Isolation of a sn-glycerol 3-phosphate: NAD oxidoreductase from spinach leaves.

An NAD-dependent glycerol 3-phosphate dehydrogenase (sn-glycerol 3-phosphate: NAD oxidoreductase; EC 1.1.1.8) has been purified from spinach leaves by a three-step procedure involving ion-exchange, gel filtration, and affinity chromatography. The enzyme has been purified over 10,000-fold to a specific activity of 38. It has a molecular weight of approximately 63,500. The pH optimum for the reduction of dihydroxyacetone phosphate is 6.8 and for glycerol 3-phosphate oxidation it is 9.5. During dihydroxyacetone phosphate reduction hyperbolic kinetics were observed when either NADH or dihydroxyacetone phosphate was the variable substrate, but concentrations of NADH greater than 150 μm were inhibitory. Michaelis constants were 0.30–0.35 mm for dihydroxyacetone phosphate and 0.01 mm for NADH. Glycerol 3-phosphate oxidation obeyed Michaelis-Menten kinetics with a K_m of 0.19 mm for NAD and 1.6 mm for glycerol 3-phosphate. The enzyme was specific for those substrates, and dihydroxyacetone, glyceraldehyde, glyceraldehyde 3-phosphate, NADPH, NADP, and glycerol were not utilized. The spinach leaf enzyme appears to be in the cytoplasm and probably functions for the production of glycerol 3-phosphate from dihydroxyacetone phosphate.     

Pyruvate dehydrogenase complex from higher plant mitochondria and proplastids: kinetics

A steady-state kinetic analysis has been performed on the pyruvate dehydrogenase complex from pea (Pisum sativum L.) mitochondria and castor bean (Ricinus communis L.) proplastids. Substrate interaction kinetics for all substrates gave parallel lines consistent with a multisite ping-pong mechanism. Product inhibition studies showed uncompetitive inhibition between acetyl-CoA and pyruvate and competitive inhibition between NADH and NAD⁺, both of which are also consistent with this mechanism. In the mitochondrial complex, acetyl-CoA showed noncompetitive inhibition versus CoA which suggests that the intermediate complex is kinetically important in the lipoamide transacetylase component of this complex. In contrast, the proplastid complex showed competitive inhibition in this interaction. NADH is a noncompetitive inhibitor versus CoA in both complexes indicating that these complexes, like the mammalian complex, may have protein-protein interactions between the second and third enzymes of the complex. Since NADH also shows noncompetitive inhibition versus pyruvate, this interaction may extend to all components of the complex. Acetyl-CoA shows noncompetitive inhibition versus NAD⁺ which may also be a result of interaction between the second and third enzymes of the complex. The limiting Michaelis constants for substrates and the inhibitor constants for both complexes were determined.     

Paradoxical activation of Leptomonas NAD-linked alpha-glycerophosphate dehydrogenase by ethidium and antrycide.

Antrycide and ethidium bromide — 2 cationic trypanocides — inhibited NAD-linked α-glycerophosphate dehydrogenase from $\text{Leptomonas}$ sp. The kinetics of enzyme inhibition was determined by Lineweaver-Burk, Dixon, or direct linear plots. Inhibition by Antrycide was noncompetitive for dihydroxyacetone phosphate in the presence of saturating Mg²⁺ or spermidine. With dihydroxyacetone phosphate at saturation, Antrycide inhibition was also noncompetitive with respect to Mg²⁺ (K_i = 115 μM) and spermidine (K_i = 85 μM). Inhibition by ethidium in the presence of saturating dihydroxyacetone phosphate, was noncompetitive for Mg²⁺ (K_i = 400 μM) but mixed for spermidine (K_i = 495 μM); inhibition was noncompetitive for dihydroxyacetone phosphate in the presence of saturating Mg²⁺ or spermidine. Rabbit-muscle α-glycerophosphate dehydrogenase was inhibited at all concentrations of Antrycide and ethidium tested, but the $\text{Leptomonas}$ enzyme was stimulated up to 3.5-fold by low concentrations of inhibitors in the absence of polyamine. New chemotherapeutic possibilities may thus be opened and an evolutionary distinction between trypanosomatid and mammalian enzyme.     

2-Mercaptoethanol as a substrate for liver alcohol dehydrogenase.

An activity was identified in a phosphate buffer extract of calf liver acetone powder which utilized 2-mercaptoethanol and NAD⁺ as substrates and formed NADH as one product. The activity responsible for catalyzing this reaction is associated with calf liver alcohol dehydrogenase based on copurification, similarity in pH optima, and similarity in response to chelating agents and other inactivating agents. Crystalline horse liver alcohol dehydrogenase also catalyzes the formation of NADH from NAD⁺ using 2-mercaptoethanol as the substrate. Although the K_m for mercaptoethanol is much lower than that for ethanol, 30 μm as compared to 0.625 mm, the maximum velocity with mercaptoethanol as the substrate is only 7% of that when ethanol is the substrate. Because of this difference in maximum velocity, 2-mercaptoethanol is an apparent competitive inhibitor with respect to ethanol with crystalline horse liver alcohol dehydrogenase, consistent with ethanol and 2-mercaptoethanol binding at the same site. The apparent K_i for 2-mercaptoethanol is 14 μm. 2-Butanethiol is a competitive inhibitor with respect to both 2-mercaptoethanol and ethanol with horse and beef liver alcohol dehydrogenases.     

Osmoregulation inDunaliella. Catalysis of the glycerol-3-phosphate dehydrogenase reaction in a chloroplast-enriched fraction ofDunaliella tertiolecta

The glycerol-3-phosphate dehydrogenase (NAD-dependent) reaction was studied in a chloroplast-enriched fraction fromDunaliella tertiolecta. The reaction has widely separated pH optima for each direction. Reduction of dihydroxyacetone phosphate proceeded with Michaelis-Menten kinetics but sigmoidal double reciprocal plots were obtained with glycerol phosphate as variable substrate. NADP served as an alternative substrate but it was somewhat less effective than NAD. The reaction was inhibited by inorganic orthophosphate and by adenine nucleotides in a manner indicative of anion inhibition. Inhibition by inorganic phosphate was competitive with DHAP and possibly also with NADH. The enzyme was activated by Na⁺ at concentrations below 200 m and inhibited at higher concentrations, the region of maximum activation being affected by substrate concentration. Inhibition by Na⁺, present as a counterion of the substrate, was evidently responsible for apparent substrate inhibition by glycerol phosphate. Several important differences were apparent between the reaction in the unfractionated chloroplast-enriched fraction and the properties of a partly purified enzyme described by Haus and Wegmann (1984a, b).In toto, the results suggest that the regulatory potential of the reaction is probably more relevant to homeostatic control of glycerol content under steady state conditions than to controlling response to water stress.Abbreviations DHAP Dihydroxyacetone phosphate - CHES 2-(N-cyclohexylamino)ethanesulphonic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid     

Reaction mechanism of aspartate transcarbamylase from mouse spleen

The reaction mechanism of aspartate transcarbamylase from mouse spleen has been determined, using steady-state kinetics, isotope-exchange experiments, inhibition studies with a transition-state analog, and product-inhibition studies. Intersecting reciprocal plots obtained when one substrate was varied against different concentrations of the second substrate indicate that the mechanism is sequential. The transition-state analog, N-(phosphonacetyl)-l-aspartate, was a powerful inhibitor of aspartate transcarbamylase, with an inhibition constant (K_i) of 2.6 × 10^?8m at 37 °C and pH 7.4 in 0.05 m Na HEPES buffer. PALA gave competitive inhibition with carbamyl phosphate and noncompetitive inhibition with l-aspartate, indicating that carbamyl phosphate must bind before aspartate for catalysis to occur. A ping-pong mechanism in which carbamyl phosphate binds first was excluded by isotope-exchange experiments, since [³²P]inorganic phosphate was not incorporated into carbamyl phosphate in the absence of aspartate. Product-inhibition studies showed that only inorganic phosphate and carbamyl phosphate gave a competitive pattern; all other combinations of substrate and product gave noncompetitive inhibition patterns when incubations were carried out at subsaturating concentrations of the second substrate. These inhibition patterns showed that carbamyl phosphate binds first, aspartate binds second, carbamyl aspartate dissociates first, and phosphate dissociates second.     

Cooperative interaction of reduced pyridine nucleotides with estrogen synthetase of human placental microsomes

The estrogen synthetase present in human placental microsomes appears to be dependent on the cooperative interaction of the reduced cofactors NADPH and NADH for optimal activity. Using steady-state concentrations of either cofactor, it was found that while the estrogen synthetase activity followed hyperbolic saturation kinetics with NADPH (K_mapp = 14 μM), the enzyme followed sigmoidal saturation kinetics when the cofactor was NADH, with the half-maximum velocity attained at a cofactor concentration of 1.1 mm. The maximum velocity obtained with NADPH as the cofactor was greater than with corresponding concentrations of NADH. Estrogen synthetase activity in the presence of NADH was not due to NADPH contamination. NADH, in the presence of small concentrations of NADPH (0.5 to 5 μm), stimulated significantly the rate of estrogen formation from androstenedione by placental microsomes and, in addition, the enzyme saturation kinetics changed from sigmoidal to hyperbolic, thus mimicking the effect of NADPH. Estrogen synthetase activity, measured in the presence of 1 mm NADH, was stimulated in a dose-dependent manner by NADPH (K_mapp = 0.4 μM NADPH) and, when the enzyme was measured in the presence of 5 μm NADPH, the activity was stimulated in a dose-dependent manner by NADH (K_mapp = 45 μM NADH). Estrogen synthetase activity measured in the presence of NADH, without and with NADPH (1 μm) remained linear both with time of incubation for approximately 15 min and with microsomal protein concentration up to 3 mg/ml. The apparent K_m of estrogen synthetase for androstenedione, when measured in the presence of NADH, was 1 μm. The synergistic interaction between NADH and NADPH in stimulating placental estrogen synthetase activity observed in vitro may, conceivably, take place in vivo in the intact placenta.     

L'alcool deshydrogénase de tomate (Lycopersicon esculentum): Étude cinétique et mécanisme d'action

In order to investigate the enzymatic mechanism of tomato alcohol dehydrogenase, kinetic studies were carried out at pH 5.8 and 9.4 for the forward and reverse reactions, respectively. Primary double reciprocal plots for several fixed concentrations of the associated substrate in all cases intersect, suggesting a sequential mechanism. Exploitation of secondary plots (slope-intercept values on the primary plots versus the reciprocals of the non-varied substrates) gives the following values: K_ms 500 μM for MeCHO, 30 μM for NADH, 2700 μM for EtOH, 12 μM for NAD⁺; K_is 40 μM for MeCHO, 3 μM for NADH, 10⁴ μM for EtOH and 45 μM for NAD⁺. The results obtained in product inhibition studies agree with an ordered bi-bi mechanism for both forward and reverse reactions. Application of Cleland's rules shows that the coenzyme was the first substrate to complex with the enzyme in both cases.     

The nature of anion inhibition of human red cell carbonic anhydrases.

We studied anionic inhibition of the reaction CO₂ + OH^?? HCO₃^? catalyzed by human red cell carbonic anhydrase B (I) and C (II), using iodide and cyanate. In the forward reaction with respect to CO₂ as the substrate, inhibition was mixed but favoring noncompetitive; the back reaction, with HCO₃^? as the substrate, yielded strict competitive kinetics. Mean inhibition constants, K_I, in the pH range 7.2–7.5 are: iodide, 0.5 mm for enzyme B and 16 mm for C; cyanate, 0.8 μm for B and 20 μm for C. When OH^? was considered as the substrate for the forward reaction, cyanate and chloride behaved as competitive inhibitors. The true inhibition constant (K_I⁰) for cyanate (calculated for infinitely low OH^?) is 0.4 μm for enzyme B and 4 μm for C. Apart from the difference in anion affinity and some 10-fold higher activity of C > B, the isozymes showed similar patterns of inhibition. Data agree with generally proposed mechanisms describing the active site as ZnH₂O with pK_a of about 7.     

NADPH-D-glyceraldehyde 3-phosphate oxidoreductase activity in muscle and other tissues of the rat

Rat muscle was found to contain a NADPH-D-glyceraldehyde 3-phosphate oxidoreductase. Of the ten tissues studied, muscle contained the highest activity at 0.90 ± 0.06 units/g. The activity was not due to L-glycerol 3-phosphate-NAD⁺ oxidoreductase utilising NADPH as a hydrogen donor, nor to coupling of the latter with a NADPH-NAD⁺ oxidoreductase. After partial inhibition of contaminating triose phosphate isomerase with glycidol phosphate, the oxidoreductase rate was faster with glyceraldehyde phosphate than with dihydroxyacetone phosphate as a substrate. Apparent K_m values of 14 μM for D-glyceraldehyde 3-phosphate and 0.7 μM for NADPH were determined.     

Glyceraldehyde-3-phosphate dehydrogenase (NADP) from Sinapis alba: Steady state kinetics

Substrate interaction and product inhibition kinetics of the forward reaction of glyceraldehyde-3-phosphate dehydrogenase (NADP) (EC 1.2.1.13) from Sinapis alba suggest an Uni Uni Uni Bi Ping Pong mechanism (NAD(P)H on, glyceraldehyde-3-phosphate off, 1,3-diphosphoglycerate on, phosphate off, NAD(P)⁺ off) with an apparent Theorell Chance displacement between 1,3-diphosphoglycerate and phosphate. The proposed mechanism predicts the existence of stable enzyme-NAD(P)⁺ and acyl-enzyme complexes as obligatory intermediates. A comparison of the present findings on the NADP-enzyme with an earlier kinetic analysis of the NAD-specific enzyme from plants (EC 1.2.1.12) by other authors shows that the kinetic mechanisms for the two enzymes, although similar in principle (both show Ping Pong kinetics), differ in some details.     

Activation of fructose 1,6-bisphosphatase in darkened intact chloroplasts by NADPH

When intact chloroplasts are incubated in the dark with dihydroxyacetone phosphate, an increase in fructose 1,6-bisphosphatase activity occurs which resembles the reductive activation observed in illuminated chloroplasts. Under optimum conditions, the activity increases to about 150 μmol · h^?1 · mg^?1 chlorophyll within 60 min. The dark activation of the enzyme is reversed by electron acceptors such as oxaloacetate, nitrite, and 3-phosphoglycerate plus ATP. Activation is most marked under strictly anaerobic conditions, being strongly inhibited by O₂. It is concluded that NADPH, generated from dihydroxyacetone phosphate in situ in the reaction catalyzed by NADP⁺-dependent glyceraldehyde phosphate dehydrogenase, can provide electrons for the reductive activation of fructose 1,6-bisphosphatase in the dark.     

Action of diethylstilbestrol on the NADH-dehydrogenase region of the respiratory chain

Diethylstilbestrol reversibly inhibits electron transfer in particle-bound mitochondrial NADH-dehydrogenase (Keilin-Hartree preparation) at a site located in the vicinity of, or superimposed on, the piericidin A- and rotenone-specific sites. At this site, diethylstilbestrol half-maximal inhibitory concentration varies from 0.2 μm (at 37 μm NADH) to 2.6 μm (at 180 μm or higher NADH concentrations). The postulated localization of the diethylstilbestrol site is supported by (a) the similar effects of the diethylstilbestrol, piericidin A, and rotenone on the kinetics of the NADH-induced changes of the 470–500-nm chromophore; (b) the lack of action of diethylstilbestrol on the NADH-ferricyanide reductase and the NADH-acetylpyridine-adenine dinucleotide transhydrogenase reactions; (c) the lack of action of diethylstilbestrol on the NADH-2,3-dimethoxy-2′-methyl-1,4-benzoquinone reaction at infinite concentration of UQ₀; (d) the similar inhibitions of the NADH-UQ₂ reaction and NADH-cytochrome b_k reaction; (e) the noncompetitive inhibition of the NADH-UQ₂ reaction; (f) the similar effects of diethylstilbestrol and rotenone on the kinetics of the NADH-UQ₀ reaction; and (g) the inhibition of the NADH-cytochrome b_k reaction, in contrast with the insensitivity of the succinate-cytochrome b_k reaction. The interaction of diethylstilbestrol with the NADH-dehydrogenase protein is supported by the summation of diethylstilbestrol, piericidin A, rotenone, and p-chloromercuribenzoate inhibitions. Diethylstilbestrol inhibition involves specific structural requirements as shown by the lesser (if any) inhibitory potency of hexestrol, diethylstilbestrol monomethyl ether, diethylstilbestrol dimethyl ether, and hexestrol dimethyl ether.     

The respiratory system of the marine bacterium Beneckea natriegens. II. Terminal branching of respiration to oxygen and resistance to inhibition by cyanide

1. Cell-free extracts of the marine bacterium Beneckea natriegens, derived by sonication, were separated into particulate and supernatant fractions by centrifugation at 150 000 × g.2. NADH, succinate, d(?)- and l(+)-lactate oxidase and dehydrogenase activities were located in the particles, with 2- to 3-fold increases in specific activity over the cell free extract. The d(?)- and l(+)-lactate dehydrogenases were NAD⁺ and NADP⁺ independent. Ascorbate-N,N,N′,N′-tetramethylphenylenediamine (TMPD) oxidase was also present in the particulate fraction; it was 7–12 times more active than the physiological substrate oxidases.3. Ascorbate-TMPD oxidase was completely inhibited by 10 μM cyanide. Succinate, NADH, d(?)-lactate and l(+)-lactate oxidases were inhibited in a biphasic manner, with 10 μM cyanide causing only 10–50 % inhibition; further inhibition required more than 0.5 mM cyanide, and 10 mM cyanide caused over 90 % inhibition. Low sulphide (5 μM) and azide (2 mM) concentrations also totally inhibited ascorbate-TMPD oxidase, but only partially inhibited the other oxidases. High concentrations of sulphide but not azide caused a second phase inhibition of NADH, succinate, d(?)-lactate and l(+)-lactate oxidases.4. Low oxidase activities of the physiological substrates, obtained by using non-saturating substrate concentrations, were more inhibited by 10 μM cyanide and 2 mM azide than high oxidase rates, yet ascorbate-TMPD oxidase was completely inhibited by 10 μM cyanide over a wide range of rates of oxidation.5. These results indicate terminal branching of the respiratory system. Ascorbate-TMPD is oxidised by one pathway only, whilst NADH, succinate, d(?)-lactate and l(+)-lactate are oxidised via both pathways. Respiration of the latter substrates occurs preferentially by the pathway associated with ascorbate-TMPD oxidase and which is sensitive to low concentrations of cyanide, azide and sulphide.6. The apparent K_m for O₂ for each of the two pathways was detected using ascorbate-TMPD and NADH or succinate plus 10 μM cyanide respectively. The former pathway had an apparent K_m of 8–17 (average 10.6) μM and the latter 2.2–4.0 (average 3.0) μM O₂.     

Yeast glutathione reductase. Steady-state kinetic studies of its transhydrogenase activity.

Yeast glutathione reductase catalyzes a pyridine nucleotide transhydrogenase reaction using either NADPH or NADH as the electron donor and thionicotinamideadenine dinucleotide phosphate as the electron acceptor. Competitive substrate inhibition of the transhydrogenase reaction by NADPH (K_i = 11 μM) is observed when NADPH is the electron donor. Competitive substrate inhibition by thionicotinamide-adenine dinucleotide phosphate (K_i = 58 μM) is observed with NADH as the electron donor. The turnover numbers of the two transhydrogenase reactions are similar and are equal to about 1% of the turnover number for the NADPH-dependent reduction of oxidized glutathione catalyzed by the enzyme. The transhydrogenase kinetics are analyzed in terms of a pingpong mechanism. It is concluded that the substrate inhibition results from formation of abortive complexes of NADPH with the reduced form of the enzyme and of thionicotinamide-adenine dinucleotide phosphate with the oxidized form of the enzyme. With NADPH as the electron donor, the apparent Michaelis constant for thionicotinamide-adenine dinucleotide phosphate is sensitive to the ionic composition of the assay medium. The data are interpreted to support the existence of a general pyridine nucleotide-binding site at the active site of the enzyme and separate from the binding site for oxidized glutathione.     

Acute hormonal control of pyruvate kinase and lactate formation in the isolated rat hepatocyte.

The activity of pyruvate kinase from the isolated rat hepatocyte was studied under conditions which allow investigation into the hormonal regulation of the enzyme. Incubating hepatocytes from fed or fasted rats with 1 μm glucagon gives approximately 60% inhibition of the enzyme activity determined at 1.6 mm P-enolpyruvate. A good correlation between the regulation of pyruvate kinase and lactate formation from 10 mm dihydroxyacetone is observed in hepatocytes from fasted rats. When hepatocytes are incubated in a Krebs-Ringer phosphate buffer, the inhibition of the pyruvate kinase activity by 1 μm glucagon is not accompanied by a marked inhibition of lactate production from fructose. Half-maximal regulation is observed at 0.26 ± 0.02 nm glucagon and 0.37 ± 0.05 nm glucagon for the enzyme and lactate formation from dihydroxyacetone respectively. Incubating hepatocytes with 10 mm l-alanine enhances inhibition of pyruvate kinase by physiological concentrations of glucagon, lowering the half-maximally effective concentration of glucagon from 0.3 nm to approximately 0.1 nm. A small but consistent inhibition of pyruvate kinase by 10 μm epinephrine is also observed and this inhibition is enhanced by 0.5 mm theophylline and by 10 mm l-alanine. The inhibition of pyruvate kinase by epinephrine both in the absence and presence of theophylline is blocked by the α-adrenergic antagonist phenoxybenzamine. The β-adrenergic blocker propranolol has no influence on the inhibition of the enzyme by epinephrine. Adenosine 3′:5′-monophosphate, N⁶O²-dibutyryl adenosine 3′:5′-monophosphate, and guanosine 3′:5′-monophosphate also inhibit glycolysis from dihydroxyacetone and modulate pyruvate kinase activity in hepatocytes from fasted rats. Oleate, ethanol, and 3-hydroxybutyrate inhibit dihydroxyacetone glycolysis, but they do not influence the activity of pyruvate kinase. The divalent metal ionophore A23187 slightly stimulates lactate synthesis from dihydroxyacetone, but it has no influence on pyruvate kinase activity.     

Steady-State Kinetics of NADH:coenzyme Q Oxidoreductase Isolated from Bovine Heart Mitochondria

Steady-state kinetics of the bovine heart NADH:coenzyme Q oxidoreductase reaction were analyzed in the presence of various concentrations of NADH and coenzyme Q with one isoprenoid unit (Q₁). Product inhibitions by NAD⁺ and reduced coenzyme Q₁ were also determined. These results show an ordered sequential mechanism in which the order of substrate binding and product release is Q₁–NADH–NAD⁺–Q₁H₂. It has been widely accepted that the NADH binding site is likely to be on the top of a large extramembrane portion protruding to the matrix space while the Q₁ binding site is near the transmembrane moiety. The rigorous controls for substrate binding and product release are indicative of a strong, long range interaction between NADH and Q₁ binding sites.     

The conversion of dihydroxyacetone phosphate to methylglyoxal and inorganic phosphate by methylglyoxal synthase.

[¹⁴C]Dihydroxyacetone phosphate labeled in either the C-1 or C-3 position was enzymatically synthesized, isolated, and utilized as a substrate for crystalline methylglyoxal synthase purified from Proteus vulgaris. After reaction with the enzyme, the methyl carbon of methylglyoxal³ was identified as CHI₃ by the iodoform reaction. The labeling pattern revealed that C-1 is dephosphorylated and reduced to the methyl group, while C-3 is oxidized to the aldehyde. Methylglyoxal was found to be noncompetitive with respect to dihydroxyacetone phosphate, while inorganic phosphate was competitive and transformed the dihydroxyacetone phosphate saturation kinetics from hyperbolic to sigmoidal. The enzyme was inactivated by freezing, and phosphate stabilized the enzyme toward both cold- and heat-induced denaturation. The phosphate moiety of the substrate appears to be required for binding, since the synthase is competitively inhibited by a variety of phosphorylated compounds but not by their nonphosphorylated counterparts. Based on these observations, and the ability of bromo- and iodoacetol phosphates to act as active-site reagents, a mechanism is proposed in which the enzyme first catalyzes the keto-enol tautomerization to the hydrogen-bonded enol which facilitates the internal oxidation-reduction and phosphoester cleavage with CO bond breakage.     

Rates and properties of endogenous cyclic photophosphorylation of isolated intact chloroplasts measured by CO2 fixation in the presence of dihydroxyacetone phosphate

1. Dihydroxyacetone phosphate in concentrations ? 2.5 mM completely inhibits CO₂-dependent O₂ evolution in isolated intact spinach chloroplasts. This inhibition is reversed by the addition of equimolar concentrations of P_i, but not by addition of 3-phosphoglycerate. In the absence of P_i, 3-phosphoglycerate and dihydroxyacetone phosphate, only about 20% of the ¹⁴C-labelled intermediates are found in the supernatant, whereas in the presence of each of these substances the percentage of labelled intermediates in the supernatant is increased up to 70–95%. Based on these results the mechanism of the inhibition of O₂ evolution by dihydroxyacetone phosphate is discussed with respect to the function of the known phosphate translocator in the envelope of intact chloroplasts.2. Although O₂ evolution is completely suppressed by dihydroxyacetone phosphate, CO₂ fixation takes place in air with rates of up to 65μ mol · mg^?1 chlorophyll · h^?1. As non-cyclic electron transport apparently does not occur under these conditions, these rates must be due to endogenous pseudocyclic and/or cyclic photophosphorylation.3. Under anaerobic conditions, the rates of CO₂ fixation in presence of dihydroxyacetone phosphate are low (2.5–7 μmol · mg^?1 chlorophyll · h^?1), but they are strongly stimulated by addition of dichlorophenyl-dimethylurea (e.g. 2 · 10^?7 M) reaching values of up to 60 μmol · mg^?1 chlorophyll · h^?1. As under these conditions the ATP necessary for CO₂ fixation can be formed by an endogenous cyclic photophosphorylation, the capacity of this process seems to be relatively high, so it might contribute significantly to the energy supply of the chloroplast. As dichlorophenyl-dimethylurea stimulates CO₂ fixation in presence of dihydroxyacetone phosphate under anaerobic but not under aerobic conditions, it is concluded that only under anaerobic conditions an “overreduction” of the cyclic electron transport system takes place, which is removed by dichlorophenyl-dimethylurea in suitable concentrations. At concentrations above 5 · 10^?7 M dichlorophenyl-dimethylurea inhibits dihydroxyacetone phosphate-dependent CO₂ fixation under anaerobic as well as under aerobic conditions in a similar way as normal CO₂ fixation. Therefore, we assume that a properly poised redox state of the electron transport chain is necessary for an optimal occurrence of endogenous cyclic photophosphorylation.4. The inhibition of dichlorophenyl-dimethylurea-stimulated CO₂ fixation in presence of dihydroxyacetone phosphate by dibromothymoquinone under anaerobic conditions indicates that plastoquinone is an indispensible component of the endogenous cyclic electron pathway.