Kinetics and mechanism of the F1 isozyme of horse liver aldehyde dehydrogenase.

The reaction mechanism of the F1 isozyme of horse liver aldehyde dehydrogenase (EC 1.2.1.3) was investigated using both steady-state and rapid kinetic techniques. Using the steady-state substrate velocity patterns, the NADH inhibition patterns at several aldehyde concentrations, and the substrate analog (adenosine diphosphoribose and chloral hydrate) inhibition patterns, the enzymic catalysis was shown to involve ordered addition of NAD followed by aldehyde. This mechanism was confirmed using the kinetics of the hydrolysis of p-nitrophenyl acetate as an indicator of the dehydrogenase substrate binding. Steady-state experiments with deuteroacetaldehyde showed the V to be unchanged, but the K_m increased (). Stopped flow experiments where E-NAD was rapidly mixed with aldehyde showed a burst of NADH formation followed by slower steady-state turnover. This result clearly indicates that the rate limiting step lies after NAD reduction. The NADH off rate (0.7 s^?1) as estimated by displacement of NADH from the E-NADH complex upon rapid addition of NAD was found to be very close to the steady-state site turnover number (0.3 s^?1). This fact and the relatively small effect of aldehyde R-group on maximal velocity suggest that the slow rate of NADH release contributes significantly to limitation of the enzyme catalytic velocity.     

Malate dehydrogenase. Kinetic studies with meso-tartrate and 2-keto-3-hydroxysuccinate, comparison of the mitochondrial and supernatant pig heart enzymes.

Initial rate, product inhibition, and isotope rate kinetic studies of pig heart mitochondrial and supernatant malate dehydrogenases, acting upon the nonphysiological substrates, meso-tartrate and 2-keto-3-hydroxysuccinate, are reported. The measured spontaneous keto-enol equilibrium for 2-keto-3-hydroxysuccinate in 0.05 m Tris-acetate (pH 8.0) at 25 °C favors the enol form, dihydroxyfumarate, with an apparent equilibrium constant of 0.036. The enzyme-catalyzed reaction favors meso-tartrate with an apparent equilibrium constant of 1.25 × 10^?6, M^?1 at pH 8.0. The mechanism apparently remains ordered bi bi for both enzymes when these nonphysiological substrates are used, and the chemical-converting hydride transfer step becomes more rate limiting for both enzymes. This conclusion is supported by $\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{V}{}_{\mathrm{<mtext>D</mtext>}}$ and $\text{(}\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{K}{}_{\mathrm{<mtext>H</mtext>}}\text{)}\text{V}{}_{\mathrm{<mtext>D</mtext>}}\text{K}{}_{\mathrm{<mtext>D</mtext>}}$ values of 2.6 and 3.1, respectively, for the mitochondrial enzyme and 1.9 and 2.9, respectively, for the supernatant enzyme.     

A Cl−/HCO3−-ATPase in the gils of Carassius Auratus. Its inhibition by thiocyanate

An ATPase is demonstrated in plasma membrane fractions of goldfish gills. This enzyme is stimulated by Cl^? and HCO₃^?, inhibited by SCN^?.Biochemical characterization shows that HCO₃^? stimulation ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 2.5}\text{mequiv./l}$) is specifically inhibited in a competitive fashion by SCN^? ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 0.25}\text{mequiv./l}$). The residual Mg²⁺-dependent activity is weakly is weakly affected by SCN^?.In the microsomal fraction chloride stimulation of the enzyme occurs in the presence of HCO₃^? ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{for chloride = 1 mequiv./l}$); no stimulation is observed in the absence of HCO₃^?. Thiocyanate exhibits a mixed type of inhibition ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 0.06}\text{mequiv./l}$) towards the Cl^? stimulation of the enzyme.Bicarbonate-dependent ATPase from the mitochondrial fraction is stimulated by Cl^?, but this enzyme has a relatively weak affinity for this substrate ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 14}\text{mequiv./l}$).     

Effects of oligomycin and quercetin on the hydrolytic activities of the (Na+ + K+)-dependent ATPase

Quercetin inhibited a dog kidney ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ preparation without affecting $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP or $\text{K}{}_{0.5}$ for cation activators, attributable to the slowly-reversible nature of its inhibition. Dimethyl sulfoxide, a selector of E₂ enzyme conformations, blocked this inhibition, while the K⁺-phosphatase activity was at least as sensitive to quercetin as the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity, all consistent with quercetin favoring E₁ conformations of the enzyme. Oligomycin, a rapidly-reversible inhibitor, decreased the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP and the $\text{K}{}_{0.5}$ for cation activators, and its inhibition was also diminished by dimethyl sulfoxide. Although oligomycin did not inhibit the K⁺-phosphatase activity under standard assay conditions, a reaction presumably catalyzed by E₂ conformations, its effects are nevertheless accommodated by a quantitative model for that reaction depicting oligomycin as favoring E₁ conformations. The model also accounts quantitatively for effects of both dimethyl sulfoxide and oligomycin on $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for substrate, and $\text{K}{}_{0.5}$ for K⁺, as well as for stimulation of phosphatase activity by both these reagents at low K⁺ but high Na⁺ concentrations.     

Stimulation by manganese and other divalent cations of the electron donation reactions of Photosystem II

Using inside-out thylakoid membranes, it has been shown that the oxidation of water and associated reduction of dichlorophenol indophenol is partially inhibited by low concentrations of cation chelators. This inhibition correlates with a removal of two manganese ions per Photosystem II reaction centre. The chelator-induced inhibition was completely reversed by the addition of low levels of Mn²⁺ ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 20 μ}\text{M}$) and higher levels of Mg²⁺ and Ca²⁺ ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 1}\text{mM}$). Other cations were not effective, indicating that the ability to overcome the inhibition did not involve a general electrostatic screening process. The degree of inhibition by chelators was greater at lower light intensities and after treatment with glutaraldehyde. In the presence of glutaraldehyde the stimulatory effect of Mn²⁺ was lost, while pretreatment with Mn²⁺ prevented the glutaraldehyde effect. These results are discussed in terms of conformational changes of the electron donation chains involving cation- (preferentially Mn-) dependent coupling between the oxygen evolving and reaction-centre complexes of Photosystem II.     

Active K+ transport in Mycoplasma mycoides var. Capri. Net and unidirectional K+ movements

Analysis of the cation composition of growing Mycoplasma mycoides var. Capri indicates that these organisms have a high intracellular K⁺ concentration ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{: 200–300}\text{mM}$) which greatly exceeds that of the growth medium, and a low Na⁺ concentration ($\text{Na}{}_{\mathrm{<mtext>i</mtext>}}{}^{\mathrm{+}}\text{: 20}\text{mM}$). Unlike $\text{Na}{}_{\mathrm{<mtext>i</mtext>}}{}^{\mathrm{+}}\text{, K}{}_{\mathrm{<mtext>i</mtext>}}{}^{\mathrm{+}}$ varies with cell aging.The K⁺ transport properties studied in washed organisms resuspended in buffered saline solution show that cells maintain a steady and large K⁺ concentration gradient across their membrane at the expense of metabolic energy mainly derived from glycolysis. In starved cells, $\text{K}{}_{\mathrm{<mtext>i</mtext>}}{}^{\mathrm{+}}$ decreases and is partially compensated by a gain in Na⁺. This substitution completely reverses when metabolic substrate is added (K⁺ reaccumulation process). Kinetic analysis of K⁺ movement in cells with steady K⁺ level shows that most of K⁺ influx is mediated by an autologous K⁺-K⁺ exchange mechanism. On the other hand, during K⁺ reaccumulation by K⁺-depleted cells, a different mechanism (a K⁺ uptake mechanism) with higher transport capacity and affinity drives the net K⁺ influx. Both mechanisms are energy-dependent.Ouabain and anoxia have no effect on K⁺ transport mechanisms; in contrast, both processes are completely blocked by dicyclohexylcarbodiimide, an inhibitor of the Mg²⁺-dependent ATPase activity.     

Kinetic alpha-deuterium isotope effects for enzymatic and nonenzymatic hydrolysis of nicotinamide-beta-riboside.

The kinetic α-secondary deuterium isotope effect, $\text{k}{}_{\mathrm{H}}\text{k}{}_{\mathrm{D}}$, for the pH-independent hydrolysis of nicotinamide riboside, yielding nicotinamide and ribose, in water at 25 ° is 1.14, establishing that this reaction proceeds with unimolecular substrate decomposition to yield a carboxonium ion, or related species, in the rate-determining step. Surprisingly, the corresponding isotope effect for the base-catalyzed decomposition of the same substrate is 1.12, a value indicating considerable sp² character at the Cl′ position in the transition state for this reaction. A similar result, $\text{k}{}_{\mathrm{H}}\text{k}{}_{\mathrm{D}}\text{= 1.15}$, was obtained for base-catalyzed hydrolysis of NAD⁺. The kinetic alpha deuterium isotope effect for the pig brain NAD glycohydrolasecatalyzed hydrolysis of nicotinamide riboside is 1.08. This value suggests that CN bond cleavage to form an intermediate carboxonium ion, or structurally related species, is at least partially rate-determining. In contrast, the corresponding value for the hydrolysis of this substrate catalyzed by Escherichia coli nicotinamide ribonucleotide glycohydrolase is very near unity, a result consistent with several interpretations including a rate-determining enzyme isomerization reaction.     

Mechanism of action of a wound-induced omega-hydroxyfatty acid:NADP oxidoreductase isolated from potato tubers (Solanum tuberosum L).

ω-Hydroxyfatty acid:NADP oxidoreductase, an enzyme involved in suberin biosynthesis, is induced by wounding potato tubers. Initial velocity and product inhibition studies with the purified enzyme suggested an ordered sequential mechanism, where NADPH is added first, followed by 16-oxohexadecanoate, and NADP is released after 16-hydroxyhexadecanoate. Substrate inhibition by NADPH was observed at concentrations higher than 0.2 mm. The inhibitory NADPH molecule competes with 16-oxohexadecanoate, indicating that it forms a dead-end complex with the E-NADPH form of the enzyme. The kinetics for the NADPH inhibition suggested that n > 1 in the rate equation $\text{v}\text{=}\text{V[NADPH]}\text{(K}{}_{\mathrm{m}}\text{+}\text{[NADPH]}\text{+}\text{[NADPH]}{}^{\mathrm{n+1}}\text{K}{}_{\mathrm{i}}\text{)}$; i.e., more than two NADPH molecules bind to enzyme. The K_m for 16-oxohexadecanoate did not change from pH 7.5 to 9.0 but increased about 10-fold from pH 9.0 to 10.0, whereas the K_m for NADPH and hexadecanal did not vary significantly in this pH range. Phenylglyoxal inactivated the enzyme; NADPH and AMP (which competes with NADPH; K_i = 1.1 mM) provided protection against such inactivation. Diethylpyrocarbonate also caused inactivation which was reversed by hydroxylamine; NADPH but not AMP protected the enzyme from this inhibition. Pyridoxal-5′-phosphate reversibly inactivated the enzyme and NaBH₄ reduction of the pyridoxal phosphate-treated enzyme resulted in irreversible inhibition; a combination of NADPH and ω-oxo C₁₆ acid provided protection against such inactivation. As the chain length of alkanals increased from C₃ to C₈, the K_m for the substrate decreased drastically from 7000 to 90μm and a further increase in chain length from C₈ to C₂₀ resulted in only a small decrease in K_m. The K_m and V for 8-oxooctanoate and 10-oxodecanoate are compared with the values obtained for 16-oxohexadecanoate. Based on these results, it is proposed that arginine acts as the binding site for NADPH, a hydrophobic crevice with lysine at the bottom forms the binding site for 16-oxohexadecanoate and histidine participates in the reaction as the proton donor.     

Synthesis of a complementary DNA to rat liver albumin mRNA.

NADPH reduces both liver microsomal cytochrome P-450 and cytochrome $\text{b}{}_5$. In the presence of CO, ferrous cytochrome P-450 can slowly transfer electrons to amaranth, an azo dye. This reaction is followed by the reoxidation of cytochrome $\text{b}{}_5$ which proceeds at essentially the same rate as does cytochrome P-450 oxidation. It is suggested that cytochrome $\text{b}{}_5$ directly reduces cytochrome P-450 in rat liver microsomes.     

The ATP-dependent reductive carboxylation of 2-oxoglutarate using cytosol from rat liver.

An enzyme system (isocitrate synthase) catalysing the conversion of 2-oxoglutarate to isocitrate has been detected in and partially purified from rat liver. The activity of this enzyme system is dependent on the presence of Mg²⁺, ATP, $\text{HCO}{}_3{}^{\mathrm{?}}$ and NADPH.The major proportion of the isocitrate synthase was found in the cytosol and its level of activity was related to the nutritional state of the animal, undergoing changes parallel to those observed in the rate of lipogenesis. During purification, the activity of the isocitrate synthase system increased relative to that of isocitrate dehydrogenase (NADP⁺).     

Localized coupling in oxidative phosphorylation by mitochondria from Jerusalem artichoke (Helianthus tuberosus)

Delocalized chemiosmotic coupling of oxidative phosphorylation requires that a single-value correlation exists between the extent of and the kinetic parameters of respiration and ATP synthesis. This expectation was tested experimentally in nigericin-treated plant mitochondria in single combined experiments, in which simultaneously respiration (in State 3 and in State 4) was measured polarographically, FΔψ (which under these conditions was equivalent to ) was evaluated potentiometrically from the uptake of tetraphenylphosphonium⁺ and the rate of phosphorylation was estimated from the transient depolarization of mitochondria during State 4-State 3-State 4 transitions. The steady-state rates of the different biochemical reactions were progressively inhibited by specific inhibitors active with different modalities on various steps of the energy-transducing process: succinate respiration was inhibited competitively with malonate or noncompetitively with antimycin A, or by limiting the rate of transport into the mitochondria of the respiratory substrate with phenylsuccinate; was dissipated by uncoupling with increasing concentrations of valinomycin; ADP phosphorylation was limited with oligomycin. The results indicate generally that when the rate of respiratory electron flow is decreased, a parallel inhibition of the rate of phosphorylation is also observed, while very limited effects can be detected on the extent of . This behavior is in marked contrast to the effect of uncoupling where the decreased rate of ATP synthesis is clearly due to energy limitation. Extending previous observations in bacterial photosynthesis and in respiration by animal mitochondria and submitochondrial particles the results indicate, therefore, that respiration tightly controls the rate of ATP synthesis, with a mechanism largely independent of . These data cannot be reconciled with a delocalized chemiosmotic coupling model.     

The effect of formate on cytochrome aa3 and on electron transport in the intact respiratory chain

1. Formate inhibits cytochrome $\text{c}$ oxidase activity both in intact mitochondria and submitochondrial particles, and in isolated cytochrome $\text{aa}{}_3$. The inhibition increases with decreasing pH, indicating that HCOOH may be the inhibitory species.2. Formate induces a blue shift in the absorption spectrum of oxidized cytochrome $\text{aa}{}_3\text{(a}{}^{\mathrm{3+}}\text{a}{}_3{}^{\mathrm{3+}}$) and in the half-reduced species ($\text{a}{}^{\mathrm{2+}}\text{a}{}_3{}^{\mathrm{3+}}$). Comparison with cyanide-induced spectral shifts, towards the red, indicates that formate and cyanide have opposite effects on the $\text{aa}{}_3$ spectrum, both in the fully oxidized and the half-reduced states. The formate spectra provide a new method of obtaining the difference spectrum of $\text{a}{}_3{}^{\mathrm{2+}}$ minus $\text{a}{}_3{}^{\mathrm{3+}}$, free of the difficulties with cyanide (which induces marked high → low spin spectral shifts in cytochrome $\text{a}{}_3{}^{\mathrm{3+}}$) and azide (which induces peak shifts of cytochrome $\text{a}{}^{\mathrm{2+}}$ towards the blue in both α- and Soret regions).3. The rate of formate dissociation from cytochrome $\text{a}{}^{\mathrm{2+}}\text{a}{}_3{}^{\mathrm{3+}}\text{-}\text{HCOOH}$ is faster than its rate of dissociation from $\text{a}{}^{\mathrm{3+}}\text{a}{}_3{}^{\mathrm{3+}}\text{-}\text{HCOOH}$, especially in the presence of cytochrome $\text{c}$. The $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ for formate inhibition of respiration is a function of the reduction state of the system, varying from 30 mM (100% reduction) to 1 mM (100% oxidation) at pH 7.4, 30 °C.4. Succinate-cytochrome $\text{c}$ reductase activity is also inhibited by formate, in a reaction competitive with succinate and dependent on [formate]².5. Formate inhibition of ascorbate plus $\text{N,N,N′,N′-}\text{tetramethyl}\text{-p-}\text{phenyl-enediamine}$ oxidation by intact rat liver mitochondria is partially released by uncoupler addition. Formate is permeable through the inner mitochondrial membrane and no differences in ‘on’ or ‘off’ inhibition rates were observed when intact mitochondria were compared with submitochondrial particles.6. NADH-cytochrome $\text{c}$ reductase activity is unaffected by formate in submitochondrial particles, but mitochondrial oxidation of glutamate plus malate is subject both to terminal inhibition at the cytochrome $\text{aa}{}_3$ level and to a slow extra inhibition by formate following uncoupler addition, indicating a third site of formate action in the intact mitochondrion.     

Non-sterol metabolism of mevalonate in vitro: artifacts and realities.

Commercial [5-¹⁴C]mevalonate is shown to contain several radioactive impurities, which give artifactually high amounts of Hyamine bound, volatile acidic radioactivity when incubated with killed or living rat renal cortex slices, as compared with [5-¹⁴C]mevalonate purified either by liquid-liquid partition chromatography or through the enzymically generated $\text{R}\text{-5-phospho-[5-}{}^{14}\text{C]mevalonate}$ by ion-exchange chromatography. The artifactual ${}^{14}\text{CO}{}_2$ results were not diluted by incubation with increasing amounts of unlabelled mevalonate, whereas the ${}^{14}\text{CO}{}_2$ and [${}^{14}\text{C}$]cholesterol produced by rat renal cortex slices incubated with purified [5-¹⁴C]mevalonate were both diluted to the same extent by unlabelled mevalonate. It is concluded that $\text{R}\text{[5-}{}^{14}\text{C]mevalonate}$ is genuinely oxidized to ${}^{14}\text{CO}{}_2\text{in}\text{vitro}$, and that purification of substrate before its use is necessary. Production of ${}^{14}\text{CO}{}_2$ and various [${}^{14}\text{C}$]lipids from purified [5-¹⁴C]mevalonate, as a function of time and substrate concentration, by renal cortex and liver slices, is described.     

Complete stoichiometry of free NADPH oxidation in liver microsomes

The stoichiometry of free NADPH oxidation in phenobarbital induced rabbit liver microsomes was measured by means of registering the rates of NADPH, H⁺ and O₂ consumption and $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ and H₂O₂ production. $\text{ΔO}{}_2{}^{\mathrm{<mtext>?</mtext>}}\text{:ΔH}{}_2\text{O}{}_2$ ratio is approximately I indicating that about half H₂O₂ results from $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ dismutation, the second half being formed directly. ΔNADPH:ΔH₂O₂ and ΔO₂:ΔH₂O₂ ratios exceed I and therefore another product of the reaction is water. The fact that the ratio (ΔNADPH-ΔH₂O₂):(ΔO₂-ΔH₂O₂) is 2 allows one to consider direct 4-electron O₂ reduction as the major way of water formation rather than endogenous substrate hydroxylation.     

Immunochemical study on the participation of cytochrome b5 in drug oxidation reactions of mouse liver microsomes.

Highly purified divalent and monovalent antibodies against cytochrome $\text{b}{}_5$, anti-$\text{b}{}_5$ immunoglobulin G (IG) and anti-$\text{b}{}_5$ Fab', were used in elucidating the role of this cytochrome in the drug-oxidizing enzyme system of mouse liver microsomes. Anti-$\text{b}{}_5$ IG strongly inhibited not only NADH-supported but also NADPH-supported oxidation of 7-ethoxycoumarin and benzo(a)pyrene, but had no inhibitory action on the oxidation of aniline. Anti-$\text{b}{}_5$ Fab' also inhibited NADH-supported and NADPH-supported benzo(a)pyrene hydroxylation. These observations indicate an essential role of cytochrome $\text{b}{}_5$ in the transfer of electrons not only from NADH but also from NADPH to cytochrome P-450 in the microsomal oxidation of some drugs, but not of aniline.     

The structure of the fluorophosphate-pyruvate kinase complex investigated by 31P relaxation rate studies

The interaction of fluorophosphate with muscle pyruvate kinase was investigated by ³¹P nuclear relaxation rate measurements. The fluorophosphate samples were highly purified and were first monitored by ¹⁹F and ³¹P relaxation rate measurements in the formation of the binary FPO₃-Mn complex. The results of the binary complex demonstrated that FPO₃^2? binds in the first coordination sphere of Mn²⁺ via the oxygen atoms but not via the fluorine. The enzyme experiments were designed under conditions where a significant fraction of the ligand is in the ternary enzyme-Mn-FPO₃ complex. These studies demonstrate that the ³¹P relaxation rate of bound FPO₃ ($\text{1}\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>m</mtext>}}\text{= 1.58 ± 0.05 × 10}{}^5\text{s}{}^{\mathrm{?1}}$) is consistent with the binding of this ligand in the first coordination sphere of enzyme-bound Mn²⁺ with an elongated Mn-O-P distance ($\text{r}{}_{\mathrm{<mtext>Mn-P</mtext>}}\text{= 3.3 ± 0.2}\text{A}\text{?}$). Such a structure is demonstrated in the ternary enzyme-Mn-FPO₃ complex, in the complex containing HCO₃^?, and in the complex also containing HCO₃^? and ADP. The data further substantiate the binding of phosphoenolpyruvate analogs in the first coordination sphere of pyruvate kinase-bound Mn²⁺.     

Studies of gastric Ca2+-stimulated adenosine triphosphatase I. characterization and general properties

Gastric microsomes do not contain any significant Ca²⁺-stimulated ATPase activity. Trypsinization of pig gastric microsomes in presence of ATP results in a significant (2–3-fold) increase in the basal (with Mg²⁺ as the only cation) ATPase activity, with virtual elimination of the K⁺-stimulated component. Such treatment causes unmaksing of a latent Mg²⁺-dependent Ca²⁺-stimulated ATPase. Other divalent cations such as Sr²⁺, Ba²⁺, Zn²⁺ and Mn²⁺ were found ineffective as a substitute for Ca²⁺. Moreover, those divalent cations acted as inhibitors of the Ca²⁺-stimulated ATPase activity. The pH optimum of the enzyme is around 6.8. The enzyme has a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 70 μM for ATP and the $\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ values for Mg²⁺ and Ca²⁺ are about $\text{4 · 10}{}^{\mathrm{?4}}\text{M}$ and 10^?7 M, respectively. Studies with inhibitors suggest the involvement of sulfhydryl and primary amino groups in the operation of the enzyme. Possible roles of the enzyme in gastric H⁺ transport have been discussed.