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An assay for determining the rate of methemoglobin reduction in hemolysates of human erythrocytes has been developed. The rates obtained by this assay, when corrected for dilution, are comparable to those obtained with intact cells. Increased ionic strength inhibits the reaction, whereas EDTA increases the rate of reduction. The rate with NADPH as electron donor is 65–70% of the rate with NADH. Added cytochrome b₅ stimulates the reaction. The assay has been used to examine erythrocytes from two methemoglobinemic sisters and their asymptomatic mother. Hemolysates of the two patients have both decreased dichlorophenolindophenol reductase activity and decreased ability to reduce methemoglobin. Hemolysates from the heterozygous mother have intermediate dichlorophenolindophenol reductase activity and intermediate methemoglobin reduction ability. The data presented in this paper indicate that the concentrations of cytochrome b₅ and cytochrome b₅ reductase determine the rate of methemoglobin reduction in hemolysates.     

Evidence that the intermediate electron acceptor,A2, in Photosystem I is a bound iron-sulfur protein

Absorption changes accompanying the formation of light-induced P-700⁺ were investigated in a highly enriched Photosystem I preparation where an intermediate electron acceptor preceding P-430 could be detected. In an enriched Photosystem I particle, light-induced reversible absorption changes observed at 700 nm in the presence of dithionite resembled those previously seen at 703 nm and 820 nm [9], thus indicating the presence of a backreaction between P-700⁺ and A^?₂. After this same Photosystem I particle was treated to denature the bound iron-sulfur centers, the photochemical changes that could be attributed to P-700 A₂ were completely lost. These results provide evidence that the intermediate electron acceptor, A₂, is a bound iron-sulfur protein. Additional studies in the 400–500 nm region with Photosystem I particles prepared by sonication indicate that the spectrum of A₂ is different from that of P-430.     

Acylresorcinols from Virola sebifera and Virola elongata

The fruits of Virola sebifera and V. elongata (Myristicaceae) contain three ω-phenylundecanoyl-substituted compounds, a 2,6-dihydroxybenzene, a 2,6-dihydroxy-4-methoxybenzene and a 3-hydroxycyclohexan-2,6-dione. Three additional 2,6-dihydroxybenzenes are substituted by hexadecanoyl, hexadec-4Z-enoyl and 8-hydroxyoctadec-4Z-enoyl groups.     

Structure–activity relationships of the aromatic site in novel anticonvulsant pyrrolo[1,2-a]pyrazine derivatives

Novel, chiral derivatives of pyrrolo[1,2-a]pyrazine with aromatic substituents at carbon C-4 were synthesized by a short synthetic sequence involving Ugi multicomponent reaction. The compounds were evaluated for their in vivo efficacy in animal models of epilepsy within the Anticonvulsant Screening Program (ASP). High activity in ‘classical’ maximal electroshock seizure (MES) and subcutaneous Metrazol (scMET) tests was characteristic for meta-substituted analogs. On the other hand, efficacy of compounds in the 6 Hz model of pharmacoresistant limbic seizures was only marginally affected by the orientation of substituents in the phenyl moiety. The most active derivative, 5a, displayed an ED₅₀ value of 32.24 mg/kg and a protective index of 6.6 (PI) in the 6 Hz test. It was also active in a pilocarpine-induced status prevention model of pharmacoresistant status epilepticus.     

6-Phosphofructo-2-kinase and D-fructose-2,6-bisphosphatase activities in mung bean seedlings

6-Phosphofructo-2-kinase (ATP: D-fructose-6-phosphate-2-phosphotransferase) and D-fructose-2,6-bisphosphatase activities have been found in extracts prepared from etiolated mung bean seedlings. The activity of 6-phosphofructo-2-kinase exhibits a sigmoidal shape in response to changes in concentrations of both substrates, D-fructose 6-phosphate and ATP (S_0.5 values of 1.8 and 1.2 mM, respectively). Inorganic orthophosphate (P_i) has a strong stimulating effect on the 2-kinase activity (A_0.5 at about 2 mM), moderately increasing the V_max and modifying the response into hyperbolic curves with K_m values of 0.4 and 0.2 mM for fructose 6-phosphate and ATP, respectively. 3-Phosphoglycerate (I_0.5 about 0.15 mM) partially inhibited the kinase activity by counteracting the P_i activation. In contrast, the activity of D-fructose-2,6-bisphosphatase (K_m 0.38 mM) is strongly inhibited by P_i (I_0.5 0.8 mM) lowering its affinity to fructose-2,6-P₂ (K_m 1.4 mM). 3-Phosphoglycerate activites the enzyme (A_0.5 at about 0.3 mM) without causing a significant change in its K_m for fructose-2,6-P₂. The activities of both of these enzymes in relationship to the metabolic role of D-fructose 2,6-bisphosphate in the germinating seed is discussed.     

Switches in 6-phosphofructo-2-kinase isoenzyme expression during rat sperm maturation

Here we analyzed Pfkfb3 and Pfkfb4 gene expression in rat testis development, isolated testicular cells and spermatozoa. Real time RT-PCR analysis during testis development showed the maximum expression of Pfkfb3 in pre-puber samples and of Pfkfb4 in adult samples. Western blot analysis showed that uPFK-2 protein, a product of Pfkfb3 gene, was present in all the cell types forming the seminiferous epithelium (Sertoli, interstitial and spermatogenic cells). In contrast, tPFK-2, a product of Pfkfb4 gene, was restricted to spermatogenic cells. Confocal analyses by indirect immunofluorescence also corroborated this expression pattern. Immunoblotting studies of isolated spermatozoa demonstrated the presence of uPFK-2 only in immature sperm and once spermatozoa became fully functional this isozyme was replaced by the testicular isozyme tPFK-2. Moreover, immunostaining confirmed that tPFK-2 was localized mainly in the acrosomal region of the sperm head and in the mid-piece of the flagellum, where other spermatogenic cell-specific glycolytic enzymes have been found.     

Correlation of redox levels of component electron carriers with total electron flux in an electron-transport system. P-700 and the photoreduction of NADP+ in chloroplast fragments

A mathematical analysis is described which measures the effects of actinic light intensity and concentration of an artificial electron donor on the steady-state light-induced redox level of a reaction-center pigment (e.g. P-700) and on the overall light-induced electron flux (e.g. reduction of NADP⁺). The analysis led to a formulation (somewhat similar to the Michaelis-Menten equation for enzyme kinetics) in which a parameter, $\text{I}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, is defined as the actinic light intensity that, at a given concentration of electron donor, renders the reaction-center pigment half oxidized and half reduced. To determine the role of a presumed reaction-center pigment, $\text{I}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ is compared with another parameter, equivalent to $\text{I}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, that is obtained independently of the reaction-center pigment by measuring the effect of actinic light intensity and concentration of electron donor on the overall electron flow.The theory was tested and validated in a model system with spinach Photosystem I chloroplast fragments by measurements of photooxidation of P-700 and light-induced reduction of NADP⁺ by reduced 2,6-dichlorophenolindophenol. A possible extension of this mathematical analysis to more general electron-transport systems is discussed.     

K+-stimulated ATPase in purified microsomes of bullfrog oxyntic cells

Differential centrifugation of oxyntic cell homogenates yielded microsomal fractions which contained large amounts of mitochondrial membrane. The presence of marker enzymes (succinate dehydrogenase and cytochrome c oxidase) indicated that mitochondrial contamination of crude microsomes ranged from 20 to 60% in different preparations. A discontinuous sucrose density gradient procedure was developed for the routine preparation of purified oxyntic cell microsomes. A K⁺-stimulated, Mg²⁺-requiring ATPase was localized in these purified membranes and coincided with the presence of a K⁺-stimulated p-nitrophenylphosphatase. Na⁺ and ouabain had no effect on the K⁺ stimulation of the microsomal ATPase. The apparent activation constant for K⁺ was approximately 1 mM at pH 7.5, the optimal pH for stimulation.An anion-sensitive ATPase has been widely studied in gastric microsomal preparations. We found that the basal microsomal ATPase (i.e. without K⁺) and the mitochondrial ATPase were inhibited by SCN^? and enhanced by HCO₃^?, however, the K⁺-stimulated component of the microsomal ATPase was virtually unaffected by these anions.     

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.     

Photooxidase system of Rhodospirillum rubrum. I. Photooxidations catalyzed by chromatophores isolated from a mutant deficient in photooxidase activity

The aerobic photooxidations of reduced 2,6-dichlorophenolindophenol and of reaction-center bacteriochlorophyll (P-870) have been investigated in membrane vesicles (chromatophores) isolated from a non-phototrophic Rhodospirillum rubrum strain. In aerobic suspensions of wild-type chromatophores, continuous light elicits an increase of the levels of 2,6-dichlorophenolindophenol and of oxidized P-870, which reach steady-state values shortly after the onset of illumination. In contrast, light induces in mutant suspensions a transient increase of the levels of 2,6-dichlorophenolindophenol and of oxidized P-870, which fall to low steady-state values within a few seconds. These observations suggest that the mutation has altered a redox constituent located on the low-potential side of the photochemical reaction center, between a pool of acceptors and oxygen.Since endogenous cyclic photophosphorylation is catalyzed by mutant chromatophores at normal rates, it appears that the constituent altered by the mutation does not belong to the cyclic electron-transfer chain responsible for photophosphorylation. However, the system which mediates the aerobic photooxidations and the cyclic system are not completely independent: endogenous photophosphorylation is inhibited by oxygen in wild-type chromatophores but not in mutant chromatophores; in addition, the inhibitor of cyclic electron flow, 2-heptyl-4-hydroxyquinoline-N-oxide, enhances the aerobic photooxidation of reduced 2,6-dichlorophenolindophenol by chromatophores from both strains.These results support a tentative branched model for light-driven electron transfer. In that model, the constituent altered in the mutant strain is located in a side electron-transfer chain which connects the cyclic acceptors to oxygen.     

Spectral analyses of extrinsic fluorescence of the nerve membrane labeled with aminonaphthalene derivatives

A fluorescence spectrophotometer was constructed to determine the emission spectrum of a nerve labeled with various fluorochromes. Using this spectrophotometer, the spectra of 2-p-toluidinylnaphthalene 6-sulfonate (2,6-TNS) and other aminonaphthalene derivatives in squid giant axons were determined at the peak of nerve excitation, as well as in the resting state of the axons. During nerve excitation the fluorescent light deriving from the 2,6-TNS-stained nerve undergoes a transient change in intensity. The spectrum of the light contributing to this change in intensity was found to be much narrower and sharper than the fluorescent spectrum of the light arising from labeled axons at rest. This narrow and sharp spectrum is interpreted as being derived from a transient variation in the polarity of the 2,6-TNS binding sites in the axon. In the Appendix, the results of a physicochemical investigation into the factors affecting the fluorescence of 2,6-TNS in vitro are described.     

Light-induced absorption changes in Photosystem I at low temperatures

Light-induced absorption changes associated with the primary photochemical reaction and dark relaxation in Photosystem I were measured at various low temperatures. A possible temperature-dependent long-range electron tunneling process was suggested to account for the unique temperature dependence of the dark decay process. The kinetics of the light-induced absorption changes are in good agreement with the light-induced EPR changes reported earlier (Ke, B., Sugahara, K., Shaw, E.R., Hansen, R. E., Hamilton, W. D. and Beinert, H. (1974) Biochim. Biophys. Acta 368, 401–408) for the same Photosystem I subchloroplast fragments at comparable temperatures.All absorption changes between 400 and 725 nm at 86 °K have identical kinetics. The light-minus-dark difference spectrum is very similar to that of P-700 at room temperature, with an additional prominent positive change at 690 nm. Possible contributions by P-430 to the blue and red spectral changes were discussed.It was demonstrated that the intensity of the measuring beam has a drastic effect on the light-induced absorption changes of Photosystem I at low temperatures. Various pretreatments of the Photosystem I fragments such as those that photochemically (or chemically) oxidize the primary donor or photoreduce the primary acceptor abolish the subsequent photochemical reaction. Continuous illumination of the Photosystem I fragments before and during freezing has the same effect.In the temperature range of ?20 to ?60 °C, an unusual counter absorption change as well as a counter EPR change were observed.     

Synthesis and antiprotozoal activity of dicationic 2,6-diphenylpyrazines and aza-analogues

Dicationic 2,6-diphenylpyrazines, aza-analogues and prodrugs were synthesized; evaluated for DNA affinity, activity against Trypanosoma brucei rhodesiense (T. b. r.) and Plasmodium falciparum (P. f.) in vitro, efficacy in T. b. r. STIB900 acute and T. b. brucei GVR35 CNS mouse models. Most diamidines gave poly(dA-dT)₂ ΔT_m values greater than pentamidine, IC₅₀ values: T. b. r. (4.8–37 nM) and P. f. (10–52 nM). Most diamidines and prodrugs gave cures for STIB900 model (11, 19a and 24b 4/4 cures); 12 3/4 cures for GVR35 model. Metabolic stability half-life values for O-methylamidoxime prodrugs did not correlate with STIB900 results.     

Ribulose-1,5-bisphosphate carboxylase-deficient plastome mutants of Oenothera

In spite of only slightly subnormal pigment contents, two plastome mutants of Oenothera (Vα, Iσ) were practically incapable of photosynthetic CO₂ fixation and another one exhibited considerably reduced photosynthesis (IVβ). While other photosynthetic enzymes were present as far as investigated, ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) activity was very low or missing altogether. As shown by gel electrophoresis, mutant IVβ contained some, though little, fraction I protein. In the other two mutants fraction I protein could not be detected. Also, neither the small nor the large subunit of ribulose-1,5-bisphosphate carboxylase could be found in these mutants. In immunodiffusion experiments with a monospecific antiserum against rye ribulose-1,5-bisphosphate carboxylase, only extracts from wild-type Oenothera produced visible precipitation lines. Still, the presence of very low levels of immunochemically reactive antigen was indicated for all three mutants. The highest level was observed in mutant IVβ. The behaviour of the mutant extracts suggested that the antigens of mutant and wild type leaves reacting with the antiserum were not identical. All mutants appeared to have a coupled electron transport system as shown by ATP measurements, light scattering and 515 nm absorption changes. Linear electron transport was possible in the mutants. Still, the photoresponse of cytochrome f and fluorescence measurements suggested altered electron transport properties in the mutants. These are interpreted to be secondary lesions of the photosynthetic apparatus caused by primary deficiency in ribulose-1,5-bisphosphate carboxylase activity. From the absence in two mutants (Vα, Iσ) of the small subunit of ribulose-1,5-bisphosphate carboxylase, which is known to be coded for by nuclear DNA and to be synthesized on cytoplasmic ribosomes, it appears that the genetic system of the plastids is capable of interfering with the genome-controlled synthesis of plastid components.