An e.p.r. study of the non-equivalence of the copper sites of caeruloplasmin.

The two Type 1 (blue) copper-binding sites of caeruloplasmin were spectroscopically differentiated by the kinetic analysis of the e.p.r. spectra during the redox cycle. One blue copper, with a hyperfine splitting constant (A parallel) of 6.8 mT, which was rapidly reduced, was not reoxidized by oxygen, whereas it was reoxidized by H2O2. The other blue copper (A parallel = 5.8 mT), which was reduced slowly, was rapidly reoxidized by either oxygen or H2O2. A conformational change of the Type 2 copper was concomitant with the fast reduction of Type 1 copper, whereas its reduction occurred during the slow phase. This sequence of events was reversed in the reoxidation step, that is, the Type 2 copper reappeared rapidly as the species with altered conformation and reverted to the symmetry typical of the native state in the slow phase. The specific reaction of a blue-copper site with the H2O2 can tentatively be related to the established ability of caeruloplasmin to prevent 'oxidative' attack of proteins and lipids.     

Kinetic studies on cytochrome c oxidase by combined epr and reflectance spectroscopy after rapid freezing.

1. Techniques and experiments are described concerned with the millisecond kinetics of EPT-detectable changes brought about in cytochrome c oxidase by reduced cytochrome c and, after reduction with various agents, by reoxidation with O2 or ferricyanide. Some experiments in the presence of ligands are also reported. Light absorption was monitored by low-temperature reflectance spectroscopy. 2. In the rapid phase of reduction of cytochrome c oxidase by cytochrome c (less than 50 ms) approx. 0.5 electron equivalent per heme a is transferred mainly to the low-spin heme component of cytochrome c oxidase and partly to the EPR-detectable copper. In a slow phase (less than 1 s) the copper is reoxidized and high-spin ferric heme signals appear with a predominant rhombic component. Simultaneously the absorption band at 655 nm decreases and the Soret band at 444 nm appears between the split Soret band (442 and 447 nm) of reduced cytochrome a. 3. On reoxidation of reduced enzyme by oxygen all EPR and optical features are restored within 6 ms. On reoxidation by O2 in the presence of an excess of reduced cytochrome c, states can be observed where the low-spin heme and copper signals are largely absent but the absorption at 655 nm is maximal, indicating that the low-spin heme and copper components are at the substrate side and the component(s) represented in the 655 nm absorption at the O2 side of the system. On reoxidation with ferricyanide the 655 nm absorption is not readily restored but a ferric high-spin heme, represented by a strong rhombic signal, accumulates. 4. On reoxidation of partly reduced enzyme by oxygen, the rhombic high-spin signals disappear within 6 ms., whereas the axial signals disappear more slowly, indicating that these species are not in rapid equilibrium. Similar observations are made when partly reduced enzyme is mixed with CO. 5. The results of this and the accompanying paper are discussed and on this basis an assignment of the major EPR signals and of the 655 nm absorption is proposed, which in essence is that published previously (Hartzell, C.R., Hansen, R.E. and Beinert, H. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2477-2481). Both the low-spin (g=o; 2.2; 1.5) and slowly appearing high-spin (g=6; 2) signals are attributed to ferric cytochrome a, whereas the 655 nm absorption is thought to arise from ferric cytochrome a3, when it is present in a state of interaction with EPR-undectectable copper. Alternative possibilities and possible inconsistencies with this proposal are discussed.     

The kinetic mechanism of beef kidney D-aspartate oxidase

The mechanism of action of the flavoprotein D-aspartate oxidase (EC 1.4.3.1) has been investigated by steady-state and stopped flow kinetic studies using D-aspartate and O2 as substrates in 50 mM KPi, 0.3 mM EDTA, pH 7.4, 4 degrees C. Steady-state results indicate that a ternary complex containing enzyme, O2, and substrate (or product) is an obligatory intermediate in catalysis. The kinetic parameters are turnover number = 11.1 s-1, Km(D-Asp) = 2.2 x 10(-3) M, Km(O2) = 1.7 x 10(-4) M. Rapid reaction studies show that 1) the reductive half reaction is essentially irreversible with a maximum rate of reduction of 180 s-1; 2) the free reduced enzyme cannot be the species which is reoxidized during turnover since its reoxidation by oxygen (second order rate constant equal to 5.3 x 10(2) M-1 s-1) is too slow to be of relevance in catalysis; 3) reduced enzyme can bind a ligand rapidly and be reoxidized as a complex at a rate faster than that observed for the free reduced enzyme; 4) the rate of reoxidation of reduced enzyme by oxygen during turnover is dependent on both O2 and D-aspartate concentrations (second order rate constant of reaction between O2 and reduced enzyme-substrate complex equal to 6.2 x 10(4) M-1 s-1); and 5) the rate-limiting step in catalysis occurs after reoxidation of the enzyme and before its reduction in the following turnover. A mechanism involving reduction of enzyme by substrate, dissociation of product from reduced enzyme, binding of a second molecule of substrate to the reduced enzyme, and reoxidation of the reduced enzyme-substrate complex is proposed for the enzyme-catalyzed oxidation of D-aspartate.     

Evidence for superoxide-dependent reduction of Fe3+ and its role in enzyme-generated hydroxyl radical formation.

This report describes studies yielding additional evidence that superoxide anion (O2) production by some biological oxidoreductase systems is a potential source of hydroxyl radical production. The phenomenon appears to be an intrinsic property of certain enzyme systems which produce superoxide and H2O2, and can result in extensive oxidative degradation of membrane lipids. Earlier studies had suggested that iron (chelated to maintain solubility) augmented production of the hydroxyl radical in such systems according to the following reaction sequence: O2 + Fe3+ leads to O2 + Fe2+ Fe2+ + H2O2 leads to Fe3+ + HO-+OH-. The data reported below provide additional support for the occurrence of these reactions, especially the reduction of Fe3+ by superoxide. Because the conditions for such reactions appear to exist in animal tissues, the results indicate a mechanism for the initiation and promotion of peroxidative attacks on membrane lipids and also suggest that the role of antioxidants in intracellular metabolism may be to inhibit initiation of degradative reactions by the highly reactive radicals formed extraneously during metabolic activity. This report presents the following new information: (1) Fe3+ is reduced to Fe2+ during xanthine oxidase activity and a significant part of the reduction was oxygen dependent. (2) Mn2+ appears to function as an efficient superoxide anion scavenger, and this function can be inhibited by EDTA. (3) The O2-dependent reduction of Fe3+ to Fe2+ by xanthine oxidase activity is inhibited by Mn2+, which, in view of statement 2 above, is a further indication that the reduction of the iron involves superoxide anion. (4) Free radical scavengers prevent or reverse the Fe3+ inhibiton of cytochrome c3+ reduction by xanthine oxidase. (5) The inhibition of xanthine oxidase-catalyzed reduction of cyt c3+ by Fe3+ does not affect uric acid production by the xanthine oxidase system. (6) The reoxidation of reduced cyt c in the xanthine oxidase system is markedly enhanced by Fe3+ and is apparently due to enhanced HO-RADICAL formation since the Fe3+-stimulated reoxidation is inhibited by free radical scavengers, including those with specificity for the hydroxyl radical.     

Redox cycling of resorufin catalyzed by rat liver microsomal NADPH-cytochrome P450 reductase

The O-dealkylation of 7-alkoxyresorufins to the highly fluorescent compound, resorufin (7-hydroxyphenoxazone), provides a rapid, sensitive, and convenient assay of certain forms of liver microsomal cytochrome P450. The results of this study indicate that NADPH-cytochrome P450 reductase catalyzes the reduction of resorufin (and the 7-alkoxyresorufins) to a colorless, nonfluorescent compound(s). The reduction of resorufin by NADPH-cytochrome P450 reductase was supported by NADPH but not NADH, and was not inhibited by dicumarol, which established that the reaction was not catalyzed by contaminating DT-diaphorase (NAD[P]H-quinone oxidoreductase). In addition to the rate of reduction, the extent of reduction of resorufin was dependent on the concentration of NADPH-cytochrome P450 reductase. The maintenance of steady-state levels of reduced resorufin required the continuous oxidation of NADPH, during which molecular O2 was consumed. When NADPH was completely consumed, the spectroscopic and fluorescent properties of resorufin were fully restored. These results indicate that the reduction of resorufin by NADPH-cytochrome P450 reductase initiates a redox cycling reaction. Stoichiometric measurements revealed of 1:1:1 relationship between the amount of NADPH and O2 consumed and the amount of H2O2 formed (measured fluorometrically). The amount of O2 consumed during the redox cycling of resorufin decreased approximately 50% in the presence of catalase, whereas the rate of O2 consumption decreased in the presence of superoxide dismutase. These results suggest that, during the reoxidation of reduced resorufin, O2 is converted to H2O2 via superoxide anion. Experiments with acetylated cytochrome c further implicated superoxide anion as an intermediate in the reduction of O2 to H2O2. However, the ability of reduced resorufin to reduce acetylated cytochrome c directly (i.e., without first reducing O2 to superoxide anion) precluded quantitative measurements of superoxide anion formation. Superoxide dismutase, but not catalase, increased the steady-state level of reduced resorufin and considerably delayed its reoxidation. This indicates that superoxide anion is not only capable of reoxidizing reduced resorufin, but is considerably more effective than molecular O2 in this regard. Overall, these results suggest that NADPH-cytochrome P450 reductase catalyzes the one-electron reduction of resorufin (probably to the corresponding semiquinoneimine radical) which can either undergo a second, one-electron reduction (presumably to the corresponding dihydroquinoneimine) or a one-electron oxidation by reducing molecular O2 to superoxide anion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Oxygen Toxicity and the Superoxide Dismutase

Oxygen caused an increase in the amount of superoxide dismutase in Escherichia coli B but not in Bacillus subtilis. E. coli B cells, induced by growth under 100% O(2), were much more resistant to the lethal effects of 20 atm of O(2) than were cells which contained the low uninduced level of this enzyme. In contrast, B. subtilis, which could not respond to O(2) by increasing its content of superoxide dismutase, remained equally sensitive to hyperbaric O(2) whether grown under 100% O(2) or areobically. The catalase in these organisms exhibited a reciprocal response to oxygen. Thus, the catalase of E. coli B was not induced by O(2), whereas that of B. subtilis was so induced. These results are consistent with the view that superoxide dismutase is an important component of the defenses of these organisms against the toxicity of oxygen, whereas their catalases are of secondary importance in this respect. The ability of streptonigrin to generate O(2) (-), by a cycle of reduction followed by spontaneous reoxidation, has been verified in vitro. It is further observed that E. coli B which contain the high induced level of superoxide dismutase were more resistant to the lethality of this antibiotic, in the presence of oxygen, than were E. coli B which contained the low uninduced level of this enzyme. This difference between induced and uninduced cells was eliminated by the removal of O(2). These results are consistent with the proposal that the enhanced lethality of streptonigrin under aerobic conditions may relate to its in vivo generation of O(2) (-) by a cycle of reduction and spontaneous reoxidation. In toto, these observations lend support to the hypothesis that O(2) (-) is an important agent of oxygen toxicity and that superoxide dismutase functions to blunt the threat posed by this reactive radical.     

Pulse-radiolysis studies on the interaction of one-electron reduced species with blue oxidases. Reduction of native and type-2-copper-depleted Vietnamese-lacquer-tree and Japanese-lacquer-tree laccases.

The interactions of one-electron reduced metronidazole (ArNO2.-) and O2.- with native and Type-2-copper-depleted Vietnamese- and Japanese-lacquer-tree laccases were studied in aqueous solution at pH 6.0 and 7.4 by using the technique of pulse radiolysis. On reaction with ArNO2.-, in the absence of O2, the holo- and the Type-2-copper-depleted proteins accept, with reduction of Type 1 copper, 2 and 1 reducing equivalents respectively. On reaction with O2.- of both holo- and Type-2-copper-depleted Vietnamese-lacquer-tree laccase, almost complete reduction of Type 1 copper was observed and, after completion of the reaction, some (less than 20%) reoxidation of Type 1 copper occurs. Reduction of Type 1 copper of the laccases by these one-electron donors occurs via a bimolecular step; however, the rate of reduction of Vietnamese-lacquer-tree laccase is over 10 times that of Japanese-lacquer-tree laccase. It is inferred that electrons enter the protein via Type 1 copper with, in the case of the holoprotein, subsequent rapid intramolecular transfer of 1 reducing equivalent within the protein. Furthermore it is suggested that intra-molecular electron transfer to Type 3 copper atoms is slow and, in the case of Type-2-copper-depleted protein, may not occur. This slow process may partially account for the variation of the catalytic activities of 'blue' oxidases.     

Nonenzymatic NADPH-dependent reduction of 2,6-dichlorophenol-indophenol

The reduction of 2,6-dichloroindophenol (DCIP) by direct interaction with NADPH was studied. The results indicate that reduction proceeds via a direct electron transfer from NADPH to DCIP, with no oxygen consumption, and a rate constant of k = 4.69 M-1.s-1. The reduced DCIP can rapidly transfer its electrons to potassium ferricyanide (K3Fe(CN)6) or ferricytochrome c, but not to nitro blue tetrazolium. Superoxide dismutase inhibits DCIP reduction in an oxygen-dependent manner by favoring the reoxidation of the reduced DCIP. We therefore conclude DCIP is not suitable for detecting O2- when the nucleotides NADH or NADPH are present.     

Two antigenic sites of tissue transglutaminase.

The immunization of rabbits with purified guinea pig liver transglutaminase resulted in the appearance of two antibody populations against the enzyme: one which reacted only with the Ca2+-enzyme complex and another which reacted with the intact as well as the Ca2+-enzyme. The Ca2+-induced confomrational change of the enzyme molecule exposes a new antigenic determinant which initiates the production of a specific antibody population. When the glutamine substrate of the enzyme was a dipeptide, the result of the interaction of the Ca2+-enzyme and its isolated specific antibody was an apparent activation of the catalytic activity. However, when protein substrates were used, an inhibition was observed. The characterization of the mechanism of the activation and the inhibition has led to the conclusion that the consequence of the interaction of Ca 2+-enzyme and its specific antibody is not only a limited steric hindrance of the active center but, besides that, a stabilization of the otherwise labile Ca2+-enzyme. The other antibody population reacts with both forms of the enzyme and its inhibitory effect, which has been observed in each assay, could be due to a prevention of the Ca2+-induced formation of the active enzyme.     

Reduction and destruction rates of nitroxide spin probes

A series of nitroxides was tested for rates of one-electron reduction in a chemical, a photochemical, and two biological systems by ESR assays. In all cases, piperidine and hydropyridine nitroxides were reduced consistently more rapidly than pyrroline and pyrrolidine nitroxides. Substituents on the nitroxides also affected reduction rates, although not as greatly as ring structure. One of the reduction systems, consisting of the photosensitizer FMN and the photoreductant EDTA, was used to study both anaerobic reduction and O2-dependent reoxidation of some of the nitroxides. Reduced piperidine and hydropyridine nitroxides were also oxidized more rapidly than the reduced pyrroline and pyrrolidine nitroxides. Reoxidation subsequent to reduction was partially inhibited by superoxide dismutase, indicating that superoxide radicals are involved in the process. Even after prolonged reoxidation, not all of the probe molecules were returned to their oxidized form, implying an irreversible "destruction" of the spin probe concomitant with its chemical reduction. Probe destruction was studied more specifically with a photochemical system for generating methyl radicals, which showed that these carbon-centered radicals destroyed different nitroxides at rates which were much less influenced by the nitroxide structures than one-electron reduction was.     

Kinetics of the reoxidation of succinate dehydrogenase.

The reoxidation phase of the catalytic cycle of succinate dehydrogenase was studied in Complex II preparations' by the rapid freeze-electron paramagnetic resonance (epr) technique. With the synthetic water-soluble Q₁ analog, 2,3-dimethoxy-5-methyl-6-pentyl-1, 4-benzoquinone (DPB), as the oxidant, the observed reoxidation of the epr-detectable components, previously reduced with dithionite or succinate, came to completion within a few milliseconds, well within the turnover time of the enzyme. Only ~80% of Fe-S center 1 and the HiPIP (the high-potential cluster) Fe-S center reacted rapidly with DPB, however; similarly incomplete reactions were observed previously in our studies of the reduction of the enzyme by succinate. The subsequent addition of ferricyanide, which appears to act as a chemical oxidant in these experiments, caused immediate reoxidation of the Fe-S centers and of the free radical. Ferricyanide and phenazine methosulfate (PMS) reoxidized all epr-detectable components in Complex II as well as in reconstitutively active, soluble preparations in' <6 ms, even at 0°C. Thus, reoxidation of the purified enzyme by PMS cannot be rate-limiting. Carboxamides and thenoyltrifluoroacetone inhibit strongly the reoxidation of the Fe-S center 1 and the HiPIP center by DPB, but not their reduction by succinate. These and other data suggest that these inhibitors block electron transport from the dehydrogenase to the Q pool on the O₂-side of the HiPIP center, but there is no evidence that they combine directly with the iron. A recent report that Wurster's blue reacts with soluble succinate dehydrogenase much more rapidly than does PMS could not be confirmed. The two oxidants react at equal rates with the purified soluble enzyme before and after it has been reincorporated into membranes.     

The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain. III. A minimal model

A steady-state kinetic investigation of the effect of K+ on the Na+-enzyme activity of the (Na+ + K+)-ATPase in broken membrane preparations is reported. Analysis of the kinetic patterns obtained, together with the results reported in the first two articles of this series permit the following conclusions. 1. K+ inhibits the Na+-enzyme (the enzyme activity measured at micromolar substrate concentrations in the presence of Na+). The inhibition of non-competitive at low and competitive at higher K+ concentrations and is enhanced by free Mg2+. 2. The results indicate that the Na+-enzyme at steady-state tends to be accumulated in an enzyme-potassium complex when K+ is added. 3. The enzyme-potassium complex, in turn, binds Mg2+ in a dead-end fashion. The dissociation constant for the enzyme-K-Mg complex, estimated from the data, is 7.2 mM. The same value was obtained earlier for the Mg2+ inhibition constant of the substrate-free form of the (Na+ + K+)-enzyme (the enzyme activity measured with Na+ and K+ and at millimolar substrate concentrations) suggesting that the two constants describe the same equilibrium. 4. On the basis of the known (optimal) activity of the (Na+ + K+)-ATPase, relative to that of the Na+-ATPase, a rate constant condition is found which must be met if the Post-Albers kinetic scheme is to satisfy the data. Kinetic data for the phosphoenzyme indicate that this condition is not satisfied. 5. On the basis of the kinetic results a model for the hydrolytic action of (Na+ + K+)-ATPase is proposed. This model encompasses the Post-Albers scheme but contains two distinctive hydrolysis cycles (an 'Na+-enzyme cycle' and a '(Na+ + K+)-enzyme cycle') with widely different affinities for the substrates. Only one of the cycles (the Na+-enzyme cycle) involves acid-stable phosphorylated enzyme intermediates at discernible steady-state concentrations. Which of the two main cycles is predominant in any particular system is determined by the concentration of ligands and substrates. 6. According to this scheme, an enzyme preparation may exhibit both a high (Na+-enzyme) and a low ((Na+ + K+)-enzyme) substrate affinity, without the necessity of assigning more than one substrate site to a particular enzyme unit at any one time.     

The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain. I. Substrate identity

A detailed steady-state kinetic investigation of the hydrolysis of ATP catalyzed by (Na+ + K+)-ATPase is reported. The activity was studied in the presence of (i) Na+ (130 mM), K+ (20 mM) and micromolar ATP concentrations and Na+ (150 mM) the ('Na+-enzyme'). The data obtained lead to the following results: 1. The action of each enzyme may be described by a simple kinetic mechanism with one (Na+-enzyme) or two ((Na+ + K+)-enzyme) dead-end Mg complexes. 2. For both enzymes, both MgATP and free ATP are substrates, with Mg2+, in the latter case, as the second substrate. 3. For each enzyme, the complete set of kinetic constants (seven for the Na+-enzyme, eight for the (Na+ + K+)-enzyme) are determined from the data. 4. For each enzyme it is shown that, in the alternate substrate mechanism obtained, the ratio of net steady-state flux along the 'MgATP pathway' to that of the 'ATP-Mg pathway' increases linearly with the concentration of free Mg2+. The parameters of this function are determined from the data. As a result of this, at high (greater than 3 mM) free Mg2+ concentrations the alternate substrate mechanism degenerates into a 'limiting' kinetic mechanism, with MgATP as the (essentially) sole substrate, and Mg2+ as an uncompetitive (Na+-enzyme) or non-competitive ((Na+ + K+)-enzyme) inhibitor.     

Site of participation of cytochrome b5 in hepatic microsomal fatty acid chain elongation. Electron input in the first reduction step

The present study provides strong evidence for the involvement of rat liver microsomal cytochrome b5 in the first reduction step of fatty acid chain elongation. The rate of reoxidation of NADH-reduced microsomal cytochrome b5 was markedly stimulated (up to 3-fold) by the addition of increasing concentrations of beta-ketohexadecanoyl-CoA (1-8 microM). A quantitative analysis of product formation, the effect of cyanide, and anaerobiosis completely exclude the possibility that desaturase activity accounted for the beta-ketohexadecanoyl-CoA-induced stimulation of the cytochrome b5 reoxidation rate. Using liver microsomes from untreated rats, the beta-keto substrate was found to stimulate the rate of reoxidation of cytochrome b5 by 30%. However, when liver microsomes from fat-free diet rats were employed the stimulation was more than 3-fold, suggesting that the beta-ketoacyl-CoA reductase is inducible by a high carbohydrate, fat-free diet. This study also provides evidence for the noninvolvement of cytochrome b5 in the terminal reaction step (second reduction step of chain elongation), which is catalyzed by the trans-2-enoyl-CoA reductase. Although trans-2-hexadecenoyl-CoA significantly stimulated the NADH-reduced cytochrome b5 reoxidation rate under aerobic conditions, it did not have any stimulatory effect under anaerobic conditions. One interpretation of these results is that the trans-2-hexadecenoyl-CoA is substrate for the microsomal delta 9 desaturase system. Consistent with this conclusion was the fact that the trans-2-hexadecenoyl-CoA inhibited the liver microsomal delta 9 desaturation of stearoyl-CoA to oleoyl-CoA.     

A new oxygenase, 2-nitropropane dioxygenase of Hansenula mrakii. Enzymologic and spectrophotometric properties.

2-Nitropropane dioxygenase, purified to homogeneity from Hansenula mrakii (IFO 0895), has a molecular weight of approximately 62,000 and consists of two subunits nonidentical in molecular weight (39,000 and 25,000). Stoichiometrical studies and the results obtained with 18O2 showed that 2 atoms of molecular oxygen are incorporated into 2 molecules of acetone formed from 2-nitropropane. In addition to 2-nitropropane, nitroethane, 3-nitro-2-pentanol, and 1-nitropropane are oxidatively dentrified. The enzyme, which exhibits absorption maxima at 274, 370, 415, and 440 nm and a shoulder at 470 nm, contains 1 mol of FAD and 1 g atom of non-heme iron per mol of enzyme. The enzyme-bound FAD is reduced by 2-nitropropane under anaerogic conditions, but the enzyme-bound Fe3+ is not affected. The introduction of oxygen to the reduced form of enzyme causes reoxidation of the enzyme. The bound FAD and Fe3+ are reduced by the addition of nitromethane, which is not a substrate, under anaerobic conditions. The aerobic dialysis of the enzyme treated with nitromethane causes reoxidation of only the Fe2+. Sodium dithionite also reduces both the enzyme-bound FAD and Fe3+ under anaerobic conditions. When the enzyme is dialyzed against 10 mM potassium phosphate buffer (pH 7.0) immediately after reduction by dithionite, the absorption spectrum similar to that of the native enzyme appeared with concomitant restoration of approximately 80% of the activity. The enzyme activity is significantly inhibited by pyrocatechol-3,5-disulfonate disodium salt, 8-hydroxyquinoline, reducing agents such as 2-mercaptoethanol, and HgCl2. The Michaelis constants are as follows: 2-nitropropane (2.13 X 10(-2) M), nitroethane (2.43 X 10(-2) M), 3-nitro-2-pentanol (6.8 X 10(-3) M), 1-nitropropane (2.56 X 10(-2) M), and oxygen (3.03 X 10(-4) M, with 2-nitropropane).     

Mechanistic studies of glutamine synthetase from Escherichia coli. An integrated mechanism for biosynthesis, transferase, ATPase reaction.

The mechanism of biosynthetic, transferase, ATPase, and transphosphorylation reactions catalyzed by unadenylylated glutamine synthetase from E. coli was studied. Activation complex(es) involved in the biosynthetic reaction are produced in the presence of either Mg2+ or Mn2+ ; however, with the Mn2+-enzyme inhibition by the product, ADP, is so great that the overall forward biosynthetic reaction cannot be detected with the known assay methods. Binding studies show that substrates (except for NH3 and NH2OH which are not reported here) can bind to the enzyme in a random manner and that binding of the ATP-glutamate, ADP-Pi or ADP-arsenate pairs is strongly synergistic. Inhibition and binding studies show that the same binding site is utilized for glutamate and glutamine in biosynthetic and transferase reactions, respectively, and that a common nucleotide binding site is used for all reactions studied. Studies of the reverse biosynthetic reaction and results of fluorescent titration experiments suggest that both arsenate and orthophosphate bind at a site which overlaps the gamma-phosphate site of nucleoside triphosphate. In the reverse biosynthetic and transferase reactions, ATP serves as a substrate for the Mn2+-enzyme but not for the Mg2+-enzyme. The ATP supported transferase activity of Mn2+-enzyme is probably facilitated by the generation of ADP through ATP hydrolysis. When AMP was the only nucleotide substrate added, it was converted to ATP with concomitant formation of two equivalents of glutamate, under the reverse biosynthetic reaction conditions, and no ADP was detected. The reversibility of 180 transfer between orthophosphate and gamma-acyl group of glutamate was confirmed. ATPase activity of Mg2+ and Mn2+ unadenylylated enzymes is about the same. Both enzymes forms catalyze transphosphorylation reactions between various purine nucleoside triphosphates and nucleoside diphosphates under biosynthetic reaction conditions. The data are consistent with the hypothesis that a single active center is utilized for all reactions studied. Two stepwise mecanisms that could explain the results are discussed.     

The existence of atypical blood group galactosyltransferase which causes an expression of A2 character in A1B red blood cells

It is generally accepted that the blood group subtypes A1 and A2 expressions are controlled by two different blood group N-acetylgalactosaminyl-transferases, that is, A1-enzyme and A2-enzyme, respectively, and that the two types of enzymes are governed by the allelic A1 and A2 genes. The observed frequencies of blood types in Caucasians are compatible to this model. However, the subtype A2 character is far more frequently observed in AB red cells than in A red cells in some black and Oriental populations. Two black blood samples with phenotype A2B contained A1-enzyme, but not A2-enzyme, and exhibited several times higher B-enzyme activity than control AB and B blood. The kinetic properties, that is, pH-activity profile and Km for UDP-Gal, of the B-enzyme from these two A2B subjects differed from that of control B-enzyme. In these two cases, therefore, the A2 character was not caused by the subactive A2-enzyme, but because of an insufficient formation of the A-substances in red cell membranes presumably caused by the competition between the A1-enzyme and the super active atypical B-enzyme at the common H-sites. The results suggest that the B gene can be subdivided into usual B1 and atypical B2, and that not only A2B subjects but also A1B2 subjects could express A2 character in their red cells. The B2 gene may be common in certain black and Oriental populations.     

Ubiquinone at center N is responsible for triphasic reduction of cytochrome b in the cytochrome bc(1) complex.

We have examined the pre-steady state reduction kinetics of the Saccharomyces cerevisiae cytochrome bc(1) complex by menaquinol in the presence and absence of endogenous ubiquinone to elucidate the mechanism of triphasic cytochrome b reduction. With cytochrome bc(1) complex from wild type yeast, cytochrome b reduction was triphasic, consisting of a rapid partial reduction phase, an apparent partial reoxidation phase, and a slow rereduction phase. Absorbance spectra taken by rapid scanning spectroscopy at 1-ms intervals before, during, and after the apparent reoxidation phase showed that this was caused by a bona fide reoxidation of cytochrome b and not by any negative spectral contribution from cytochrome c(1). With cytochrome bc(1) complex from a yeast mutant that cannot synthesize ubiquinone, cytochrome b reduction by either menaquinol or ubiquinol was rapid and monophasic. Addition of ubiquinone restored triphasic cytochrome b reduction, and the duration of the reoxidation phase increased as the ubiquinone concentration increased. When reduction of the cytochrome bc(1) complex through center P was blocked, cytochrome b reduction through center N was biphasic and was slowed by the addition of exogenous ubiquinone. These results show that ubiquinone residing at center N in the oxidized cytochrome bc(1) complex is responsible for the triphasic reduction of cytochrome b.     

Pathway and regulation of erythritol formation in Leuconostoc oenos.

It was recently observed that Leuconostoc oenos GM, a wine lactic acid bacterium, produced erythritol anaerobically from glucose but not from fructose or ribose and that this production was almost absent in the presence of O2. In this study, the pathway of formation of erythritol from glucose in L. oenos was shown to involve the isomerization of glucose 6-phosphate to fructose 6-phosphate by a phosphoglucose isomerase, the cleavage of fructose 6-phosphate by a phosphoketolase, the reduction of erythrose 4-phosphate by an erythritol 4-phosphate dehydrogenase and, finally, the hydrolysis of erythritol 4-phosphate to erythritol by a phosphatase. Fructose 6-phosphate phosphoketolase was copurified with xylulose 5-phosphate phosphoketolase, and the activity of the latter was competitively inhibited by fructose 6-phosphate, with a Ki of 26 mM, corresponding to the Km of fructose 6-phosphate phosphoketolase (22 mM). These results suggest that the two phosphoketolase activities are borne by a single enzyme. Extracts of L. oenos were also found to contain NAD(P)H oxidase, which must be largely responsible for the reoxidation of NADPH and NADH in cells incubated in the presence of O2. In cells incubated with glucose, the concentrations of glucose 6-phosphate and of fructose 6-phosphate were higher in the absence of O2 than in its presence, explaining the stimulation by anaerobiosis of erythritol production. The increase in the hexose 6-phosphate concentration is presumably the result of a functional inhibition of glucose 6-phosphate dehydrogenase because of a reduction in the availability of NADP.     

Serratia marcescens-Lytic Enzyme Produced by Micromonospora sp. Strain No. 152

A strain of Micromonospora sp. producing a lytic enzyme toward Serratia marcescens was isolated from soil. The lytic enzyme, called 152-enzyme, was purified from the culture filtrate by salting-out with ammonium sulfate, DEAE-cellulose column chromatography, and gel filtration on Sephadex G-75. The molecular weight of 152-enzyme was 17,000 and the isoelectric point was pH 7.3. The 152-enzyme showed lytic activity toward S. marcescens, Pseudomonas aeruginosa, Proteus vulgaris, Escherichia coli, and Bacillus subtilis, but was completely intert toward Staphylococcus aureus. The enzyme also showed caseinolytic activity. The lytic and caseinolytic activities of 152-enzyme were maximum around pH 11.0 and at 60°C. Both activities were inhibited by DFP and API-2c. Liberation of amino groups from cell walls of P. aeruginosa by incubation with 152-enzyme suggested that the enzyme was a kind of cell wall-lytic peptidase.