtubule association was fast at 5.7 μm⁻¹ s⁻¹, followed by rate-limiting ADP release at 12.8 s⁻¹. ATP binding at 7.5 μm⁻¹ s⁻¹ was followed by an ATP-promoted isomerization at 84 s⁻¹ to form the intermediate poised for ATP hydrolysis, which then occurred at 33 s⁻¹. ATP hydrolysis was required for dissociation of the microtubule·KIF3AB complex, which was observed at 22 s⁻¹. The dissociation step showed an apparent affinity for ATP that was very weak (K_½,ATP at 133 μm). Moreover, the linear fit of the initial ATP concentration dependence of the dissociation kinetics revealed an apparent second-order rate constant at 0.09 μm⁻¹ s⁻¹, which is inconsistent with fast ATP binding at 7.5 μm⁻¹ s⁻¹ and a K_d,ATP at 6.1 μm. These results suggest that ATP binding per se cannot account for the apparent weak K_½,ATP at 133 μm. The steady-state ATPase K_m,ATP, as well as the dissociation kinetics, reveal an unusual property of KIF3AB that is not yet well understood and also suggests that the mechanochemistry of KIF3AB is tuned somewhat differently from homodimeric processive kinesins.     

Functional and Biochemical Characterization of Cucumber Genes Encoding Two Copper ATPases CsHMA5.1 and CsHMA5.2

Plant copper P_1B-type ATPases appear to be crucial for maintaining copper homeostasis within plant cells, but until now they have been studied mostly in model plant systems. Here, we present the molecular and biochemical characterization of two cucumber copper ATPases, CsHMA5.1 and CsHMA5.2, indicating a different function for HMA5-like proteins in different plants. When expressed in yeast, CsHMA5.1 and CsHMA5.2 localize to the vacuolar membrane and are activated by monovalent copper or silver ions and cysteine, showing different affinities to Cu⁺ (K_m ∼1 or 0.5 μm, respectively) and similar affinity to Ag⁺ (K_m ∼2.5 μm). Both proteins restore the growth of yeast mutants sensitive to copper excess and silver through intracellular copper sequestration, indicating that they contribute to copper and silver detoxification. Immunoblotting with specific antibodies revealed the presence of CsHMA5.1 and CsHMA5.2 in the tonoplast of cucumber cells. Interestingly, the root-specific CsHMA5.1 was not affected by copper stress, whereas the widely expressed CsHMA5.2 was up-regulated or down-regulated in roots upon copper excess or deficiency, respectively. The copper-induced increase in tonoplast CsHMA5.2 is consistent with the increased activity of ATP-dependent copper transport into tonoplast vesicles isolated from roots of plants grown under copper excess. These data identify CsHMA5.1 and CsHMA5.2 as high affinity Cu⁺ transporters and suggest that CsHMA5.2 is responsible for the increased sequestration of copper in vacuoles of cucumber root cells under copper excess.     

Control of logarithmic growth rates of tobacco callus tissue by cytokinins

The growth rates of tobacco callus tissues on media containing 10⁻⁶ to 10 μm 6-(γ,γ-dimethylallylamino)purine (2iP) were measured. At concentrations of 10⁻⁴ μm and above growth rates were exponential and dependent on cytokinin concentration. At 2iP concentrations of 10⁻⁴ to 0.33 μm, the exponential rate was maintained for 4 to 5 doublings of fresh and dry weight. After this period a linear phase, resulting in approximately 1 doubling of weight, occurred. The growth of tissues on media containing higher than 0.33 μm 2iP was exponential for only about 15 days. At the end of this time, and well before they achieved half their final weight, they exhibited growth which was less rapid than logarithmic but more rapid than linear. Comparisons with zeatin, 6-benzylaminopurine and kinetin indicated that, although the maximum growth rates obtained with relatively high concentrations (0.1-1 μm) were similar, the naturally occurring cytokinins, 2iP and zeatin, promoted faster rates at lower concentrations (10⁻³-10⁻² μm) than did 6-benzylaminopurine and kinetin.     

H-ATPase Activity from Storage Tissue of Beta vulgaris: III. Modulation of ATPase Activity by Reaction Substrates and Products

Two distinct membrane fractions containing H⁺-ATPase activity were prepared from red beet. One fraction contained a H⁺-ATPase activity that was inhibited by NO₃⁻ while the other contained a H⁺-ATPase inhibited by vanadate. We have previously proposed that these H⁺-ATPases are associated with tonoplast (NO₃⁻-sensitive) and plasma membrane (vanadate-sensitive), respectively. Both ATPase were examined to determine to what extent their activity was influenced by variations in the concentration of ATPase substrates and products. The substrate for both ATPase was MgATP²⁻, and Mg²⁺ concentrations in excess of ATP had only a slight inhibitory effect on either ATPase. Both ATPases were inhibited by free ATP (i.e. ATP concentrations in excess of Mg²⁺) and ADP but not by AMP. The plasma membrane ATPase was more sensitive than the tonoplast ATPase to free ATP and the tonoplast ATPase was more sensitive than the plasma membrane ATPase to ADP.

Inhibition of both ATPases by free ATP was complex. Inhibition of the plasma membrane ATPase by ADP was competitive whereas the tonoplast ATPase demonstrated a sigmoidal dependence on MgATP²⁻ in the presence of ADP. Inorganic phosphate moderately inhibited both ATPases in a noncompetitive manner.

Calcium inhibited the plasma membrane but not the tonoplast ATPase, apparently by a direct interaction with the ATPase rather than by disrupting the MgATP²⁻ complex.

The sensitivity of both ATPases to ADP suggests that under conditions of restricted energy supply H⁺-ATPase activity may be reduced by increases in ADP levels rather than by decreases in ATP levels per se. The sensitivity of both ATPases to ADP and free ATP suggests that modulation of cytoplasmic Mg²⁺ could modulate ATPase activity at both the tonoplast and plasma membrane.

     

Inability of tributyltin-induced chloride/hydroxyl exchange to stimulate calcium transport in mitochondria isolated from flight muscle of the sheep blowfly Lucilia cuprina

Tributyltin in the concentration range 1–4μm failed to stimulate Ca²⁺ transport by Lucilia flight-muscle mitochondria in a medium containing KCl and respiratory substrate but devoid of P_i, despite its promotion of a rapid Cl⁻/OH⁻ exchange. When 2mm-P_i was present, concentrations of tributyltin greater than 1μm inhibited the initial rate of Ca²⁺ transport and induced efflux of the ion from the mitochondria in Cl⁻- or NO₃⁻-containing media. Lower concentrations had little effect. Oligomycin added at up to 10μg/mg of mitochondrial protein had no effect on Ca²⁺ transport. By contrast, approx. 0.3μm-tributyltin completely inhibited respiration supported by α-glycerophosphate in either the presence or absence of added ADP. The data suggest that tributyltin can inhibit Ca²⁺ transport in Lucilia flight-muscle mitochondria other than by facilitating a Cl⁻/OH⁻ exchange or producing an oligomycin-like effect.     

Nitric-oxide Dioxygenase Function of Human Cytoglobin with Cellular Reductants and in Rat Hepatocytes

Cytoglobin (Cygb) was investigated for its capacity to function as a NO dioxygenase (NOD) in vitro and in hepatocytes. Ascorbate and cytochrome b₅ were found to support a high NOD activity. Cygb-NOD activity shows respective K_m values for ascorbate, cytochrome b₅, NO, and O₂ of 0.25 mm, 0.3 μm, 40 nm, and ∼20 μm and achieves a k_cat of 0.5 s⁻¹. Ascorbate and cytochrome b₅ reduce the oxidized Cygb-NOD intermediate with apparent second order rate constants of 1000 m⁻¹ s⁻¹ and 3 × 10⁶ m⁻¹ s⁻¹, respectively. In rat hepatocytes engineered to express human Cygb, Cygb-NOD activity shows a similar k_cat of 1.2 s⁻¹, a K_m(NO) of 40 nm, and a k_cat/K_m(NO) (k′_NOD) value of 3 × 10⁷ m⁻¹ s⁻¹, demonstrating the efficiency of catalysis. NO inhibits the activity at [NO]/[O₂] ratios >1:500 and limits catalytic turnover. The activity is competitively inhibited by CO, is slowly inactivated by cyanide, and is distinct from the microsomal NOD activity. Cygb-NOD provides protection to the NO-sensitive aconitase. The results define the NOD function of Cygb and demonstrate roles for ascorbate and cytochrome b₅ as reductants.     

Partial Purification of the NADH-Nitrate Reductase Complex from Chlorella pyrenoidosa

The nitrate reductase complex from Chlorella pyrenoidosa has been purified by a procedure which includes as main steps, ammonium sulfate fractionation, polyethylene glycol treatment, and DEAE-cellulose chromatography. The Michaelis constants for NADH, FAD, and NO₃⁻ in the NADH-nitrate reductase assay are 10 μm, 2.6 μm, and 0.23 mm, respectively. Heat treatment exerts varying effects on the enzymatic activities associated with the nitrate reductase complex.     

Calcium Regulates Molecular Interactions of Otoferlin with Soluble NSF Attachment Protein Receptor (SNARE) Proteins Required for Hair Cell Exocytosis

Mutations in otoferlin, a C2 domain-containing ferlin family protein, cause non-syndromic hearing loss in humans (DFNB9 deafness). Furthermore, transmitter secretion of cochlear inner hair cells is compromised in mice lacking otoferlin. In the present study, we show that the C2F domain of otoferlin directly binds calcium (K_D = 267 μm) with diminished binding in a pachanga (D1767G) C2F mouse mutation. Calcium was found to differentially regulate binding of otoferlin C2 domains to target SNARE (t-SNARE) proteins and phospholipids. C2D–F domains interact with the syntaxin-1 t-SNARE motif with maximum binding within the range of 20–50 μm Ca²⁺. At 20 μm Ca²⁺, the dissociation rate was substantially lower, indicating increased binding (K_D = ∼10⁻⁹) compared with 0 μm Ca²⁺ (K_D = ∼10⁻⁸), suggesting a calcium-mediated stabilization of the C2 domain·t-SNARE complex. C2A and C2B interactions with t-SNAREs were insensitive to calcium. The C2F domain directly binds the t-SNARE SNAP-25 maximally at 100 μm and with reduction at 0 μm Ca²⁺, a pattern repeated for C2F domain interactions with phosphatidylinositol 4,5-bisphosphate. In contrast, C2F did not bind the vesicle SNARE protein synaptobrevin-1 (VAMP-1). Moreover, an antibody targeting otoferlin immunoprecipitated syntaxin-1 and SNAP-25 but not synaptobrevin-1. As opposed to an increase in binding with increased calcium, interactions between otoferlin C2F domain and intramolecular C2 domains occurred in the absence of calcium, consistent with intra-C2 domain interactions forming a “closed” tertiary structure at low calcium that “opens” as calcium increases. These results suggest a direct role for otoferlin in exocytosis and modulation of calcium-dependent membrane fusion.     

Anion-sensitive, h-pumping ATPase in membrane vesicles from oat roots

H⁺-pumping ATPases were detected in microsomal vesicles of oat (Avena sativa L. var Lang) roots using [¹⁴C]methylamine distribution or quinacrine fluorescent quenching. Methylamine (MeA) accumulation into vesicles and quinacrine quench were specifically dependent on Mg,ATP. Both activities reflected formation of a proton gradient (ΔpH) (acid inside) as carbonyl cyanide m-chlorophenylhydrazone, nigericin (in the presence of K⁺), or gramicidin decreased MeA uptake or increased quinacrine fluorescence. The properties of H⁺ pumping as measured by MeA uptake were characterized. The K_mapp for ATP was about 0.1 millimolar. Mg,GTP and Mg, pyrophosphate were 19% and 30% as effective as Mg,ATP. MeA uptake was inhibited by N,N′-dicyclohexylcarbodiimide and was mostly insensitive to oligomycin, vanadate, or copper. ATP-dependent MeA was stimulated by anions with decreasing order of potency of Cl⁻ > Br⁻ > NO₃⁻ > SO₄²⁻, iminodiacetate, benzene sulfonate. Anion stimulation of H⁺ pumping was caused in part by the ability of permeant anions to dissipate the electrical potential and in part by a specific requirement of Cl⁻ by a H⁺ -pumping ATPase. A pH gradient, probably caused by a Donnan potential, could be dissipated by K⁺ in the presence or absence of ATP. MeA uptake was enriched in vesicles of relatively low density and showed a parallel distribution with vanadate-insensitive ATPase activity on a continuous dextran gradient. ΔpH as measured by quinacrine quench was partially vanadate-sensitive. These results show that plant membranes have at least two types of H⁺ -pumping ATPases. One is vanadate-sensitive and probably enriched in the plasma membrane. One is vanadate-resistant, anion-sensitive and has many properties characteristic of a vacuolar ATPase. These results are consistent with the presence of electrogenic H⁺ pumps at the plasma membrane and tonoplast of higher plant cells.     

Effect of l-Canavanine on Nitrate Reductase in Corn Roots

l-Canavanine inhibits the appearance of nitrate reductase (NADH-nitrate oxidoreductase, EC 1.6.6.1) in both root tips and mature root sections of corn (Zea mays L.). Ten-fold more canavanine was required to cause a 50% reduction in the level of nitrate reductase activity (NRA) in root tips than in mature root sections. For example with one particular batch of seeds 500 μm canavanine was effective in root tips whereas only 50 μm was required in mature root sections. In root tips arginine (1 mm) completely reversed the effect of 1 mm canavanine. In mature root sections higher concentrations of arginine (approximately 5 mm) were required for a complete reversal of the canavanine effect. Additions of canavanine to roots after a period of 3 hours with 5 mm KNO₃ resulted in a loss of NRA. NO₃⁻ protected nitrate reductase from this inactivation in both root tip and mature root sections.     

Modification of logarithmic growth rates of tobacco callus tissue by gibberellic Acid

Logarithmic growth rates (either fresh or dry weight basis) of tobacco callus tissues grown on 10⁻⁴ to 10⁻¹ μm cytokinin are increased if gibberellic acid (10⁻³-2 μm) is incorporated into the medium. At higher (1-10 μm) cytokinin concentrations gibberellic acid has little effect on growth rate but extends the duration of logarithmic growth. The gibberellic acid effect is noticeable only after one weight doubling, is dependent on concentration, and occurs when either glucose or sucrose is used as carbon source. The gibberellic acid response includes a decrease in percentage of dry weight relative to control tissues. The maximum dry weight yield, although achieved sooner than controls, does not differ appreciably from yields of tissue not treated with gibberellic acid.     

Nitrogen metabolism of soybeans: I. Effect of tungstate on nitrate utilization, nodulation, and growth

The effects of N source (6 mm nitrogen as NO₃⁻ or urea) and tungstate (0, 100, 200, 300, and 400 μm Na₂ WO₄) on nitrate metabolism, nodulation, and growth of soybean (Glycine max [L.] Merr.) plants were evaluated. Nitrate reductase activity and, to a lesser extent, NO₃⁻ content of leaf tissue decreased with the addition of tungstate to the nutrient growth medium. Concomitantly, nodule mass and acetylene reduction activity of NO₃⁻-grown plants increased with addition of tungstate to the nutrient solution. In contrast, nodule mass and acetylene reduction activity of urea-grown plants decreased with increased nutrient tungstate levels. The acetylene reduction activity of nodulated roots of NO₃⁻-grown plants was less than 10% of the activity of nodulated roots of urea-grown plants when no tungstate was added. At 300 and 400 μm tungstate levels, acetylene reduction activity of nodulated roots of NO₃⁻-grown plants exceeded the activity of comparable urea-grown plants.     

H-ATPase Activity from Storage Tissue of Beta vulgaris: I. Identification and Characterization of an Anion-Sensitive H-ATPase

Microsomal membranes isolated from red beet (Beta vulgaris L.) storage tissue were found to contain high levels of ionophore-stimulated ATPase activity. The distribution of this ATPase activity on a continuous sucrose gradient showed a low density peak (1.09 grams per cubic centimeter) that was stimulated over 400% by gramicidin and coincided with a peak of NO₃⁻-sensitive ATPase activity. At higher densities (1.16-1.18 grams per cubic centimeter) a shoulder of gramicidin-stimulated ATPase that coincided with a peak of vanadate-sensitive ATPase was apparent. A discontinuous sucrose gradient of 16/26/34/40% sucrose (w/w) was effective in routinely separating the NO₃⁻-sensitive ATPase (16/26% interface) from the vanadate-sensitive ATPase (34/40% interface). Both membrane fractions were shown to catalyze ATP-dependent H⁺ transport, with the transport process showing the same differential sensitivity to NO₃⁻ and vanadate as the ATPase activity.

Characterization of the lower density ATPase (16/26% interface) indicated that it was highly stimulated by gramicidin, inhibited by KNO₃, stimulated by anions (Cl⁻ > Br⁻ > acetate > HCO₃⁻ > SO₄²⁻), and largely insensitive to monovalent cations. These characteristics are very similar to those reported for tonoplast ATPase activity and a tonoplast origin for the low density membrane vesicles was supported by comparison with isolated red beet vacuoles. The membranes isolated from the vacuole preparation were found to possess an ATPase with characteristics identical to those of the low density membrane vesicles, and were shown to have a peak density of 1.09 grams per cubic centimeter. Furthermore, following osmotic lysis the vacuolar membranes apparently resealed and ATP-dependent H⁺ transport could be demonstrated in these vacuole-derived membrane vesicles. This report, thus, strongly supports a tonoplast origin for the low density, anion-sensitive H⁺-ATPase and further indicates the presence of a higher density, vanadate-sensitive, H⁺-ATPase in the red beet microsomal membrane fraction, which is presumably of plasma membrane origin.

     

Human DNA Polymerase ν Catalyzes Correct and Incorrect DNA Synthesis with High Catalytic Efficiency

DNA polymerase ν (pol ν) is a low fidelity A-family polymerase with a putative role in interstrand cross-link repair and homologous recombination. We carried out pre-steady-state kinetic analysis to elucidate the kinetic mechanism of this enzyme. We found that the mechanism consists of seven steps, similar that of other A-family polymerases. pol ν binds to DNA with a K_d for DNA of 9.2 nm, with an off-rate constant of 0.013 s⁻¹and an on-rate constant of 14 μm⁻¹ s⁻¹. dNTP binding is rapid with K_d values of 20 and 476 μm for the correct and incorrect dNTP, respectively. Pyrophosphorylation occurs with a K_d value for PP_i of 3.7 mm and a maximal rate constant of 11 s⁻¹. Pre-steady-state kinetics, examination of the elemental effect using dNTPαS, and pulse-chase experiments indicate that a rapid phosphodiester bond formation step is flanked by slow conformational changes for both correct and incorrect base pair formation. These experiments in combination with computer simulations indicate that the first conformational change occurs with rate constants of 75 and 20 s⁻¹; rapid phosphodiester bond formation occurs with a K_eq of 2.2 and 1.7, and the second conformational change occurs with rate constants of 2.1 and 0.5 s⁻¹, for correct and incorrect base pair formation, respectively. The presence of a mispair does not induce the polymerase to adopt a low catalytic conformation. pol ν catalyzes both correct and mispair formation with high catalytic efficiency.     

myo-Inositol homeostasis in foetal rabbit lung

In several species, lung maturation is accompanied by a decline in the phosphatidylinositol content of lung surfactant and a concomitant increase in its phosphatidylglycerol content. To examine the possibility that this developmental change is influenced by the availability of myo-inositol, potential sources of myo-inositol for the developing rabbit lung were investigated. On day 28 of gestation the myo-inositol content of foetal rabbit lung tissue (2.3±0.5μmol/g of tissue) was not significantly different from that of adult lung tissue but the activity of d-glucose 6-phosphate:1l-myo-inositol 1-phosphate cyclase (cyclase) in foetal lung tissue (81.0±9.0nmol·h⁻¹·g of tissue⁻¹) was higher than that found in adult lung tissue (23.2±1.0nmol·h⁻¹·g of tissue⁻¹). Day 28 foetal rabbit lung tissue was found also to take up myo-inositol by a specific, energy-dependent, Na⁺-requiring mechanism. Half-maximal uptake of myo-inositol by foetal rabbit lung slices was observed when the concentration of myo-inositol in the incubation medium was 85μm. When the myo-inositol concentration was 1mm (but not 100μm) the addition of glucose (5.5mm) stimulated myo-inositol uptake. myo-Inositol uptake was observed also in adult rabbit lung and was found to be sub-maximal at the concentration of myo-inositol found in adult rabbit serum. The concentration of myo-inositol in the serum of pregnant adult rabbits (47.5±5.5μm) was significantly lower than that of non-pregnant adult female rabbits (77.9±9.2μm). On day 28 of gestation the concentration of myo-inositol in foetal serum (175.1±12.0μm) was much less than on day 25, but more than that found on day 30. A transient post-partum increase in the concentration of myo-inositol in serum was followed by a rapid decline. Much of the myo-inositol in foetal rabbit serum probably originates from the placenta, where on day 28 of gestation a high cyclase activity (527±64nmol·h⁻¹·g of tissue⁻¹) was measured. The gestational decline in serum myo-inositol concentration, together with the decreasing cyclase activity of the lungs, is consistent with the view that maturation of the lungs is accompanied by decreased availability of myo-inositol to this tissue.     

Inhibition of purine phosphoribosyltransferases of Ehrlich ascites-tumour cells by 6-mercaptopurine

1. The formation of adenosine 5′-phosphate, guanosine 5′-phosphate and inosine 5′-phosphate from [8-¹⁴C]adenine, [8-¹⁴C]guanine and [8-¹⁴C]hypoxanthine respectively in the presence of 5-phosphoribosyl pyrophosphate and an extract from Ehrlich ascites-tumour cells was assayed by a method involving liquid-scintillation counting of the radioactive nucleotides on diethylaminoethylcellulose paper. The results obtained with guanine were confirmed by a spectrophotometric assay which was also used to assay the conversion of 6-mercaptopurine and 5-phosphoribosyl pyrophosphate into 6-thioinosine 5′-phosphate in the presence of 6-mercaptopurine phosphoribosyltransferase from these cells. 2. At pH 7·8 and 25° the Michaelis constants for adenine, guanine and hypoxanthine were 0·9 μm, 2·9 μm and 11·0 μm in the assay with radioactive purines; the Michaelis constant for guanine in the spectrophotometric assay was 2·6 μm. At pH 7·9 the Michaelis constant for 6-mercaptopurine was 10·9 μm. 3. 25 μm-6-Mercaptopurine did not inhibit adenine phosphoribosyltransferase. 6-Mercaptopurine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 4·7 μm) and hypoxanthine phosphoribosyltransferase (K_i 8·3 μm). Hypoxanthine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 3·4 μm). 4. Differences in kinetic parameters and in the distribution of phosphoribosyltransferase activities after electrophoresis in starch gel indicate that different enzymes are involved in the conversion of adenine, guanine and hypoxanthine into their nucleotides. 5. From the low values of K_i for 6-mercaptopurine, and from published evidence that ascites-tumour cells require supplies of purines from the host tissues, it is likely that inhibition of hypoxanthine and guanine phosphoribosyltransferases by free 6-mercaptopurine is involved in the biological activity of this drug.     

Alkylguanidine inhibition of ion absorption in oat roots

The effect of various alkylguanidines on ion absorption and energy metabolism in oat (Avena sativa cv. Goodfield) roots has been investigated. Of several alkylguanidines tested, octylguanidine was the most effective inhibitor of both K⁺ and Cl⁻ absorption by excised roots. At 225 μm octylguanidine, the transport of both ions was inhibited within 60 seconds and to a similar extent. Octylguanidine inhibited mitochondrial oxidative phosphorylation and mitochondrial adenosine 5′-triphosphatase (ATPase). The plasma membrane ATPase was also inhibited if the membranes were diluted and pretreated with Triton X-100.     

The use of potential-sensitive cyanine dye for studying ion-dependent electrogenic renal transport of organic solutes. Spectrophotometric measurements

Renal transport of four different categories of organic solutes, namely sugars, neutral amino acids, monocarboxylic acids and dicarboxylic acids, was studied by using the potential-sensitive dye 3,3′-diethyloxadicarbocyanine iodide in purified luminal-membrane and basolateral-membrane vesicles isolated from rabbit kidney cortex. Valinomycin-induced K⁺ diffusion potentials resulted in concomitant changes in dye–membrane-vesicle absorption spectra. Linear relationships were obtained between these changes and depolarization and hyperpolarization of the vesicles. Addition of d-glucose, l-phenylalanine, succinate or l-lactate to luminal-membrane vesicles, in the presence of an extravesicular>intravesicular Na⁺ gradient, resulted in rapid transient depolarization. With basolateral-membrane vesicles no electrogenic transport of d-glucose or l-phenylalanine was observed. Spectrophotometric competition studies revealed that d-galactose is electrogenically taken up by the same transport system as that for d-glucose, whereas l-phenylalanine, succinate and l-lactate are transported by different systems in luminal-membrane vesicles. The absorbance changes associated with simultaneous addition of d-glucose and l-phenylalanine were additive. The uptake of these solutes was influenced by the presence of Na⁺-salt anions of different permeabilities in the order: Cl⁻>SO₄²⁻>gluconate. Addition of valinomycin to K⁺-loaded vesicles enhanced uptake of d-glucose and l-phenylalanine in the presence of an extravesicular>intravesicular Na⁺ gradient. Gramicidin or valinomycin plus nigericin diminished/abolished electrogenic solute uptake by Na⁺- or Na⁺+K⁺-loaded vesicles respectively. These results strongly support the presence of Na⁺-dependent renal electrogenic transport of d-glucose, l-phenylalanine, succinate and l-lactate in luminal-membrane vesicles.