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.     

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.     

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.     

Protein crystal’s shape and polymorphism prediction within the limits resulting from the exploration of the Miyazawa–Jernigan matrix

A computer study of the prediction of the protein crystal’s shape and polymorphism of crystal’s structures within the limits resulting from the exploration of the Miyazawa–Jernigan matrix is presented. In this study, a coarse-graining procedure was applied to prepare a two-dimensional growth unit, where instead of full atom representation of the protein a two-type (hydrophobic–hydrophilic, HP) aminoacidal representation was used. The interaction energies between hydrophobic ($E_{\text{HH}}$) aminoacids were chosen from the well-known HP-type models ($E_{\text{HH}}\in [-4\text{,}-3\text{,}-2.3\text{,}-1]$), whereas interaction energies between hydrophobic and hydrophilic aminoacids ($E_{\text{HP}}$) as well as interaction energies between hydrophilic aminoacids ($E_{\text{PP}}$) were chosen from the range: $<-1\text{,}1>$, but not all values from this range fulfiled limitations resulting from the exploration of the Miyazawa–Jernigan matrix. Exploring every positively vetted combinations of energy interactions a polymorphism of the unit cell was observed what led to the fact that different final crystal’s shapes were obtained.     

The effects of fasting on swimming performance in juvenile qingbo (Spinibarbus sinensis) at two temperatures

We measured the following variables to investigate the effects of fasting and temperature on swimming performance in juvenile qingbo (Spinibarbus sinensis): the critical swimming speed (U_crit), resting metabolic rate ${(\dot{\mathrm{M}}O}_{\mathrm{2rest}})$ and active metabolic rate ${(\dot{\mathrm{M}}O}_{2active})$ of fish fasting for 0 (control), 1, 2 and 4 weeks at low and high acclimation temperatures (15 and 25 °C). Both fasting treatment and temperature acclimation had significant effects on all parameters measured (P<0.05). Fasting at the higher temperature had a negative effect on all measured parameters after 1 week (P<0.05). However, when acclimated to the lower temperature, fasting had a negative effect on U_crit until week 2 and on ${(\dot{\mathrm{M}}O}_{\mathrm{2rest}})$, ${(\dot{\mathrm{M}}O}_{\mathrm{2active}})$ and metabolic scope (MS, ${(\dot{\mathrm{M}}O}_{\mathrm{2active}})$–${(\dot{\mathrm{M}}O}_{\mathrm{2rest}})$) until week 4 (P<0.05). The values of all parameters at the lower temperature were significantly lower than those at the higher temperature in the identical fasting period groups except for ${(\dot{\mathrm{M}}O}_{\mathrm{2rest}})$ of the fish that fasted for 2 weeks. The relationship between fasting time (T) and U_crit was described as U_crit(15)=−0.302T²−0.800T+35.877 (r=0.781, n=32, P<0.001) and U_crit(25)=0.471T²−3.781T+50.097 (r=0.766, n=32, P<0.001₎ at 15 and 25 °C, respectively. The swimming performance showed less decrease in the early stage of fasting but more decrease in the later stage at the low temperature compared to the high temperature, which might be related to thermal acclimation time, resting metabolism, respiratory capacity, energy stores, enzyme activity in muscle tissue and energy substrate utilization changes with fasting between low and high temperatures. The divergent response of the swimming performance to fasting in qingbo at different temperatures might be an adaptive strategy to seasonal temperature and food resource variation in their habitat.     

The effect of encapsulated fine grain sediment and test morphology on the resistance of planktonic foraminifera to dissolution

The tests of planktonic foraminifera recovered from deep-sea sediment are commonly observed to encapsulate fine grain carbonate sediment within their chambers. In sediment below the lysocline, the interstitial water within the chambers may not be as corrosive as the seawater in contact with the outer surface of the test due to slow continuous dissolution of the encapsulated carbonate. As a consequence, the pore walls of the foram dissolve more slowly than the outer surface of the test.Using published dissolution rate measurements for foraminifera and deep-sea sediment, the effect of diffusional reduction of pore wall dissolution was quantitatively estimated with a one-dimensional model for the steady state condition where diffusional flux out of the foraminifer's pores is balanced by the dissolution flux from the encapsulated fines and pore walls. The diffusional effect is found to principally depend on the structural parametric ratioT_wd_T/f, whereT_w is the wall thickness,d_T the test diameter andf is the test porosity. In the case of adult planktonic foraminifera, the ratio of the pore wall to outer surface dissolution flux is predicted to vary between 60% for the thin-walled porous species and 30% for thick-walled tests.Incorporation of the predicted pore to outer surface flux ratios into the morphologic index equation of Adelseck (1978) results in a very good prediction (73% of the variation) of the solution index of Berger (1975) obtained from ranking species counts from core tops. A simple empirical equation which may be useful for prediction of the resistance of extinct microfossils was found as follows:

$R=\frac{T_w}{\lambda \{1+[A_w/(A_s?A_p)][.74?.27log?(T_w{d}_T/f)]\}}$

is the measured ratio of pore wall area to outer surface of the test, andT_wd_T/f is in units of 10⁴ μm².     

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.