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The formate dehydrogenase involved in electron transport from formate to fumarate in Vibrio succinogenes.

1. The formate dehydrogenase of Vibrio succinogenes, which is involved in electron transport with fumarate as terminal acceptor, was solubilized with Triton X-100 and purified some 200-fold by means of chromatography on hydroxyapatite, sucrose-density-gradient centrifugation and chromatography on DEAE-Sephadex. Gel filtration failed to increase the specific acitivity of the enzyme while gel electrophoresis in the presence of dodecylsulfate revealed that 73% of the protein of the preparation consisted of a polypeptide of Mr 110 000. The Mr of the functional enzyme was found to be 263 000 on the basis of the Stokes radius (5.8 nm) and the sedimentation coefficient (11.3 S). 2. The preparation contained 9 micronmol molybdenum/g protein and about 170 mumol iron-sulfur/g protein. The contents of b and c cytochromes varied and were lower than that of molybdenum. The low-potential cytochrome b [Kröger, A. and Innerhofer, A. (1976) Eur. J. Biochem. 69, 497-506] present in the preparation was reduced by formate. 3. The preparation catalyzed the reduction of a variety of dyes by formate, but not of NAD, FMN, ferredoxin or oxygen. The reduction of CO2 or bicarbonate by reduced methyl viologen was not catalyzed. The reaction with benzyl viologen obeyed the rate law consistent with a ping-pong mechanism. The Km for formate was 1.5 mM at infinite concentration of benzyl viologen while that for benzyl viologen was 0.53 mM at infinite formate concentration. Enzymic activity was inhibited by azide, KCN and HgCl2, but not by 4-chloromercuriphenylsulfonate or 2-(n-nonyl)-4-hydroxyquinoline-N-oxide, both of which inhibit overall electron transport. The inhibition by azide was competitive with formate; the Ki was 45 micron. 4. The midpoint potential of the low-potential cytochrome b of the membrane fraction was shifted -40 mV by the presence of 2-(n-nonyl)-4-hydroxyquinoline-N-oxide. 5. It is concluded that the formate dehydrogenase of V. succinogenes is isolated as a dimer consisting of two identical subunits of Mr 110,000, each of which carries one atom of molybdenum and iron-sulfur groups. The low-potential cytochrome b is the direct acceptor for the electrons of formate dehydrogenase in the electron transport of formate-fumarate reduction of V. succinogenes. Inhibition of electron transport of the membrane fraction between formate dehydrogenase and menaquinone by 2-(n-nonyl)-4-hydroxyquinoline-N-oxide [Kröger, A. and Innerhofer, A. (1976) Eur. J. Biochem. 69, 487-495] is caused by the inhibitor binding to the low-potential cytochrome b.

Aldehyde Oxidoreductases

Formation of the formate-nitrate electron transport pathway from inactive components in Escherichia coli.

When Escherichia coli was grown on medium containing 10 mM tungstate the formation of active formate dehydrogenase, nitrate reductase, and the complete formate-nitrate electron transport pathway was inhibited. Incubation of the tungstate-grown cells with 1 mM molybdate in the presence of chloramphenicol led to the rapid activation of both formate dehydrogenase and nitrate reductase, and, after a considerable lag, the complete electron transport pathway. Protein bands which corresponded to formate dehydrogenase and nitrate reductase were identified on polyacrylamide gels containing Triton X-100 after the activities were released from the membrane fraction and partially purified Cytochrome b1 was associated with the protein band corresponding to formate dehydrogenase but was not found elsewhere on the gels. When a similar fraction was prepared from cells grown on 10 mM tungstate, an inactive band corresponding to formate dehydrogenase was not observed on polyacrylamide gels; rather, a new faster migrating band was present. Cytochrome b1 was not associated with this band nor was it found anywhere else on the gels. This new band disappeared when the tungstate-grown cells were incubated with molybdate in the presence of chloramphenicol. The formate dehydrogenase activity which was formed, as well as a corresponding protein band, appeared at the original position on the gels. Cytochrome b1 was again associated with this band. The protein band which corresponded to nitrate reductase also was severely depressed in the tungstate-grown cells and a new faster migrating band appeared on the polyacrylamide gels. Upon activation of the nitrate reductase by incubation of the cells with molybdate, the new band diminished and protein reappeared at the original position. Most of the nitrate reductase activity which was formed appeared at the original position of nitrate reductase on gels although some was present at the position of the inactive band formed by tungstate-grown cells. Apparently, inactive forms of both formate dehydrogenase and nitrate reductase accumulate during growth on tungstate which are electrophoretically distinct from the active enzymes. Activation by molybdate results in molecular changes which include the reassociation of cytochrome b1 with formate dehydrogenase and restoration of both enzymes to their original electrophoretic mobilities.

Aldehyde Oxidoreductases

Rosette formation between human lymphocytes and sheep erythrocytes. Inhibition of rosette formation by specific glycopeptides.

Rosette formation with unsensitized sheep erythrocytes is a characteristic of human thymus dependent lymphocytes. Release of glycopeptides from the sheep erythrocyte by trypsin reduces rosette formation. These tryptic glycopeptides inhibit rosette formation by untrypsinized sheep erythrocytes; this suggests that rosetting is mediated by erythrocyte surface glycopeptides. To investigate the molecular nature of this interaction, we examined the abilities of various model compounds to act as haptenic inhibitors of rosette formation. Inhibition is given by glycopeptides bearing oligosaccharide units rich in sialic acid, galactose, N-acetylglucosamine, and mannose linked to asparagine residues through glycosylamine bonds. Among compounds tested, fetuin glycopeptide is most effective, but human transferrin glycopeptide and human erythrocyte glycopeptide I also inhibit rosette formation. Other compounds including human erythrocyte glycopeptide II, human IgG glycopeptide, lacto-N-neotetraose, 3'- and 6'-sialyllactose show no significant inhibition. Neither sialic acid, galactose, manose, nor N-acetyl-glucosamine alone inhibits rosette formation. Stepwise degradation of fetuin glycopeptide established the galactose residues as important determinants of inhibitory activity. Fetuin glycopeptide blocks rosette formation when added to a suspension of human lymphocytes and sheep erythrocytes or when preincubated with human lymphocytes, but not when preincubated with sheep erythrocytes. Studies of the binding of [3H] fetuin glycopeptide to normal lymphocytes demonstrate 7.5 x 10(6) saturable binding sites per cell. No saturable binding of this compound to sheep erythrocyte membranes is observed. Compared to normals, lymphocytes from patients with chronic lymphatic leukemia demonstrate decreased fetuin glycopeptide binding with a mean of 0.9 x 10(6) sites per cell. This decreased binding correlates with the impaired ability of these cells to form rosettes. The data suggest that fetuin glycopeptide inhibits rosette formation by binding to the thymus-dependent cell where competition occurs with sheep erythrocytes for specific lymphocyte surface receptors.

Animals

Differentiation between Clostridium acidiurici and Clostridium cylindrosporum on the basis of specific metal requirements for formate dehydrogenase formation.

The formate dehydrogenases of Clostridium acidiurici and of C. cylindrosporum coupled the oxidation of formate with the reduction of viologen dyes. The basal activity level was about 0.85 mumoles/min X mg of protein for both species. The level of formate dehydrogenase of C. acidiurici increased 12-fold when 10(-7) M tungstate and selenite were present during growth. Molybdate exerted no effect. On the other hand, molybdate and selenite were required to increase the formate dehydrogenase of C. cylindrosporum, and tungstate exhibitedan antagonistic effect in this organism. Growth on hypoxanthine generally depended on the addition of bicarbonate. Supplementation with tungstate and selenite accellerated growth of C. acidiurici and increased again the level of formate dehydrogenase. The addition of both, molybdate and selenite was necessary to initiate growth of C. cyclindrosporum and to form an active formate dehydrogenase. The differences in the requirement for metal ion supplementation to form high levels of formate dehydrogenase and their involvement in hypoxanthine degradation can be used to differentiate between C. acidiurici and C. cylindrosporum.

Aldehyde Oxidoreductases

[Genetic control of the formation of plasmid F'. I. Effect of recA- and seg-2 mutations in an Hfr donor strain on the character of plasmid F' formation].

Assuming the similarity of the processes of illegitimate recombination, such as deletion formation, with the process of F' plasmid formation, we have undertaken the study of the influence of recA- and seg- alleles of Hfr donor on the F' plasmid formation. The data obtained demonstrate the strong influence of donor genotype on the frequency of F' plasmid formation and on the nature of F' plasmids formed, thus demonstrating that the most of F' plasmids have been formed via recombination in Hfr donor cells. The recA- mutation decreased the total yield of F' plasmids selected using both proximal and distal Hfr markers and affected drastically the distribution of the F' plasmids inheriting different proximal unselected markers. The existence of recA-dependent and recA-independent modes of F' plasmid formation was demonstrated. The Escherichia coli chromosome contains regions which involve preferentially in recA-dependent (between proA and gal, and clockwise from gal) or recA-independent (between leu and proA, and the region counterclockwise from argE) recombination. The seg-2 mutation causes only partial block of both recA-dependent and recA-independent recombination pathways, however it causes dramatic decrease of genetic exchanges leading to the formation of the type II F' plasmids. Both seg- and recA- mutations decrease the frequency of the formation of Tra+ F' transconjugants. The percent of Tra- transconjugants, which remain sensitive to MS2 and Q beta donor specific phages, also drops significantly under the influence of the recA- and seg- alleles. Thus, the recombination involving the F structure in wild type strains and seg- mutants occures preferentially in the points of F outside the regions essential for transfer and sensitivity to male specific phages, while in recA- and recA-ges- strains the points inside these regions (tra operon) frequently involved in F' plasmid looping out. There exist more strict correlation between the fertility and sensitivity to phage Q beta than to phage MS2.

Chromosome Deletion

CO2 reduction to formate by NADH catalysed by formate dehydrogenase from Pseudomonas oxalaticus.

The direct reduction of CO2 to formate is catalysed by formate: NAD oxidoreductase in the presence of substrate amounts of NADH. Proof for this reaction is supplied by the detection of a CO2-dependent NADH oxidation, and by the identification of [14c] formate as the product of a NADH-dependent reduction of [14c]carbonate. The enzyme-catalysed CO2 reduction by NADH attains the equilibrium predicted by thermodynamic considerations, a state which is also reached from the formate side. The Michaelis constant for CO2 is about 40 mM indicating the low affinity of the enzyme for this substrate. The corresponding value for formate is 0.1 mM. Under the special conditions employed the enzyme catalyses the formate oxidation about 30 times faster than the CO2 reduction. That CO2 and not HCO3- is the active species in the reduction was shown by comparing the ph dependency of the velocities of the forward and back reactions and by observing the kinetics of CO2 reduction during the simultaneous attainment of the CO2-HCO3- equilibrium.

Aldehyde Oxidoreductases

Pyridine-2, 6-dicarboxylic acid (dipicolinic acid) formation in Bacillus subtilis. II Non-enzymatic and enzymatic formations of dipicolinic acid from alpha, epsilon-diketopimelic acid and ammonia.

Non-enzymatic formation of dipicolinic acid (DPA) from diketopimelic acid and ammonia was clearly demonstrated using a new method for DPA analysis. The reaction rates of DPA formation were almost the same under aerobic and anaerobic conditions. Nearly equimolecular quantities of DPA and tetrahydrodipicolinic acid were detected in spontaneous reaction mixture. The spontaneous reaction seemed to be due to dismutation of dihydrodipicolinic acid, resulting in DPA and tetrahydrodipicolinic acid. The apparent optimum pH of the spontaneous reaction was 8.2 and the maximal rate of DPA formation was observed with a 1 : 4 molar ratio of diketopimelic acid to ammonia. The rate of the spontaneous reaction was stimulated by ferrous sulfate, FMN, and riboflavin. Dihydrodipicolinate reductase catalyzes the reduction of dihydrodipicolinate, prepared from pyruvate and aspartic beta-semialdehyde, with NADPH as reductant. The reductase was isolated from Bacillus subtilis, and found to stimulate DPA formation from diketopimelic acid and ammonia. The enzymatic DPA formation was absolutely dependent on oxygen, and optimum pH was 6.4. The catalytic action of the enzyme was similar to that of the oxidase. Possible mechanisms of DPA formation from diketopimelic acid and ammonia are proposed.

Aerobiosis

[Formation of spontaneous and immune rosettes by the cells of the thymus and other formations of the rabbit lymphoid system].

Only T1--RFC (rosette-forming cells) are revealed in the thymus of nonimmunized rabbits. Their number is 2--2.5 times less than in the palatine tonsils, submaxillary lymph nodes and the spleen. T2--RFC are present in these lymphoid formations. There is an increase in the T1--RFC in the thymus after the intravenous immunization of rabbits with sheep erythrocytes. In other lymphoid formations the correlation of the population of cells of the thymus origin altered as a result of increase in the number of T2--RFC. B--RFC accumulated in considerable amounts. Dynamics of T2 and B--RFC accumulation in the lymphoid formations corresponded to the highest antibody titres in the rabbit blood. In the formation of primary immune response the amount of the T1 and T2-RFC in the formations of rabbit lymphoid system depended on the dose of the antigen.

Animals

Role of the chlC gene in formation of the formate-nitrate reductase pathway in Escherichia coli.

Five temperature-sensitive chlC mutants were isolated from Escherichia coli by the technique of localized mutagenesis. All of the mutants produced severely reduced levels of both nitrate reductase and formate dehydrogenase when grown at 43 degrees C. In three of the mutants, the nitrate reductase activity produced at the permissive temperature was shown to be thermolabile compared with the activity produced by the parent wild-type strain, both in membrane preparations and in preparations released from the membrane by deoxycholate. In each case, formate dehydrogenase activity was similar to the wild-type activity in its stability to heat. It is concluded that the chlC gene codes for at least one of the polypeptide chains of nitrate reductase and that the chlC mutations affect indirectly the formation of formate dehydrogenase.

Aldehyde Oxidoreductases

[Correlation between bioelectrical processes of the cortex, thalamus, midbrain reticular formation during formation of a defensive conditioned reflex in rabbits].

EEG power spectra of the sensorimotor area of the neocortex, the dorsal hippocampus, midbrain reticular formation and anteroventral thalamic nucleus, as well as corresponding coherence functions and phase spectra, undergo changes during formation and performance of defensive conditioned reflex in rabbit. The conclusion is draen that in the process of conditioning a morphofunctional system of brain structures is established including the above mentioned formations. Their functional integration occurs on the basis of theta-rhythm. The execution of a conditioned act requires isorhythmicity of electrical processes within the theta-range in the studied structures and an adequate level of their excitability, which is manifested in the dominance of 6,0 c/s frequency.

Animals

A contribution to the formation mechanism of calcium oxalate urinary calculi. II. In vitro experiments concerning the theory of the formation of Whewellite and Weddellite urinary calculi.

In vitro investigations of the formation of Whewellite or Weddellite are described. By means of different precipitation models the influence of cationic minerals on the formation of Weddellite could be observed. The possible conversion of Weddellite into Whewellite in vivo is demonstrated by in vitro experiments. A theory of the formation of Weddellite or Whewellite urinary calculi is developed on the basis of the results obtained.

Calcium

Follicle formation in the embryonic chick thyroid. III. Initiation of follicle formation.

It has been proposed that the follicular spaces in the thyroid form by either the coalescence of intracellular droplets or by separation of cell apices by secretion into the extracellular space. On the basis of examination of thyroid primordia in early chick embryos this study provides evidence that in the chick, at least, follicle formation conforms to the second model. The first indications of change in the chick thyroid is the appearance of interdigitations of the cell apices. These interdigitations form microvilli as the two surfaces become separated and the follicular space is established. Vesicles with two types of contents can be identified in proximity with the cell surface during follicle formation, but it is not clear if either the dense particulate or the more electron-lucid materials that they contain actually enter the follicular space. Neither removal of the pituitary gland by decapitation nor inhibition of collagen synthesis and a concomitant failure of the invasion of capsular mesenchyme prevents the initiation of normal follicle formation.

Animals

Mechanism of the formation of megamitochondria induced by copper-chelating agents. I. On the formation process of megamitochondria in cuprizone-treated mouse liver.

Processes of the formation of cuprizone-induced megamitochondria in mouse liver have been studied in detail by electron microscopy. The earliest change observed was the presence of large intramitochondrial granules. The next stage was the formation of myelin figures by which mitochondria were apparently connected. The third stage was characterized by megamitochondria connected with each other by their outer membranes. Continuity of mitochondria were further examined by serial sections, and megamitochondria were proved to be connected to each other far more frequentlythan expected on one plane of section. A model for the mechanism of megamitochondrial formation is proposed based on electron microscopic evidences, involving the fusion of mitochondrial membranes. Possibility is also discussed that cuprizone-induced megamitochondria may fuse to one single branching mitochondrion.

Adrenal Glands

Reaginic antibody formation in the mouse. VII. Depression of the ongoing IgE antibody formation by suppressor T cells.

The ongoing IgE antibody formation against ovalbumin (OA) in high responder mice was depressed by i.v. injections of either native or urea-denatured ovalbumin (UD-OA). Adoptive transfer experiments to determine the helper function of spleen cells from the treated animals showed that helper function for both IgE and IgG antibody responses diminished after treatment. Evidence was obtained that treatment suppressed the expansion of IgE-G memory cells. When the same treatment with OA or UD-OA was given to OA-primed mice before the appearance of IgE antibody in their serum, OA-specific splenic suppressor T cells were demonstrable. Thus, the transfer of splenic T cells from treated mice into normal mice suppressed the primary IgE and IgG antibody responses of the recipeints to DNP-OA. It was also found that the transfer of the splenic T cells from UD-OA-treated mice into OA-primed mice depressed ongoing IgE antibody formation in the recipients. The results suggested strongly that the decrease of helper function and the depression of ongoing IgE antibody formation by repeated injections of UD-OA was caused by generation of antigen (OA)-specific suppressor T cells.

Animals

[Processes of spore formation and gramicidin C formation by Bacillus brevis var. G.B].

Correlation between gramidicin C biosynthesis and sporulation in the process of Bac. brevis var. G.B. cultivation under various aeration conditions was studied. It was shown that biosynthesis of gramicidin C was characteristic of the young cells and its level was the highest during the culture active growth. The time of the sporulating forms appearance depended on the aeration rate which defined the quantitative composition of the population during the phase of the culture active growth and the stationary phase. Under the optimal aeration conditions the spore formation started during the phase of the culture active growth after some decrease in the maximum level of the cell productivity with respect to the antibiotic. When the aeration rate was increased the spore formation was shifted to later periods of the culture development, i.e. the stationary phase and the phase of the cell autolysis, the gap between the highest levels of gramicidin C buosynthesis and the beginning of sporulation being increased. Under certain aeration conditions the spore formation was not observed, while gramicidin C was synthesized. A conclusion has been made that there is no correlation between gramacidine C biosynthesis and sporualtion in Bacillus brevis var. G.B.

Air

A contribution to the formation mechanism of calcium oxalate urinary calculi. I. Stabilising urinary constituents in the formation of weddellite.

25 to 30% of calcium oxalate urinary calculi consist of the metastable Weddellite crystal phase. By fractionation of urine it was found that mineral substances are stabilising factors. The stability was checked in dry condition at room temperature at 38 degrees C and at 110 degrees C. These results could be confirmed by precipitation from synthetic solutions. Mg, Zn, Ni, Co, Mn and Cu individually, and above all in combination, promote the formation of Weddellite. The formation of mixed crystal phases must be considered one of the main factors for the stabilisation of Weddelite in the urinaty calculus.

Calcium

Methemoglobin formation and binding to blood constituents as indicators for the formation, availability and reactivity of activated metabolites derived from trans-4-aminostilbene and related aromatic amines.

trans-4-Aminostilbene derivatives exhibit higher acute and chronic toxicity than 4-aminobibenzyl derivatives. Yet, trans-4-aminostilbene produced less methemoglobin in female Wistar rats than 4-aminobibenzyl. This cannot be explained by differences in N-oxidation since trans-4-nitrosostilbene was also less efficient than 4-nitrosobibenzyl. The fate of intravenously injected, highly and specifically 3H-labeled trans-4-aminostilbene, cis-4-aminostilbene, 4-aminobibenzyl, trans-4-nitrosostilbene and 4-nitrosobibenzyl was investigated. The results indicate that trans-4-aminostilbene and 4-aminobibenzyl are N-oxidized to a similar extent and primary activation products of trans-4-aminostilbene appear even faster in the blood. However, intermediates originating during methemoglobin formation are more reactive and covalently bind to hemoglobin 2--3 times as much with trans-stilbene as compared to bibenzyl derivatives. As a consequence the availability of these intermediates in the cyclic process and thus methemoglobin formation is reduced. Therefore, binding to hemoglobin rather than levels of methemoglobin appears to be an indicator for the availability and reactivity of some activated aromatic amine metabolites.

Animals

Intracellular mechanisms of the formation of homo- and heteropolymeric isozymes. I. Kinetics of the formation of the heteropolymeric isozyme of malate dehydrogenase in parasexual hybrids of two Acetabularia species.

The isozyme pattern of malate dehydrogenase (MDH) of Acetabularia crenulata and A. mediterranea is characterized by heterogeneity in different regions of the cytoplasm of both algae, as well as by species specificity. The formation of the heteropolymeric MDH isozyme is restricted to a definite region of the cytoplasm of heterokaryons and nuclear-cytoplasmic A.crenulata-A.mediteranea hybrids at different stages of their development. The data obtained suggest that the concentrations of the free subunits of MDH, coded for by homologous genes, are unevenly distributed in the cytoplasm of hybrid cells. The heteropolymeric MDH isozyme in these cells is presumably the result of the de novo synthesis of isozyme subunits. This seems plausible inasmuch as no exchange occurs between the homopolymeric MDH isozymes of both parental types in the cytoplasm. The formation of the heteropolymeric MDH isozyme is tentatively related to the spatial compartmentalization of the mRNAs of homologous genes coding for the MDH subunits.

Acetabularia