Nitrogen fixation in cultured cowpea Rhizobia: inhibition and regulation of nitrogenase activity.

Nitrogenase activity in agar cultures of cowpea rhizobia, strain 32H1, was rapidly inhibited by $\text{NH}{}_4{}^{\mathrm{+}}$ but this was relieved by increased O₂ tension. Inhibition was more rapid than that caused by inhibitors of protein synthesis and was not relieved by methionine sulfoximine or methionine sulfone. Under conditions were nitrogenase activity was inhibited by $\text{NH}{}_4{}^{\mathrm{+}}$, glutamine synthetase and glutamate synthase were substantially unaffected. Glutamate dehydrogenase was undetected in either nitrogenase active or $\text{NH}{}_4{}^{\mathrm{+}}$ inhibited cultures. These results indicate that $\text{NH}{}_4{}^{\mathrm{+}}$ inhibition of nitrogenase activity in strain 32H1 is not effected through glutamine synthetase regulation of nitrogenase synthesis.     

Regulation of glutamate synthase from Bacillussubtilis by Glutamine

Glutamate synthase, an important enzyme in the assimilation of ammonia, was measured in cultures of $\text{Bacillus}\text{subtilis}$ grown with different nitrogen sources. An attempt was made to correlate the specific activity to the intracellular levels of five metabolites of glutamate metabolism: aspartate, glutamate, glutamine, alanine and $\text{NH}{}_4{}^{\mathrm{+}}$. An inverse relationship was found between the activity of glutamate synthase and the pool level of glutamine. We propose that the intracellular concentration of glutamine is an important element in controlling the level of glutamate synthase.     

ColE1 hybrid plasmids containing Escherichia coli genes involved in the biosynthesis of glutamate and glutamine

The Clarke-Carbon bank of Escherichia coli strains carrying ColE1 hybrid plasmids was screened for complementation of gdh, gltB, and glnA mutations affecting nitrogen metabolism in E. coli. Plasmids which complemented each one of these mutations were isolated. In every case, the plasmids conferred to otherwise mutant cells the capacity to synthesize the corresponding wild-type enzymes: glutamate dehydrogenase, glutamate synthase, and glutamine synthetase (GS), respectively. For three representative plasmids, endonuclease restriction maps were constructed. One of the plasmids, pACR1, which complemented glnA mutations, including the glnA21::Tn5 insertion, was deemed to carry the glnA⁺ allele. GS synthesis by pACR1 $\text{glnA}{}^{\mathrm{+}}\text{glnA20}$ heterozygous merodiploids was subjected to repression by growth on 15 mm NH₄⁺ and had a twofold high derepressed level than wild-type (glnA⁺) haploid cells when grown on 0.5 mm NH₄⁺ or on glutamate as only nitrogen sources. The presence of glutamine as sole nitrogen source promoted repressed GS synthesis in the $\text{glnA}{}^{\mathrm{+}}\text{glnA20}$ merodiploids. By contrast, glutamine allowed almost fully derepressed synthesis of GS in glnA⁺ haploid cells.     

Effects of L-methionine-DL-sulphoximine on the assimilation of newly fixed NH3, acetylene reduction and heterocyst production in Anabaena cylindrica.

The addition of exogenous L-methionine-DL-sulphoximine (MSO) to N₂-fixing cultures of the blue-green alga Anabaena cylindrica results in over half of the newly fixed NH₃ being released into the medium. MSO also inhibits glutamine synthetase (GS) activity, has negligible effect on alanine dehydrogenase activity, and glutamate dehydrogenase activity under N₂-fixing conditions is negligible. In the presence of MSO, intracellular pools of glutamate and glutamine decrease, those of aspartate and alanine + glycine show little change, and the NH₃ pool increases. MSO alleviates the inhibitory effect of exogenous NH₄⁺ on nitrogenase synthesis and heterocyst production. The results suggest that in N₂-fixing cultures of A. cylindrica the primary NH₃ assimilating pathway involves GS, and probably glutamate synthase (GOGAT), and that the repressor of nitrogenase synthesis and heterocyst production is not NH₄⁺ but is GS, GOGAT, or a product of their reactions.     

Effects of L-methionine-DL-sulfoximine and beta-N-oxalyl-L-alpha, beta-diaminopropionic acid on nitrogenase biosynthesis and activity in Rhodopseudomonas capsulata.

Inhibitors of glutamine synthetase cause derepression of nitrogenase biosynthesis in the presence of $\text{NH}{}_4{}^{\mathrm{+}}$ in the photosynthetic bacterium Rhodopseudomonas capsulata. A new derepressor of nitrogenase biosynthesis, β-N-oxalyl-L-α,β-diaminopropionic acid (ODAP), is here compared with the widely used L-methionine-DL-sulfoximine (MSX). With both compounds, a quantitative correlation has been observed between inhibition of glutamine synthetase and derepression of nitrogenase biosynthesis. We also find that both MSX and ODAP inhibit nitrogenase activity in vivo in R. capsulata. The latter effect seems to be indirect and related to the previously reported reversible inhibition of nitrogenase activity in vivo by $\text{NH}{}_4{}^{\mathrm{+}}$. As a control it was observed that neither $\text{NH}{}_4{}^{\mathrm{+}}$ nor MSX nor ODAP inhibit nitrogenase activity in vivo in Clostridium pasteurianum.     

Equilibrium constants for association of guanidinium and ammonium ions with oxyanions: The effect of changing basicity of the oxyanion

Using guanidinium and n-butylammonium cations (C⁺) as models for the positively charged side chains in arginine and lysine, we have determined the association constants with various oxyanions by potentiometric titration. For a dibasic acid, H₂A, three association complexes may exist: $\text{K}{}_{\mathrm{<mtext>1M</mtext>}}\text{=}\text{[CHA]}\text{[C}{}^{\mathrm{+}}\text{]}\text{[HA}{}^{\mathrm{?}}\text{]}$; $\text{K}{}_{\mathrm{<mtext>1D</mtext>}}\text{=}\text{[CA}{}^{\mathrm{?}}\text{]}\text{[C}{}^{\mathrm{+}}\text{]}\text{[A}{}^{\mathrm{2?}}\text{]}$; $\text{K}{}_{\mathrm{<mtext>2D</mtext>}}\text{=}\text{[C}{}_2\text{A]}\text{[C}{}^{\mathrm{+}}\text{]}\text{[CA}{}^{\mathrm{?}}\text{]}$. For guanidinium ion and phosphate, K_1M = 1.4, K_1D = 2.6, and K_2D = 5.1. The data for carboxylates indicate that the basicity of the oxyanion does not affect the association constant: acetate, pK_a = 4.8, K_1M = 0.37; formate, pK_a = 3.8, K_1M = 0.32; and chloroacetate, pK_a = 2.9, K_1M = 0.43, all with guanidinium ion. Association constants are also reported for carbonate, dimethylphosphinate, benzylphosphonate, and adenylate anions.     

The effect of ammonium ions upon isolated reactions of the glycolytic scheme

In the glycolytic system derived from rat brain acetone powder, ammonium ion has been found to stimulate three different reactions: (a) the transphosphorylase reaction from phosphoenolpyruvate, (b) the phosphohexokinase reaction, and (c) the hexokinase reaction. The transphosphorylases are affected differently depending upon whether adenosine diphosphate or adenylic acid is the phosphate acceptor; in the case of the latter, the dependency upon $\text{NH}{}_4{}^{\mathrm{+}}$ is particularly marked. A highly active myokinase is present in these extracts and its activity influences the transphosphorylase reaction to a considerable extent. The phosphohexokinase reaction is stimulated to a greater extent by $\text{NH}{}_4{}^{\mathrm{+}}$ than is the hexokinase reaction. In contrast to these reactions which require the participation of the adenylic system, triose phosphate oxidase activity is uninfluenced by the presence of $\text{NH}{}_4{}^{\mathrm{+}}$.     

Respiration-driven proton translocation with nitrite and nitrous oxide in Paracoccus denitrificans

(1) $\text{H}$⁺/electron acceptor ratios have been determined with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing endogenous substrates during reduction of O₂, NO^?₂ or N₂O. Under optimal H⁺-translocation conditions, the ratios $\text{H}{}^{\mathrm{+}}\text{O}$, $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were 6.0–6.3, 4.02, 5.79 and 3.37, respectively. (2) With ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as exogenous substrate, addition of NO^?₂ or N₂O to an anaerobic cell suspension resulted in rapid alkalinization of the outer bulk medium. $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were ?0.84, ?2.33 and ?1.90, respectively. (3) The $\text{H}{}^{\mathrm{+}}\text{oxidant}$ ratios, mentioned in item 2, were not altered in the presence of $\text{valinomycin}\text{K}{}^{\mathrm{+}}$ and the triphenylmethylphosphonium cation. (4) A simplified scheme of electron transport to O₂, NO^?₂ and N₂O is presented which shows a periplasmic orientation of the nitrite reductase as well as the nitrous oxide reductase. Electrons destined for NO^?₂, N₂O or O₂ pass two H⁺-translocating sites. The $\text{H}{}^{\mathrm{+}}\text{electron}$ acceptor ratios predicted by this scheme are in good agreement with the experimental values.     

Soluble and enzymatically stable (Na++K+)-ATPase from mammalian kidney consisting predominantly of protomer αβ-units: Preparation,assay and reconstitution of active Na+, K+transport

Soluble $\text{(Na}{}^{\mathrm{+}}\text{+K}{}^{\mathrm{+}}\text{)-ATPase}$ consisting predominantly of αβ-units with $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ below 170 000 was prepared by incubating pure membrane-bound $\text{(Na}{}^{\mathrm{+}}\text{+K}{}^{\mathrm{+}}\text{)-ATPase}$ (35–48 μmol P_i/min per mg protein) from the outer renal medulla with the non-ionic detergent dodecyloctaethyleneglycol monoether (C₁₂E₈). $\text{(Na}{}^{\mathrm{+}}\text{+K}{}^{\mathrm{+}}\text{)-ATPase}$ and potassium phosphatase remained fully active in the detergent solution at C₁₂E₈/protein ratios of 2.5–3, at which 50–70% of the membrane protein was solubilized. The soluble protomeric $\text{(Na}{}^{\mathrm{+}}\text{+K}{}^{\mathrm{+}}\text{)-ATPase}$ was reconstituted to Na⁺, K⁺ pumps in phospholipid vesicles by the freeze-thaw sonication procedure. Protein solubilization was complete at C₁₂E₈/protein ratios of 5–6, at the expense of partial inactivation, but $\text{(Na}{}^{\mathrm{+}}\text{+K}{}^{\mathrm{+}}\text{)-ATPase}$ and potassium phosphatase could be reactivated after binding of C₁₂E₈ to Bio-Beads SM2. At C₁₂E₈/protein ratios higher than 6 the activities were irreversibly lost. Inactivation could be explained by delipidation. It was not due to subunit dissociation since only small changes in sedimentation velocities were seen when the C₁₂E₈/protein ratio was increased from 2.9 to 46. As determined immediately after solubilization, $\text{S}{}_{\mathrm{<mtext>20,</mtext><mtext>w</mtext>}}$ was 7.4 S for the fully active $\text{(Na}{}^{\mathrm{+}}\text{+K}{}^{\mathrm{+}}\text{)-ATPase}$, 7.3 S for the partially active particle, and 6.5 S for the inactive particle at high C₁₂E₈/protein ratios. The maximum molecular masses determined by analytical ultracentrifugation were 141 000–170 000 dalton for these protein particles. Secondary aggregation occurred during column chromatography, with formation of enzymatically active $\text{(αβ)}{}_2\text{-}\text{dimers}$ or $\text{(αβ)}{}_3\text{-}\text{trimers}$ with $\text{S}{}_{\mathrm{<mtext>20,</mtext><mtext>w</mtext>}}\text{=10–12}$ S and apparent molecular masses in the range 273 000–386 000 daltons. This may reflect non-specific time-dependent aggregation of the detergent micelles.     

N(4)-amino-and N(4)-hydroxycytosines as base analogue mutagens

N(4)-Aminocytosine [N(4)NH₂C] and N(4)-amino-2′-deoxycytidine [N(4)NH₂dC] are highly mutagenic for Escherichia coli and phage φ 80 but not for T₄. There is some evidence that they are incorporated into the φ 80 DNA but $\text{[}{}^{14}\text{C}\text{]-N(4)}\text{NH}{}_2\text{C}$ could not be detected in the bacterial DNA.N(4)-Hydroxy-5,6-dihydro-6-hydroxylaminodeoxycytidine (di-NHOH-dC) is mutagenic for φ 80 and E. coli, but N(4)-hydroxydeoxycytidine [N(4)OH-dC] only has a strong inactivating effect.     

Studies of the nucleotide-binding sites on the mitochondrial F1-ATPase through the use of a photoactivable derivative of adenylyl imidodiphosphate

(1) $\text{N-4-}\text{Azido}\text{-2-}\text{nitrophenyl}\text{-γ-[}{}^3\text{H]aminobutyryl-Ado}\text{PP[}\text{NH}\text{]P(}\text{NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P)}$ a photoactivable derivative of 5-adenylyl imidodiphosphate (AdoPP[NH]P), was synthesized. (2) Binding of ³H]NAP₄-AdoPP[NH]P to soluble ATPase from beef heart mitochrondria (F₁) was studied in the absence of photoirradiation, and compared to that of $\text{[}{}^3\text{H]Ado}\text{PP[}\text{NH}\text{]P}$. The photoactivable derivative of AdoPP[NH]P was found to bind to F₁ with high affinity, like AdoPP[NH]P. Once $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ had bound to F₁ in the dark, it could be released by AdoPP[NH]P, ADP and ATP, but not at all by NAP₄ or AMP. Furthermore, preincubation of F₁ with unlabeled AdoPP[NH]P, ADP, or ATP prevented the covalent labeling of the enzyme by $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ upon photoirradiation. (3) Photoirradiation of F₁ by $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ resulted in covalent photolabeling and concomitant inactivation of the enzyme. Full inactivation corresponded to the binding of about 2 mol $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}\text{mol F}{}_1$. Photolabeling by NAP₄-AdoPP[NH]P was much more efficient in the presence than in the absence of MgCl₂. (4) Bound $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ was localized on the α- and β-subunits of F₁. At low concentrations (less than 10 μM), bound $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ was predominantly localized on the α-subunit; at concentrations equal to, or greater than 75 μM, both α- and β-subunits were equally labeled. (5) The extent of inactivation was independent of the nature of the photolabeled subunit (α or β), suggesting that each of the two subunits, α and β, is required for the activity of F₁. (6) The covalently photolabeled F₁ was able to form a complex with aurovertin, as does native F₁. The ADP-induced fluorescence enhancement was more severely inhibited than the fluorescence quenching caused by ATP. The percentage of inactivation of F₁ was virtually the same as the percentage of inhibition of the ATP-induced fluorescence quenching, suggesting that fluorescence quenching is related to the binding of ATP to the catalytic site of F₁.     

Nuclear magnetic resonance study of the rate of electron transfer between cytochrome c and iron hexacyanides

The rates of electron exchange between ferricytochrome c (C^III)3 and ferrocytochrome c (C^II) were observed as a function of the concentrations of ferrihexacyanide (Fe^III) and ferrohexacyanide (Fe^II) by monitoring the line widths of several proton resonances of the protein. Addition of Fe^II to C^III homogeneously increased the line widths of the two downfield paramagnetically shifted heme methyl proton resonances to a maximal value. This was interpreted as indicating the formation of a stoichiometric complex, C^III·Fe^II, in the over-all reaction:

$\text{C}{}^{\mathrm{III}}\text{+}\text{Fe}{}^{\mathrm{II}}\text{?}\text{k}{}_{\mathrm{?1}}\text{k}{}_1\text{C}{}^{\mathrm{III}}\text{·}\text{Fe}{}^{\mathrm{II}}\text{?}\text{k}{}_{\mathrm{?2}}\text{k}{}_2\text{C}{}^{\mathrm{II}}\text{·}\text{Fe}{}^{\mathrm{III}}\text{?}\text{k}{}_{\mathrm{?3}}\text{k}{}_3\text{C}{}^{\mathrm{III}}\text{+}\text{Fe}{}^{\mathrm{II}}$

Values for $\text{k}{}_1\text{k}{}_{\mathrm{?1}}\text{= 0.4 × 10}{}^3\text{m}{}^{\mathrm{?1}}\text{and}\text{k}{}_2\text{= 208}\text{s}{}^{\mathrm{?1}}$, respectively, were calculated from the maximal change in line width observed at pH 7.0 and 25 °C. Changes in the line width of C^III in the presence of Fe^II and either KCl or Fe^III suggest that complexation is principally ionic, that Fe^III and Fe^II compete for a common site. Addition of saturating concentrations of Fe^III to C^III produced only minor changes in the nuclear magnetic resonance spectrum of C^III suggesting that complexation occurs on the protein surface.Addition of Fe^III to C^II in the presence of excess Fe^II (to retain most of the protein as C^II) increased the line width of the methyl protons of ligated methionine 80. A value for k_?2 ≈ 2.08 × 10⁴ s^?1 was calculated from the dependence of linewidth on the concentration of Fe^II at 24 °C. These rates are shown to be consistent with the over-all rates of reduction and oxidation previously determined by stopped flow measurements, indicating that k₂ and k_?2 were rate limiting. From the temperature dependence the enthalpies of activation are 7.9 and 15.2 kcal/mol for k₂ and k_?2, respectively.     

Interaction of lymphocytes and macrophage cell line cells (M1 cells). I. Functional maturation and appearance of Fc receptors im M1 cells

M₁ cells, which are cell line cells established from myeloid leukemia cells of the SL strain mouse, can differentiate from blast cells ($\text{M}{}_1{}^{\mathrm{?}}$) to mature macrophages ($\text{M}{}_1{}^{\mathrm{+}}$) within 48 hr, when they are cultured with conditioned medium (CM) obtained from murine embryonic fibroblasts. While $\text{M}{}_1{}^{\mathrm{?}}$ cells have no phagocytic activity nor Fc receptor (FcR), $\text{M}{}_1{}^{\mathrm{+}}$ cells possess both characteristics. The appearance of FcR is temperature-dependent and inhibited by a metabolic inhibitor, cycloheximide. FcR on $\text{M}{}_1{}^{\mathrm{+}}$ cells is resistant to trypsin and pronase. $\text{M}{}_1{}^{\mathrm{+}}$ cells improve the viability of macrophage-depleted SL splenic lymphocytes and restore the in vitro secondary plaque forming cell response of macrophage-depleted spleen cells to particulate and soluble antigens. $\text{M}{}_1{}^{\mathrm{?}}$ cells lack this macrophage-substituting capacity. Mm₁ cells, mutant cells from M₁ cells, having FcR and higher phagocytic activity than $\text{M}{}_1{}^{\mathrm{+}}$ cells, are also devoid of this capacity.     

Effect of N-alkyl-N,N,N-trimethylammonium ions on phosphatidylcholine model membrane structure as studied by 31P-NMR

The interaction of |C_nH_2n+1N⁺(CH₃)₃| · I^? (n = 3, 6, 9, 12, 14, 16 or 18) with egg-yolk phosphatidylcholine-water dispersions has been studied by ³¹P-NMR spectroscopy. It is shown that the effective anisotropy of ³¹P chemical shift (?Δσ_eff) of the lamellar phospholipid liquid-crystalline phase L_α increases with increasing concentration and alkyl chain length of the drug. Addition of $\text{|}\text{C}{}_6\text{H}{}_{13}\text{N}{}^{\mathrm{+}}\text{(CH}{}_3\text{)}{}_3\text{| ·I}{}^{\mathrm{?}}$ or |C₉H₁₉N⁺(CH₃)₃|·I^? to the phospholipid-water dispersion at a molar ratio ammonium salt:phospholipid > 0.8 induces in the dispersion a structure with an effective isotropic phospholipid motion. This structure is unstable and slowly transforms into the hexagonal phase. These effects have not been observed in phospholipid-water dispersions mixed with the ammonium derivatives with the longer alkyl chains n  12, 14, 16 or 18. It is proposed that these results might explain the effects of the investigated drugs on the nerve, muscle and bacterial cells.     

The insect brain (Na+ + K+)-ATPase Binding of ouabain in the hawk moth, Manduca sexta

(1) A quantitative study has been made of the binding of ouabain to the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ in homogenates prepared from brain tissue of the hawk moth, Manduca sexta. The results have been compared to those obtained in bovine brain microsomes. (2) The insect brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ will bind ouabain either in the presence of Mg²⁺ and P_i, (‘Mg²⁺, P_i’ conditions) or in the presence of Na⁺, Mg²⁺, and an adenine nucleotide (‘nucleotide’ conditions) as is the case for the bovine brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$. The binding conditions did not alter the total number of receptor sites measured at high ouabain concentrations in either tissue. (3) Potassium ion decreases the affinity (increases the $\text{K}{}_{\mathrm{<mtext>D</mtext>}}$) of ouabain to the M. sexta brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ under both binding conditions. However, ouabain binding is more sensitive to K⁺ inhibition under the nucleotide conditions. In bovine brain ouabain binding is equally sensitive to K⁺ inhibition under the both conditions. (4) The enzyme-ouabain complex has a rate of dissociation that is 10-fold faster in the M. sexta preparation than in the bovine brain preparation. Because of this, the M. sexta ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ has a higher $\text{K}{}_{\mathrm{<mtext>D</mtext>}}$ for ouabain binding and is less sensitive to inhibition by ouabain than the bovine brain enzyme. (5) This data supports the hypothesis that two different conformational states of the M. sexta ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ can bind ouabain.     

Stimulating effects of monovalent cations on activator-dissociated and activator-associated states of Ca2+-ATPase in human erythrocytes

The additional activation by monovalent cations of the $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-dependent}$ ATPase (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied.The Ca²⁺-ATPase occurs in two different states. In the A-state the enzyme is virtually free of protein activator and the kinetics of Ca²⁺ activation is characterized by low apparent Ca²⁺ affinity and low maximum activity. In the B-state the enzyme is associated with activator and the kinetics is characterized by high Ca²⁺ affinity and high maximum activity.At optimum concentrations of Ca²⁺ the additional activation of the B-state by K⁺, NH₄⁺, Na⁺ and Rb⁺ exceeded the corresponding activations of the A-state, and half-maximum activations by K⁺, NH₄⁺, and Na⁺ were achieved at lower concentrations in the B-state than in the A-state. Li⁺ and Cs⁺ activated the two states almost equally but maximum activation was obtained at lower cation concentrations in the B-state than in the A-state.The activation of the B-state by the various cations decreased in the order $\text{K}{}^{\mathrm{+}}\text{> NH}{}_4{}^{\mathrm{+}}\text{> Na}{}^{\mathrm{+}}\text{= Rb}{}^{\mathrm{+}}\text{> Li}{}^{\mathrm{+}}\text{= Cs}{}^{\mathrm{+}}$. The A-state was activated almost equally by K⁺, Na⁺, NH₄⁺, and Rb⁺ and to a smaller extent by Li⁺ and Cs⁺.At sub-optimum concentrations of Ca²⁺ high concentrations of monovalent cations (100 mM) activated the Ca²⁺-ATPase equally in the A-state and the B-state. In the absence of Ca²⁺ the monovalent cations inhibited the Mg²⁺-dependent ATPase in both types of membranes. This dependence on Ca²⁺ indicates that the monovalent cations interact with the Ca²⁺ sites in the B-state.The results suggest that K⁺ or Na⁺, or both, contribute to the regulation of the Ca²⁺ pump in erythrocytes.     

Photosystem II energy coupling in chloroplasts with H2O2 as electron donor

NH₂OH-treated, non-water-splitting chloroplasts can oxidize H₂O₂ to O₂ through Photosystem II at substantial rates (100–250 μequiv · h^?1 · mg^?1 chlorophyll with 5 mM H₂O₂) using 2,5-dimethyl-p-benzoquinone as an electron acceptor in the presence of the plastoquinone antagonist dibromothymoquinone. This H₂O₂ → Photosystem II → dimethylquinone reaction supports phosphorylation with a $\text{P}\text{e}{}_2$ ratio of 0.25–0.35 and proton uptake with $\text{H}{}^{\mathrm{+}}\text{e}$ values of 0.67 (pH 8)–0.85 (pH 6). These are close to the $\text{P}\text{e}{}_2$ value of 0.3–0.38 and the $\text{H}{}^{\mathrm{+}}\text{e}$ values of 0.7–0.93 found in parallel experiments for the H₂O → Photosystem II → dimethylquinone reaction in untreated chloroplasts. Semi-quantitative data are also presented which show that the donor → Photosystem II → dibromothymoquinone (→O₂) reaction can support phosphorylation when the donor used is a proton-releasing reductant (benzidine, catechol) but not when it is a non-proton carrier (I^?, ferrocyanide).     

Studies on (K+ + H+)-ATPase IV. Effects of phospholipase C treatment

(1) The total phospholipid content of a gradient purified ($\text{K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}$)-ATPase preparation from pig gastric mucosa is 105 μmol per 100 mg protein, and consists of 29% sphingomyelin, 29% phosphatidylcholine, 28% phosphatidylethanolamine, 10% phosphatidylserine and 4% phosphatidylinositol. The cholesterol content corresponds to 50 μmol per 100 mg protein. (2) Treatment with phospholipase C (from Clostridium welchii and Bacillus cereus) results in an immediate decrease of the phosphate content. Up to 50% of the phospholipids are hydrolyzed by each phospholipase C preparation alone, without further hydrolysis by increased phospholipase concentration or prolonged incubation time. Combined treatment with the two phospholipase C preparations, sequentially or simultaneously, hydrolyzes up to 65% of the phospholipids. (3) The ($\text{K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}$)-ATPase and K⁺ stimulated $\text{p-}\text{nitrophenylphosphatase}$ activities are decreased proportionally with the total phospholipid content, indicating that these enzyme activities are dependent on phospholipids. (4) Phospholipase C treatment does not change optimal pH, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for ATP and temperature dependence of the gastric ($\text{K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}$)-ATPase, but slightly decreases the $\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ value for K⁺. (5) Phospholipase C treatment lowers the $\text{Ado}\text{PP[}\text{NH}\text{]P}$ binding and phosphorylation capacities, suggesting that inactivation occurs primarily on the substrate binding level. (6) Most of the results can be understood by assuming that hydrolysis of the phospholipids by phospholipase C leads to aggregation of the membrane protein molecules and complete inactivation of the aggregated ATPase molecules.