Purification and properties of purine hydroxylase II from Aspergillus nidulans

Purine hydroxylase II from Aspergillus nidulans has been purified to near homogeneity. The enzyme has a pI of 5.7, a molecular weight of 300,000, and two subunits with molecular weight of 153,000 each. The enzyme contains 2 FAD, 2 molybdenum atoms, and 4 (2 Fe-2S) iron-sulfur centers per molecule and exhibits broad specificity for reducing and oxidizing substrates. Among the more notable characteristics are the ability to oxidize hypoxanthine and nicotinic acid but not xanthine and virtually complete inactivity with oxygen. Moreover, while the enzyme is inactivated by borate and methanol, it is very resistant to cyanide and arsenite and it not inactivated by allopurinol. At infinite concentrations of reducing and oxidizing substrates, the Km for hypoxanthine was 119 microM, for nicotinic acid was 136 microM, and for NAD+ was 525 microM.     

Optical and electron paramagnetic resonance spectroscopic studies on purine hydroxylase II from Aspergillus nidulans

Purine hydroxylase II from Aspergillus nidulans contains a molybdenum cofactor very similar to that found in a number of other molybdenum-containing hydroxylases. (A. nidulans contains two purine hydroxylases, I and II, related to each other by possession of a common cofactor and overlapping substrate specificity.) Addition of reducing substrates effects bleaching of the visible absorption spectrum of the enzyme, the decrease in absorbance at 450 nm being linearly proportional to that at 550 nm. No increase in absorption at longer wavelengths was observed during such titrations. Electron paramagnetic resonance studies of reduced samples of native and modified enzyme species showed the presence of a number of Mo(V) signals (gav = 1.97), exhibiting H hyperfine coupling, comparable to those in the corresponding enzymes from other sources. The enzyme possesses two non-heme-iron-sulfur centers, one (Fe2S2)I with gav less than 2.0 and the other (Fe2S2)II with gav greater than 2.0. The flavin radical signal observed at pH 7.8 had a linewidth of 1.5 mT, indicating it to be the anionic form FAD- . In this respect purine hydroxylase II is unique among all molybdenum-containing hydroxylases studied to date.     

Some properties of a deoxyribonuclease isolated from Aspergillus niger

A deoxyribonuclease, isolated from the mycelia of Aspergillus niger, grown as surface cultures on a liquid medium, was partially purified and had an optimum pH of 9.5 and an optimum temperature of 37°C. The enzyme was strongly activated by Mg²⁺ while Mn²⁺, Ca²⁺ or Co²⁺ activated the enzyme to lesser extents. Thiol reagents, mercaptoethanol and dithiothreitol (DTT) activated the enzyme. S-Adenosylmethionine at low concentration (2.8?5.0 × 10^?2 mM) activated the enzyme but at a higher concentration (11.5 × 10^?2 mM) and above it inhibited the enzyme. The effect of EDTA on the enzyme was variable. The enzyme had both ATP-dependent and independent activities, the former usually being higher. ATP could be replaced by CTP or GTP. The nucleoside diphosphates ADP, CDP and GDP could replace ATP but they were not as effective as the triphosphates.     

RNase-sensitive glucocorticoid-receptor complexes from HELA cell nuclei

Dexamethasone-receptor complexes can be extracted from HeLa cell nuclei by mild sonication. These complexes can account for 800-1000 binding sites/cell, and are indistinguishable from salt-extractable ones as judged by sucrose gradient centrifugation in the presence of 0.3 M KCl, showing a sedimentation coefficient of 3.6 S. These complexes, however, broadly sediment in the 7 to 3.6 S region of low salt sucrose gradients. Enzymatic treatment of soluble extracts from nuclear sonicates shows that RNA is associated to dexamethasone-receptor complexes.     

Immobilization of proteins on partially hydrolyzed agarose beads

Treatment of agarose beads with mild acid (0.2 M HCl, 55 degrees C, several hours) hydrolyzes some of the glycosidic bonds between D-galactosyl residues and 3,6-anhydro-L-galactosyl residues, and thus produces aldehydo-groups useful for immobilization of amino compounds by reductive amination with NaCNBH3. More than 20 mg (0.3 mumol) of bovine serum albumin could be coupled per gram of partially hydrolyzed agarose beads. Arthrobacter neuraminidase immobilized by this method was useful for desialylation of sialyl glycoconjugates, and was found not to leach from the gel and to be much more thermostable than the free enzyme.     

Immobilization of Clostridium thermocellum cells on bituminous coal particles

The immobilization isotherms of Clostridium thermocellum cells on bituminous coal particles of approximately 0.15- to 0.18-mm diameter were experimentally measured at 60, 45, and 30 degrees C with a pH value of 7.0 and with pH values of 6.0 and 5.0 at 60 degrees C. The immobilization data were correlated into Langmuir forms and their characteristic coefficients were obtained. A method to obtain thermodynamic quantities delta G, delta H, and delta S for the immobilization is also demonstrated.     

Enzymatic transfer of mannose from guanosine diphosphate mannose to yeast mannan-protein complexes

The radioactive products derived from transfer of [¹⁴C]mannose residues from GDP-[¹⁴C]mannose to endogenous acceptors of a Hansenula holstii particulate enzyme preparation have been solubilized by Pronase digestion. From this soluble mixture, glycopeptides containing [¹⁴C]mannose have been purified and have been shown by β-elimination-reduction experiments to contain radioactive mannose and oligosaccharides of mannose linked to serine and threonine residues. Radioactive macromolecular complexes of mannan-protein were extracted from the particulate enzyme fraction with hot, neutral citrate buffer. These components contained variable quantities of protein, mannose, and phosphate. The more neutral components were reduced in size by Pronase digestion and yielded glycopeptides similar to those obtained by direct Pronase digestion of the particulate fraction.     

Synthesis and characterization of tertiary phosphine Pd(0) complexes

The synthesis of PdL_n complexes (L = tertiary phosphine; n = 2,3,4) is reported. The coordination number results to be a function of the steric hindrance of the phosphine. Two-coordinate, 14-electron complexes, have been isolated with bulky phosphines (PPrⁱ₃, P (cyclohexyl)₃, PBu^t₂Ph). The behaviour in solution of the PdL_n complexes has been studied by ¹³C NMR spectroscopy.     

Interactions between polynucleotides and platinum (II) complexes

Reaction of either $\text{cis}$ or $\text{trans}$ Pt(NH₃)₂Cl₂ with poly(A) in dilute aqueous solution leads to quantitative precipitation of the polymer at Pt/nucleotide ratios above 0.5. It is proposed that at ratios less than this, intramolecular binding of one Pt to two bases is favored; at higher ratios, intermolecular cross-linking becomes important and precipitation results. The absence of isomer selectivity in precipitation implies that the biological specificity of the $\text{cis}$ form results from a process other than cross-linking of polynucleotide strands. Other observations suggest that the coordinated ammonia of nucleotide-platinum(II) ammine complexes may be unusually labile.     

Proton chemical shifts in fluorocinnamate-chymotrypsin complexes

Binding of cinnamate or fluorocinnamate anions to α-chymotrypsin is accompanied by chemical shift changes at each proton of the cinnamate structure. The direction and magnitude of these shifts are consistent with the expected binding of these inhibitors at the active site of the enzyme. The protein-induced fluorine chemical shift effects at each position in the aromatic ring are compared to the shift effects observed when a hydrogen occupies the same position. There is no discernible relation between the proton and fluorine chemical shifts, leading to the conclusion that those factors dominantly responsible for the shift effects are different for the two sets of data.     

Stoichiometries of electron transport complexes in spinach chloroplasts

The stoichiometric relationship among photosystem II complexes, photosystem I complexes, cytochrome b/f complexes, high-potential cytochrome b-559, and chlorophyll in spinach chloroplasts has been determined. Two features of this data stand out, in contrast to currently proposed stoichiometries in which the ratio of photosystem II to photosystem I is reported to be 2:1 and the chlorophyll to reaction center ratio to be as low as 260:1. Using a variety of techniques it was found that the stoichiometry of photosystem II:photosystem I:cytochrome b/f complex was 1:1:1, within 10%, and that the ratio of total chlorophyll to these components was 600:1, also within 10%. A ratio of two high-potential cytochrome b-559 molecules per 640 chlorophyll, or two molecules per photosystem II reaction center, was found. These ratios were remarkably constant regardless of the time of year or the source of the spinach. The concentration of photosystem II complexes was determined using a pH electrode to measure the flash-induced proton release resulting from water oxidation. The photosystem I reaction center concentration was measured by two different techniques that compared favorably. In the first method a pH electrode was used to measure the amount of flash-induced proton consumption associated with the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive oxidation of N,N,N',N'- tetramethylphenylenediamine , resulting in the production of hydrogen peroxide. In the second method the amount of P700 oxidized by far-red light was determined using dual-wavelength spectroscopy. The concentration of the cytochrome b/f complex was determined assuming 1 mol of cytochrome f per complex. The concentration of cytochrome f was measured spectroscopically by its light-induced turnover and by chemical difference spectra. The concentration of high-potential cytochrome b-559 was determined by chemical difference spectra. In addition to these studies, the light-induced absorbance change exhibiting a peak at 323 nm that has been attributed to the reduction of the primary quinone acceptor of photosystem II has been investigated. This measurement frequently has been used to quantitate the photosystem II to chlorophyll ratio. However, in view of these results it is argued that this technique significantly overestimates the photosystem II concentration.     

Multiple forms of chlorophyll-protein complexes from a thermophilic cyanobacterium Synechococcus sp

Eight chlorophyll-proteins were resolved from the thylakoid membranes, or digitonin particles, of a thermophilic cyanobacterium Synechococcus sp. by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Six chlorophyll-proteins with slower electrophoretic mobilities were shown to be P700-chlorophyll a-protein complexes (CP1), whereas faster-moving proteins (CP2) were related to photosystem 2. Extraction of CP1 complexes from the membranes with different detergent/chlorophyll ratios and reelectrophoresis of extracted CP1 complexes indicated that the chlorophyll-proteins are closely interrelated with each other; any CP1 complex could be transformed to other CP1 complexes with faster electrophoretic mobilities. This, together with the Ferguson plot and the polypeptide composition, showed that six CP1 complexes are different in terms of polypeptide composition, oligomerization, SDS-binding, or conformation of the proteins but represent, in the order of increasing electrophoretic mobility, increasing degree of modification of the native P700-chlorophyll a-protein.     

Cellulase and ethanol production from cellulose by Neurospora crassa

Two strains of Neurospora crassa have been identified which utilize cellulase and produce extracellular cellulase [see 1,4-(1,3; 1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and β-d-glucosidase [β-d-glucoside glucohydrolase, EC 3.2.1.21]. The activities were detected as early as 48 h in the culture broth. These cultures also fermented d-glucose, d-xylose and cellulosic materials to ethanol as the major product of fermentation. The conversion of cellulose to ethanol was >60%, indicating the potential of using Neurospora for the direct conversion of cellulose to ethanol.     

Colorimetric determination of agarose-immobilized proteins by formation of copper-protein complexes

A simple, reliable, and sensitive colorimetric procedure for the determination of proteins bound to agarose is described. The procedure utilizes the capacity of diethyldithiocarbamate to react with cupric ions resulting in a complex of dark yellow color. The extent of reduction in the color, due to chelation of Cu2+ by the immobilized proteins, indicates the amount of protein. The optimum conditions for the determination of immobilized proteins are investigated. The formation of Cu2+-protein complex proceeds stepwise until enough excess of Cu2+ is present to form the final complex. The reliability of the procedure requires that all the protein species, samples and standards, are in the final Cu2+-protein complex form. A comparative study on the determination of different proteins bound to agarose using this method as well as other methods, such as protein balance, modified Lowry reaction, and direct ultraviolet spectrophotometry, is described. The method is substantially more reliable, accurate, and simple than previously described methods.     

Inhibition of vitamin K-dependent carboxylase by metal ions and metal complexes: a reassessment

The vitamin K-dependent enzymatic carboxylation of glutamyl residues in blood protein precursors and in synthetic peptides is inhibited in vitro by transition metal complexes. Some authors suggested it is a result of metal ions interaction with intermediary oxygenated species. Using an oxygraph we have observed increases in the rate of oxygen utilization in the carboxylating system containing reduced vitamin K after addition of some transition metal ions and complexes. Kinetic studies indicate that, although oxygen utilization is increased by the addition of Cu2+, Fe3+, and hematin, the initial rate of carboxylation is not affected. The rate of carboxylation rapidly decreases at oxygen concentrations below 50 microM and reaches zero when oxygen is depleted. UV spectroscopy revealed simultaneous acceleration of the conversion of vitamin K hydroquinone into the parent quinone. The magnitude of these effects, as well as carboxylation inhibition, depends on the oxidation potential of the complexed ion and its lipophilicity. Addition of stable Mn parallel ion, which has no inhibitory effect on carboxylation, does not increase the rate of oxygen utilization nor the hydroquinone oxidation. The results suggest that inhibition of carboxylation by transition metals is mainly due to depletion of the necessary components (oxygen, vitamin K hydroquinone) of the carboxylating system rather than quenching of activated, oxygen-containing intermediates.     

Immunological and circular dichroism studies of maleylated f-1 (A) histone and complexes with DNA

Complexes of calf thymus f-1 (A) histone and homologous DNA were examined by circular dichroism. The maleylation of f-1 (A) produces a polypeptide with decreased ability to modify the circular dichroism spectrum of f-1 (A)-DNA complexes. By the introduction of two to three maleyl groups per f-1 (A) molecule, the alteration of the DNA CD spectrum is reduced by nearly half compared to that induced by the native nonmaleylated f-1 (A). Similarly maleylation reduces the serological reactivity of the histone, i.e., the reaction of the maleylated f-1 (A) with specific complement fixing f-1 (A) antibodies. On the other hand, moderate maleylation of f-1 (A) improves the cross-inhibition of the f-2b-anti-f-2b reaction by native f-1 (A) while extensively maleylated f-1 (A) is inert with respect to the same reaction. These results are interpreted in terms of possible conformational changes induced in f-1 (A) by maleylation, partially due to decreasing the histone net charge and perhaps as well as removal of specific site charges necessary for correct binding and interaction. Such an interpretation is consistent with the altered CD spectrum of maleylated f-1 (A) (i.e., a decreased and slightly red-shifted [θ]₁₉₈) and moreover explains why maleylation of two to three lysines per f-1 (A) molecule hinders simultaneously the very different DNA-histone and histone-complement fixing antibody interactions.     

Fluorine chemical shifts in complexes of sodium trifluoralkylsulfates with reduced proteins

Equilibrium dialysis experiments were carried out for several proteins, reduced with dithioerythritol, in aqueous buffer and the anionic surfactants, sodium 12,12,12-trifluorododecylsulfate or sodium 13,13,13-triflourotridecylsulfate, with surfactant concentrations above the critical micelle concentration. Fluorine chemical shifts were determined for each retentate and dialysate solution. The results show that most of the proteins bind 3.2 ± 0.4 millimoles of fluorinates surfactant per gram. In every case the chemical shift of the bound detergent ions is very near that found for micelles, suggesting that the bound ions form micelle-like aggregates.     

The scavenging of superoxide radical by manganous complexes: in vitro

Dialyzable manganese has been shown to be present in millimolar concentrations within cells of Lactobacillus plantarum and related lactic acid bacteria. This unusual accumulation of Mn appears to serve the same function as Superoxide dismutase (SOD), conferring hyperbaric oxygen and Superoxide tolerance on these SOD-free organisms. The form of the Mn in the lactic acid bacteria and the mechanisms whereby it protects the cell from oxygen damage are unknown. This report examines the mechanisms by which Mn catalytically scavenges O₂^?, both in the xanthine oxidase/cytochrome c SOD assay and in a number of in vitro systems relevant to the in vivo situation. In all the reaction mixtures examined, Mn(II) is first oxidized by O₂^? to Mn(III), and H₂O₂ is formed. In pyrophosphate buffer the Mn(III) thus formed is re-reduced to Mn(II) by a second O₂^?, making the reaction a true metal-catalyzed dismutation like that catalyzed by SOD. Alternatively, if the reaction takes place in orthophosphate or a number of other buffers, the Mn(III) is preferentially reduced largely by reductants other than O₂^?, such as thiols, urate, hydroquinone, or H₂O₂. H₂O₂, a common product of the lactic acid bacteria, reacted rapidly with Mn(III) to form O₂, apparently without intermediate O₂ release. Free hexaquo Mn(II) ions were shown by electron spin resonance spectroscopy and activity assays in noncomplexing buffers to be poorly reactive with O₂^?. In contrast, Mn(II) formed complexes having a high catalytic activity in scavenging O₂^? with a number of organic acids, including malate, pyruvate, propionate, succinate, and lactate, with the Mn-lactate complex showing the greatest activity.