Physicochemical Properties and Amino Acid Composition of Alkaline Proteinase from Aspergillus sojae

Some physicochemical properties and amino acid composition of alkaline proteinase from Aspergillus sojae were found to be as follows: The isoelectric point was at pH 5.1. The molecular weight was 25,500 using the Sheraga-Mandelkern’s formula, based upon the values of the sedimentation coefficient $(s_{20,w}^{°}=\text{?}2.82\text{?}\text{S})$, the intrinsic viscosity ([η] = 0.027 dl/g), and the partial specific volume $({\displaystyle \overline{V}}\text{?}=\text{?}0.726\text{?}\text{ml}/\text{g})$. The enzyme contains 16.8% of nitrogen and is composed of 250 residues of amino acid; Asp₃₁ Glu₁₉, Gly₂₇, Ala₃₂, Val₁₈, Leu₁₄, Ile₁₄, Ser₂₈, Thr₁₈, ${\text{(}{\displaystyle \stackrel{}{{\displaystyle \stackrel{?}{\text{Cys C}}}}}\text{ys})}_1$, Met₂, Pro₆, Phe₇, Tyr₈, Trp₂, His₅, Lys₁₄, Arg₃, (amide-NH₃)₂₀.     

Studies on Fish-killing Components of Callicarpa candicans

A fish-killing component, which was named callicarpone, C₂₀H₂₈O₄, m.p. 111°C, ${[\alpha ]}_{\text{D}}^{{23}^{°}}$ ?180° (chloroform) was isolated from the leaf of Callicarpa candicans and was found to have conjugated carbonyl groups, double bond and one hydroxyl group. The toxicity to fish was evaluated to be as strong as rotenone and ten times stronger than sodium pentachlorophenoxide.

It was deduced from the spectral data of callicarpone (I), the tetraol (IV), the chlorohydrin (VII) and the anhydro-diacetyl derivative (II) that I was a tricarbocyclic diterpene having an ene-l,4-dione and an α-hydroxy-isopropyl attached to an oxide ring. The structure of the rearranged product obtained by treatment with sodium carbonate was established to be IX by converting it to 11-methoxy-ferruginol methyl ether. As this rearrangement was assumed to be initiated by proton abstraction by base, the structure I was required for callicarpone to explain the formation of IX.     

Sclerone,a New Metabolite of Sclerotinia sclerotiorum (LIB) DE BARY

Sclerone, mp 138~140°C, C₁₀H₁₀O₃ was isolated from the culture filtrate of Sclerotinia sclerotiorum. This is optically active, ${[\alpha ]}_D^{20}?30°$, and gave 1,5-naphthalenediol on pyrolysis. Oxidation with MnO₂ yielded juglone. From the chemical and physical evidences, the structure was determined to be 4,5-dihydroxy-(4S)-3,4-dihydro-1(2H)-naphthalenone.     

Analysis of Amylose-borate Interaction by Frontal Gel Filtration

Amylose-borate interaction has been analyzed by frontal gel chromatography, using the constituent velocity data alone. The constituent Velocity equation was reformulated in terms of elution volume for a type of interacting system described by$\text{A}+i\text{B}={\text{AB}}_i(i=1,2,3\dots n)$

Detailed examination of the binding data indicates that, in the complex formation between amylose and borate, this type of equilibria operates predominantly, if not solely. Use of the constituent elution volume equation enabled us, for the first time, to evaluate the association constant (K) and number of binding site pertaining to this system, i.e., K = 4.9 10² and n = 1. There was no evidence indicating the occurrence of the formation of inclusion complex.     

On the Treewidths of Graphs of Bounded Degree

In this paper, we develop a new technique to study the treewidth of graphs with bounded degree. We show that the treewidth of a graph G = (V, E) with maximum vertex degree d is at most (1−Ce−4.06d)|V| for sufficiently large d, where C is a constant.     

Kinetics of H2O2-driven catalysis by a lytic polysaccharide monooxygenase from the fungus Trichoderma reesei

Owing to their ability to break glycosidic bonds in recalcitrant crystalline polysaccharides such as cellulose, the catalysis effected by lytic polysaccharide monooxygenases (LPMOs) is of major interest. Kinetics of these reductant-dependent, monocopper enzymes is complicated by the insoluble nature of the cellulose substrate and parallel, enzyme-dependent, and enzyme-independent side reactions between the reductant and oxygen-containing cosubstrates. Here, we provide kinetic characterization of cellulose peroxygenase (oxidative cleavage of glycosidic bonds in cellulose) and reductant peroxidase (oxidation of the reductant) activities of the LPMO TrAA9A of the cellulose-degrading model fungus Trichoderma reesei. The catalytic efficiency (kcat/Km(H2O2)) of the cellulose peroxygenase reaction (k_cat = 8.5 s⁻¹, and Km(H2O2)=30μM) was an order of magnitude higher than that of the reductant (ascorbic acid) peroxidase reaction. The turnover of H₂O₂ in the ascorbic acid peroxidase reaction followed the ping-pong mechanism and led to irreversible inactivation of the enzyme with a probability of 0.0072. Using theoretical analysis, we suggest a relationship between the half-life of LPMO, the values of kinetic parameters, and the concentrations of the reactants.     

On the Adjacent Eccentric Distance Sum Index of Graphs

For a given graph G, ε(v) and deg(v) denote the eccentricity and the degree of the vertex v in G, respectively. The adjacent eccentric distance sum index of a graph G is defined as ξsv(G)=∑v∈V(G)ε(v)D(v)deg(v), where D(v)=∑u∈V(G)d(u,v) is the sum of all distances from the vertex v. In this paper we derive some bounds for the adjacent eccentric distance sum index in terms of some graph parameters, such as independence number, covering number, vertex connectivity, chromatic number, diameter and some other graph topological indices.     

New chemistry based on reduction of homobinuclear bis(μ-phosphido) metal complexes

Studies are reported on the chemical reduction of the homobinuclear bis(μ-phosphido) metal complexes ${(CO)}_3\stackrel{︹}{Fe{(\mu -P{R}_2)}_{2}F}e{(CO)}_3$ (R = Ph or Me), ${(NO)}_2-\stackrel{︹}{Fe{(\mu -PP{h}_2)}_{2}F}e{(NO)}_2$ and ${(CO)}_4\stackrel{︹}{M{(\mu -PP{h}_2)}_{2}M}{(CO)}_4$ (M = Mo or W). Two reduction pathways have been observed which result in different two-electron transformations: (1) with Na or LiAlH₄, electron transfer to yield the corresponding symmetric dianions of the type L_nM(μ-PR₂)₂ML_n^2? without metalmetal bond and (2) with M′BR′₃H(M′ = Li, Na, or K; R′ = Et or sec-Bu), hydride transfer to give monoanionic complexes of the type $L_n\stackrel{︹}{M(\mu -P{R}_2)(\mu -L)M}{L}_{n?1}{(P{R}_{2}H)}^{?}$ or $L_n\stackrel{︹}{M(\mu -P{R}_2)M}{L}_n{(P{R}_{2}H)}^{?}$ (M = Fe, Mo, or W; L = CO or NO; R = Ph or Me). The monoanionic complexes can be deprotonated with n-BuLi at ?78 °C to the corresponding unsymmetric dianions $L_n\stackrel{︹}{M(\mu -P{R}_2)(\mu -L)M}{L}_{n?1}{(P{R}_2)}^{2?}$ (M = Fe; L = CO or NO; R = Ph) or symmetric dianions L_nM(μ-PR₂)₂ML_n^2? (M = Mo or W; L = CO; R = Ph). The unsymmetric dianions isomerize on slight warming to the symmetric dianions, which undergo protonation by CF₃COOH to yield the aforementioned monoanions. Reactions of several members of these three classes of binuclear anions with CF₃COOH, alkylating reagents, 1,1-diiodohydrocarbons and metal diiodo complexes have resulted in the synthesis of new binuclear and trinuclear compounds. Examples include ${(CO)}_3(H)\stackrel{︹}{Fe(\mu -PP{h}_2)Fe}{(CO)}_3(PP{H}_{2}H)$, ${(CO)}_3\stackrel{︹}{Fe(\mu -PP{h}_2)(\mu -C(R)O)Fe}{(CO)}_2(PP{h}_{2}R)$ (R = Me, Et, n-Pr, or i-Pr), ${(CO)}_4\stackrel{︹}{M{(\mu -PP{h}_2)}_{2}M}{(CO)}_3(C(R)Ome)$ (M = Mo or W; R = Me or Ph), ${(CO)}_2({\eta }^3?C_3{H}_5)\stackrel{︹}{Fe(\mu ?PP{h}_2)?F}e{(CO)}_3(PP{h}_2{C}_3{H}_5)$, ${(CO)}_4\stackrel{︹}{M{(\mu ?PP{h}_2)}_{2}M}{(CO)}_3(C(R)Ome)$, ${(NO)}_2\stackrel{︹}{Fe(\mu ?C{H}_2)(\mu ?P{h}_{2}PPP{h}_2)Fe}{(NO)}_2$, and Fe₂Co(η⁵-C₅H₅)(CO)(NO)₄(μ-PPh₂)₂. Synthetic and mechanistic studies on these reactions are presented.     

Reactive Nitrogen Species-Dependent Effects on Soybean Chloroplasts

Nitric oxide (NO) generation by soybean (Glycine max, var ADM 4800) chloroplasts was studied by electron paramagnetic resonance (EPR) spin-trapping technique.¹ Both nitrite and L-arginine (arg) are the required substrates for enzymatic activities considered as possible sources of NO in plants. Soybean chloroplasts showed a NO production of 3.2 ± 0.2 nmol min⁻¹ mg⁻¹ protein in the presence of 1 mM NaNO₂. Chloroplasts incubated with 1 mM arg showed a NO production of 0.76 ± 0.04 nmol min⁻¹ mg⁻¹ protein. This production was inhibited when chloroplasts were incubated in presence of NOS-inhibitors L-NAME and L-NNA. In vitro exposure of chloroplasts to a NO-donor (GSNO) decreased both ascorbyl radical content and the activity of ascorbate peroxidase, without modification of the total ascorbate content. Exposure of the isolated chloroplasts to a NO-donor decreased lipid radical content in membranes, however, incubation in the presence of 25 µM peroxynitrite (ONOO⁻) led to an increase in lipid-derived radicals (34%). The effect of ONOO⁻ on protein oxidation was determined by western blotting, showing an increase in carbonyl content either in stroma or thylakoid proteins as compared to control. Taken as a whole, NO seems to be an endogenous metabolite in soybean chloroplasts and reactive nitrogen species could exert either antioxidant or prooxidant effects on chloroplasts, since both a decreased lipid radical content in membranes and a decrease in the activity of ascorbate peroxidase were observed after exposure to a NO donor.Key Words: ascorbate, ascorbate peroxidase, chloroplasts, nitric oxide, peroxynitriteThe origin of nitric oxide (NO) in plants under aerobic conditions is currently under study. Although plants with low level of arginine shows arg-stimulated NO accumulation,² the mechanism for arginine-dependent NO synthesis in plants is still unknown, because the detection of an animal-type NOS remains elusive to date.^3,4 Even though assimilatory nitrate reductase is an enzymatic source of NO, its role in vivo would be limited by both its cytosolic localization which difficult the availability for nitrite, and the relative high K_m for nitrite (100 µM).⁵Chloroplasts have been previously marked as NO sources based in nonquantitative studies employing fluorescence microscopy^6,7 and immunogold electron microscopy.⁸ In our work we employed an specific technique (EPR, electron paramagnetic resonance with spin trap⁹) to detect NO as an endogenous metabolite and to quantify its generation in the presence of different substrates. In order to gain insight on the mechanism leading to NO production both nitrite-dependent and arg-dependent pathways were evaluated. In the presence of 1 mM arg and 0.1 mM NADPH the rate of NO generation was 0.76 ± 0.04 nmol min⁻¹ mg⁻¹ prot (arg-dependent synthesis). The synthesis of NO resulted completely blocked in the presence of arg analogs (L-NAME and L-NNA). It is important to point out that the content of arg in the chloroplasts stroma is high as compared to the content of other amino acids (56.7 ± 0.8 nmol mg⁻¹ prot), suggesting that this pathway could be operative under physiological conditions.Soybean chloroplasts showed a NO production of 3.2 ± 0.2 nmol min⁻¹ mg⁻¹ prot in the presence of 1 mM NaNO₂. Furthermore, NO generation was detected in the presence of nitrite concentrations as low as 25 mM. Since nitrite-dependent NO generation resulted inhibited by 50% by the addition of DCMU, and no NO generation was measured in the stroma fraction, thylakoidal electron transport seems to be a key feature in NO synthesis.According to this scenario and assuming that the two independent pathways for NO generation in chloroplasts are operative, the total rate of production of NO could be understood as the generation by the activity of an arg-dependent enzyme and by a NO₂⁻ dependent pathway, as indicated by eq. 1.d[NO]dt=(d[NO]dt)NOS like+(d[NO]dt)NO2−(1)Regarding the NO disappearance, from a kinetic point of view, the rate of the reaction of NO with O₂⁻ to generate peroxynitrite seems to be the main pathway, since the reaction is diffusionally controlled. Thus, the rate of disappearance of NO could be estimated from the rate of generation of ONOO⁻ (eq. 2); however, other reactions should be considered under nonphysiological conditions.−d[NO]dt=d⌊ONOO−⌋dt=k[NO][O2−](2)NO generation rate should be equal to NO consumption rate in order to keep a physiological NO steady state concentration (eq. 3)d[NO]dt=−d[NO]dt(3)Thus, replacing NO generation and disappearance rates by those rates indicated in equations 1 and 2, (d[NO]dt)NOS like+(d[NO]dt)NO2−=k[NO][O2−](4) The data obtained under unrestricted availability of substrates, indicate a generation rate of NO by the activity of a NOS-like enzyme of 13 × 10⁻⁹ M s⁻¹. Chloroplastic NO generation rate in the presence of 100 µM NO₂⁻ was 14 × 10⁻⁹ M s⁻¹. Thus, according to equation 1, the rate of generation of NO is approximately 3 × 10⁻⁸ M s⁻¹. Assuming a steady state concentration for O₂⁻ of 1 nM in chloroplasts¹⁰ and a rate constant (k) of 6.9 × 10⁹ M⁻¹ s⁻¹ for the reaction between O₂⁻ and NO,¹¹ a steady state concentration of 4 nM for NO in the chloroplast could be estimated. Since under in vivo conditions chloroplasts may content the required substrates for the NO synthesis, the assays presented here strongly suggest that a feasible NO production could take place inside the chloroplasts. However, nonsupplemented chloroplasts did not show any NO-dependent EPR signal. This observation agrees with the fact that NO steady state concentration under physiological conditions as was calculated here (4 nM) is below the EPR detection limit (500 nM).¹²Further studies should be performed to characterize NO oxidative effects on chloroplasts. Scavenging of O₂⁻ and H₂O₂ is essential for chloroplasts to maintain their ability to fix CO₂ since several enzymes in the CO₂-reduction cycle are sensitive to active oxygen species.¹³ These organelles lacking catalase, contain a significant peroxidase activity.¹⁴ H₂O₂-reduction catalized by ascorbate peroxidase (AP) lead to ascorbate oxidation and produces ascorbyl radical (A^.).¹⁵ In isolated chloroplast the content of A^.. was evaluated in DMSO based extract by EPR¹⁶. Quantification of EPR signals indicated that A^. content in control chloroplasts (123 ± 5 pmol mg⁻¹ prot) decreased after exposure to NO (Fig. 1). The total content of ascorbate, assessed by an HPLC technique¹⁷ in chloroplasts isolated from soybean leaves exposed to NO was not significantly different from the measured content in chloroplasts not exposed to the NO donor (Fig. 1). The activity of AP was significantly decreased by 48, 53 and 54% after exposure of the chloroplasts to NO-donor. Previous data suggested that AP could be inactivated by NO via oxidation of functional thiols.¹⁸ Besides, the reversible inhibition of AP could be due to the formation of Fe-nitrosyl complexes between NO and the Fe atom of the heme group, as it was previously described for NO-mediated activation of guanylate cyclase and the inhibition of cytochrome P₄₅₀ and catalase in mammals.¹⁹ The data presented here showed that in isolated chloroplasts exposed to a NO donor, there could be either a limited damage associated to the decrease in the content of A^.. or an increased cellular deterioration by the decrease in the activity of the enzyme responsible for the scavenging of H₂O₂.Open in a separate windowFigure 1Ascorbate metabolism in soybean chloroplasts after NO exposure. A^.. content (▪), ascorbate content (▪), and AP activity (*) as a function of the exposure of isolated chloroplasts to GSNO in the presence of 50 µM DTT. * = significantly different at p ≥ 0.05 from the value obtained in the absence of GSNO + 50 µM DTT.Thus, in situ generation of NO could play a protective role in preventing the oxidation of chloroplastic lipids; however, the reaction of NO with O₂⁻ leading to ONOO⁻ production may result in a potential source of damage or as it is shown here by the significant decrease of the AP activity that consumes H₂O₂. NO is a suitable candidate to modulate cellular H₂O₂ level through the chloroplast function, as an initial step to regulate complex metabolic pathways directed to activate physiological responses, defense pathways or deleterious effects in the cytosol. Furthermore, an integrated study on the effect of nitrogen reactive species is required under stress conditions to characterize the metabolic pathways involved in the resulting cellular damage.