Lag period for phototropism inPilobolus crystallinus sporangiophores

The lag period for the second positive curvature was examined inPilobolus crystallinus sporangiophores. The lag period for curvature development was 20–30 min at lower fluence rates than 6.32 nmol/m²s but greatly extended at higher fluence rates. When a 20-min symmetrical irradiation with blue light was applied before a 20-min unilateral blue light irradiation, sporangiophores bent as much as those unilaterally and continuously irradiated for 40 min. However, when a 20-min unilateral irradiation was followed by a 20-min symmetrical irradiation, sporangiophores did not show any curvature. That is, the reaction during the first 20 min of the lag period is independent of light direction. This light-direction-independent lag period is considered to be the duration required for adaptation. The lag period for phototropism was also extended when fluence rate was reduced after the start of irradiation. These results suggested that an adaptation process is involved in phototropism ofPilobolus.     

Specific Time Course of Peroxidase Oxidation in the Presence of SH-Containing Inhibitors. Comparison with the Inhibition of Polyphenoloxidase Activities

The time courses of the peroxidase reaction in presence of SH-containinginhibitors are very specific. After a lag period resulting froma transitory absence of reaction, the oxidation develops butits initial velocity is in some cases decreased. The durationof the lag period depends on the inhibitor concentration butcan also be modified by incubating the inhibitor with hydrogenperoxide or with the enzyme. With respect to the inhibitionof polyphenoloxidase activities, two kinds of action of SH-containingcompounds might be recognized. The absence of the reaction observedduring the first phase may be related to the chelation of Feions contained in the peroxidases, inducing then an inactivationof the enzymes. The beginning of the reaction, at the end ofthe lag period, might be due to the oxidation of the inhibitorSH-radicals by hydrogen peroxide or by some early oxidationproducts. The second action of the inhibitors might be due toa reduction of some early oxidation products such as quinoneswhich prevents the formation of the coloured polymer. The initialvelocity of the reaction is therefore lowered. This second phasedepends on the nature of the substrate of the peroxidase reaction. (Received February 10, 1984; Accepted August 24, 1984)     

De novo synthesis of poly(dAT) by the cell extract of a thermophile Bacillus stearothermophilus carrying a plasmid pTB913

Abstract In the absence of added template and primer, DNA synthesis activity which required dATP, dTTP, magnesium ion, and ATP was detected in the cell extracts prepared from a thermophile Bacillus stearothermophilus carrying a plasmid pTB913, but not from the strain without plasmid. Polymer synthesis was detectable only after a lag period and then proceeded at an exponential rate. The DNA synthesized in vitro was the alternating copolymer of dAMP and dTMP, poly(dAT). This reaction was very similar to the de novo DNA synthesis by DNA polymerase I of Escherichia coli, Bacillus subtilis , and Micrococcus luteus , except for the requirement of ATP and thermostability.     

Critical effect of ammonium ions on the enzymatic reaction of a novel transfructosylating enzyme for fructooligosaccharide production from sucrose

A crude transfructosylating enzyme from a new isolate Bacillus marcerans EG-7 required a lag period for catalytic activity during formation of fructooligosaccharides from sucrose. The lag period was markedly reduced by addition of various ammonium ions to the reaction mixture; 100 mM NH₄NO₃ enhanced the enzyme activity by 15 fold (at 40 h) reducing the lag-time from 25 h to 30 min. In contrast, partially purified enzyme completely removed the lag period while ammonium ions still accelerated the initial reaction rate.     

A kinetic study of the melanization pathway between L-tyrosine and dopachrome

In the pathway of melanin biosynthesis originating from L-tyrosine, the dopachrome accumulation at physiological pH is produced with a pronounced lag period, during which the level of L-dopa increases, following a sigmoidal kinetics to reach a steady-state. A kinetic model has been proposed for the overall pathway of melanization from L-tyrosine to dopachrome; it explains the lag period present during the dopachrome accumulation as well as the influence of L-tyrosine and tyrosinase over this lag period. Use of this model is also valid to explain the kinetics of L-dopa accumulation in the reaction medium, as has been tested by simulation.     

The action of o-dihydric phenols in the hydroxylation of p-coumaric acid by a phenolase from leaves of spinach beet (Beta vulgaris L.)

1. Under defined conditions, the hydroxylation of p-coumaric acid catalysed by a phenolase from leaves of spinach beet (Beta vulgaris L.) was observed to develop its maximum rate only after a lag period. 2. By decreasing the reaction rate with lower enzyme concentrations or by increasing it with higher concentrations of reductants, the length of the lag period was inversely related to the maximum rate subsequently developed. 3. Low concentrations of caffeic acid or other o-dihydric phenols abolished this lag period. With caffeic acid, the rate of hydroxylation was independent of the reductant employed. 4. Hydroxylation was inhibited by diethyldithiocarbamate, but with low inhibitor concentrations hydroxylation recovered after a lag period. This lag could again be abolished by the addition of high concentrations of caffeic acid or other o-dihydric phenols. 5. Catechol oxidase activity showed no lag period, and did not recover from diethyldithiocarbamate inhibition. 6. The purified enzyme contained 0.17-0.33% copper; preparations with the highest specific activity were found to have the highest copper content. 7. The results are interpreted to suggest that the oxidation of o-dihydric phenols converts the enzymic copper into a species catalytically active in hydroxylation. This may represent the primary function for the catechol oxidase activity of the phenolase complex. The electron donors are concerned mainly, but not entirely, in the reduction of o-quinones produced in this reaction.     

Poly(hydroxyalkanoic acid) Biosynthesis in Ectothiorhodospirashaposhnikovii: Characterization and Reactivity of a Type III PHA Synthase

Ectothiorhodospira shaposhnikovii is able to accumulate polyhydroxybutyrate (PHB) photoautotrophically during nitrogen-limited growth. The activity of polyhydroxyalkanoate (PHA) synthase in the cells correlates with PHB accumulation. PHA synthase samples collected during the light period do not show a lag phase during in vitro polymerization. Synthase samples collected in the dark period displays a significant lag phase during in vitro polymerization. The lag phase can be eliminated by reacting the PHA synthase with the monomer, 3-hydroxybutyryl-CoA (3HBCoA). The PHA synthase genes (phaC and phaE) were cloned by screening a genomic library for PHA accumulation in E. coli cells. The PHA synthase expressed in the recombinant E. coli cells was purified to homogeneity. Both sequence analysis and biochemical studies indicated that this PHA synthase consists of two subunits, PhaE and PhaC and, therefore, belongs to the type III PHA synthases. Two major complexes were identified in preparations of purified PHA synthase. The large complex appears to be composed of 12 PhaC subunits and 12 PhaE subunits (dodecamer), whereas the small complex appears to be composed of 6 PhaC and 6 PhaE subunits (hexamer). In dilute aqueous solution, the synthase is predominantly composed of hexamer and has low activity accompanied with a significant lag period at the initial stage of reaction. The percentage of dodecameric complex increases with increasing salt concentration. The dodecameric complex has a greatly increased specific activity for the polymerization of 3HBCoA and a negligible lag period. The results from in vitro polymerizations of 3HBCoA suggest that the PHA synthase from E. shaposhnikovii may catalyze a living polymerization and demonstrate that two PhaC and two PhaE subunits comprise a single catalytic site in the synthase complex.     

Catabolic Activities of Neisseria meningitidis: Utilization of Glutamate

Glutamate was catabolized at a rapid rate by Neisseria meningitidis, group B. Surprisingly, there was a lag of 5 to 30 min in respiration, but not in CO(2) production from C(1), and an appreciable amount of succinate accumulated. The eventual rapid rate of respiration was not prevented by the addition of chloramphenicol. The lag period was eliminated by combinations of substrates that favored the activity of a glutamate-oxaloacetate transaminase. It is suggested that with glutamate as the sole substrate, the reaction terminated at succinate, required only moderate O(2) uptake, and did not result in the transport of succinate to enzymatic sites. The lag period represented the time required for the accumulation of succinate and its transport to enzymatic sites by energy provided by the metabolism of the remaining glutamate. When the transaminase was operative, on the other hand, successive products of the reaction were immediately placed in contact with enzymatic sites.     

Inhibition of protein hydroperoxide formation by protein thiols

Studies on plasma and cells exposed to hydroxyl and peroxyl radicals have indicated that there are few inhibitors of protein hydroperoxide formation. We have, however, observed a small variable lag period during bovine serum albumin (BSA) oxidation by 2-2' azo-bis-(2-methyl-propionamidine) HCl (AAPH) generated peroxyl radicals, where no protein hydroperoxide was formed. The addition of free cysteine to BSA during AAPH oxidation also produced a lag phase suggesting protein thiols could inhibit protein hydroperoxide formation. The selective reduction of thiols on BSA by beta-mercaptoethanol treatment caused the appearance of a lag period where no protein hydroperoxide was formed during the AAPH mediated oxidation. Increasing free thiol concentration on the BSA increased the lag period. Protein hydroperoxide formation began when the protein thiol concentration dropped below one thiol per BSA molecule. It is unlikely that the lag period is due to gross structural alteration of the reduced protein since blocking the free thiols with N-ethyl maleimide eliminated the lag in protein hydroperoxide formation. Protein thiols were found to be ineffective in inhibiting hydroxyl radical-mediated protein hydroperoxide formation during X-ray radiolysis. Evidence is given for protein thiol oxidation occurring via a free radical mediated chain reaction with both free cysteine and protein bound thiol. The data suggest that reduced protein thiol groups can inhibit protein hydroperoxide formation by scavenging peroxyl radicals.     

Effect of different phenols on the nadh-oxidation catalysed by a peroxidase from lupin

Cell wall-bound peroxidase (EC 1.11.1.7) from lupin (Lupinus albus) shows a transition from oxidase to peroxidase activity when it oxidizes NADH. The oxidase phase represents a lag period in the time course of the reaction. This phase is phenol-dependent and responsible for hydrogen peroxide formation. Guaiacol, an assay substrate, and p-coumaric, ferulic and sinapic acids, precursors of the cinnamyl alcohols used in the lignification process affect both the length of lag period and the rate of the peroxidase phase of NADH oxidation. The effect of different phenols on the time course of the reaction is related to the efficacy (V_max/K_m ratio) of the enzyme when it is acting on them as a peroxidese.     

Inhibition of protein hydroperoxide formation by protein thiols

Abstract

Studies on plasma and cells exposed to hydroxyl and peroxyl radicals have indicated that there are few inhibitors of protein hydroperoxide formation. We have, however, observed a small variable lag period during bovine serum albumin (BSA) oxidation by 2-2′ azo-bis-(2-methyl-propionamidine) HCl (AAPH) generated peroxyl radicals, where no protein hydroperoxide was formed. The addition of free cysteine to BSA during AAPH oxidation also produced a lag phase suggesting protein thiols could inhibit protein hydroperoxide formation. The selective reduction of thiols on BSA by β-mercaptoethanol treatment caused the appearance of a lag period where no protein hydroperoxide was formed during the AAPH mediated oxidation. Increasing free thiol concentration on the BSA increased the lag period. Protein hydroperoxide formation began when the protein thiol concentration dropped below one thiol per BSA molecule. It is unlikely that the lag period is due to gross structural alteration of the reduced protein since blocking the free thiols with N-ethyl maleimide eliminated the lag in protein hydroperoxide formation. Protein thiols were found to be ineffective in inhibiting hydroxyl radical-mediated protein hydroperoxide formation during X-ray radiolysis. Evidence is given for protein thiol oxidation occurring via a free radical mediated chain reaction with both free cysteine and protein bound thiol. The data suggest that reduced protein thiol groups can inhibit protein hydroperoxide formation by scavenging peroxyl radicals.     

Chemiluminescence of Pholasin caused by peroxynitrite

The kinetics of the oxidation of Pholasin by peroxynitrite, which leads to emission of light, were studied. The reaction shows a lag phase, which is smaller at higher peroxynitrite-to-Pholasin ratios. The total light emission approximately doubles from pH 5 to 9 and decreases precipitously to half the pH 5 value at pH 10. Dioxygen and carbon dioxide accelerate the reaction course, but they do not change significantly the reaction yield. Chemiluminescence of Pholasin is suppressed by antioxidants, but no significant shift is noticed in the time at which light emission is maximal. The chemiluminescence intensity is strongly dependent on the potassium concentration, although it is not significantly affected by lithium, cesium, or magnesium; potassium decreases luminescence. The mechanism of the peroxynitrite-induced oxidation of Pholasin may start with the reversible formation of a protein-peroxynitrite intermediate, or a first oxidation product, followed in subsequent steps by decomposition and light emission. However, many questions concerning the mechanism of the light emission remain to be elucidated.     

Ionic changes associated with lead stimulation of DNA synthesis in Balb/c3T3 cells

Lead at slightly subtoxic concentrations markedly stimulated the rate of DNA synthesis in cultured animal cells. This stimulation was closely correlated with formation of a precipitate that was adsorbed and taken up by the cells under certain medium conditions. Data suggest that a precipitate-induced perturbation of the surface membrane leads to intracellular changes responsible for stimulation of DNA synthesis. Maximum stimulation of³H-thymidine incorporation by optimum concentrations of lead is delayed about 8 h compared to that in serum stimulation. In cells stimulated significantly by lead, but not in unstimu-lated cells, a reproducible rise of about 13% in intracellular magnesium occurred over a 24 h period, with an 8 h lag in the increase compared to that observed in serum stimulation. In view of the increases in intracellular magnesium consistently associated with and preceding stimulation of DNA synthesis by several different mitogens including serum and insulin, the present time-coordinated positive correlation between magnesium and DNA synthesis provides evidence for the primary involvement of this divalent cation in growth stimulation produced by lead.     

Insufficient mobilization of calcium by early breakdown of phosphatidylinositol 4,5-bisphosphate for aggregation of human platelets by collagen

When human platelets (5 X 10(8)/ml) were stimulated by a threshold concentration of collagen (2 micrograms/ml), a lag period of about 60 s was seen before the initiation of release reaction and aggregation. Breakdown of [32P]phosphatidylinositol 4,5-bisphosphate was seen within 10 s after the addition of collagen. The concentration of intracellular free Ca2+ (monitored by Quin II) rose from 80 nM to 145 nM within 10 s after stimulation by collagen. However, a lag period of about 50 s remained. The rise was not blocked by indomethacin. It was supposed that the initial Ca2+ mobilization by myo-inositol 1,4,5-trisphosphate was too small to cause aggregation. Thromboxane A2 was gradually accumulated during the lag period and then abruptly increased in parallel with aggregation. These events were completely inhibited by 10 microM indomethacin. Thus, aggregation appeared to be dependent on the generation of thromboxane A2. Addition of 25 nM A23187 at 10 s after stimulation by collagen shortened the lag period before initiation of the abrupt thromboxane A2 generation, secretion and aggregation, whereas 25 nM A23187 could not cause these reactions in the absence of collagen. Accordingly, the lag period is assumed to be required for accumulation of free Ca2+ to the threshold for aggregation of platelets. It is considered that thromboxane A2 plays a central role in Ca2+ mobilization during stimulation of human platelets by collagen.     

Differentiation between exonucleases and endonucleases and between haplotomic and diplotomic endonucleases using 3-h-dna-coated wells of plastic depression plates as substrate.

Using our new method for assaying DNases with radioactively labeled DNA bound to wells of plastic depression plates as substrate, we could distinguish between endonucleases and exonucleases and between haplotomic and diplotomic endonucleases. Oligonucleotides smaller than 30 detach from the DNA binding sites of the well into the reaction mixture. Thus, a lag period was evident before endonucleases produced small soluble oligonucleotides, while exonucleases released mononucleotides or short oligonucleotides without any lag period. Haplotomic and diplotomic endonucleases were detected because of the different rates in which they produce small soluble oligonucleotides which were expressed in different lag periods. Under conditions in which the haplotomic DNase 1 changes its mode of action to become a diplotomic enzyme, the shift was clearly detected by a change in the lag period in the well assay.     

Static and kinetic studies of calmodulin and melittin complex.

Ca2+ binding to calmodulin triggers conformational change of the protein which induces exposure of hydrophobic surfaces. Melittin has been believed to bind to Ca(2+)-bound calmodulin through the exposed hydrophobic surfaces. However, tryptophan fluorescence measurements and gel chromatography experiments with the melittin-calmodulin system revealed that melittin bound to calmodulin at zero salt concentration even in the absence of Ca2+; addition of salt removed melittin from Ca(2+)-free calmodulin. This means not only the hydrophobic interaction but also the electrostatic interaction contributes to the melittin-calmodulin binding. The fluorescence stopped-flow studies of the dissociation reaction of melittin-calmodulin complex revealed that Ca2+ removal from the complex induced a conformational change of calmodulin, resulting in reduction of the hydrophobic interaction between melittin and calmodulin, but the electrostatic interaction kept melittin still bound to calmodulin for a subsecond lag period, after which melittin dissociated from calmodulin. The fluorescence stopped-flow experiments on the dissociation reaction of complex of melittin and tryptic fragment(s) of calmodulin revealed that the lag period of the melittin dissociation reaction was attributable to the interaction between the C-terminal half of calmodulin and the C-terminal region of melittin.     

Studies on the mechanism of inhibition of amphibian oocyte adenylate cyclase by progesterone

Progesterone treatment induces the meiotic maturation of Xenopus laevis oocytes. Previous evidence indicates that this hormonal effect may be due to inhibition of oocyte adenylate cyclase. The present work studies several aspects of the mechanism of adenylate cyclase inhibition by this hormone. Forskolin greatly stimulates oocyte adenylate cyclase in the absence of guanine nucleotides and this activity is not sensitive to progesterone inhibition. In addition the forskolin-activated enzyme is not inhibited by a wide range of guanine nucleotide, in the presence or absence of hormone. The time course of cAMP synthesis catalyzed by oocyte adenylate cyclase in the presence of guanyl-5′_l-imidodiphosphate (Gpp(NH)p) shows an initial lag period that does not depend on the concentration of Gpp(NH)p. Progesterone causes a very significant increase in the hysteresis of the reaction, at least doubling the half-time of enzyme activation. The hormonal effect on the lag cannot be reversed by saturating concentrations of Gpp(NH)p. Progesterone also decreases the steady-state rates of the reaction. This effect, however, depends on the concentration of Gpp(NH)p. High concentrations of Gpp(NH)p almost completely reverse the inhibition of the steady-state rates. Progesterone does not inhibit if it is added to the reaction after the initial lag period. Guanosine-5′-O-(2-thiodiphosphate) (GDP-β-S) is an efficient competitive inhibitor of Gpp(NH)p activation of adenylate cyclase. Progesterone inhibition is observed at all concentrations of GDP-β-S and is potentiated at high ratios of GDP-β-S to Gpp(NH)p. These data indicate that progesterone inhibits by interfering with the activation of the N_s subunit of the enzyme by guanine nucleotides, rather than through a mechanism involving a separate N_i subunit.     

Steady state and pre-steady state kinetic properties of rat liver selenium-glutathione peroxidase.

The kinetic properties of partially purified rat liver selenium-glutathione peroxidase were studied under various conditions. Steady state kinetic measurements show sigmoidal saturation curves, parabolic double reciprocal plots, and Hill coefficients greater than unity. Although these kinetic results appear to show cooperative interactions between subunits, they more reflect the presence of several oxidation-reduction forms of the catalytic site. A substrate-induced transition between enzyme forms was evidence by the occurrence of a lag in the attainment of the final steady state velocity under certain preincubation conditions. This hysteretic behavior was evident only when the enzyme was incubated in the absence of reduced glutathione, the donor substrate. Thus, reduced glutathione induces the transition to the fully active form of the enzyme, a slow process requiring about 0.5 min after addition of glutathione, depending on conditions. The length, tau, of the lag period is dependent on the concentrations of enzyme and glutathione, but to a first approximation, this lag period is independent of the concentration of the hydroperoxide acceptor substrate. The lag period is also relatively independent of the nature of the hydroperoxide species. A model for the transition process that is compatible with these observations and with the possible oxidation-reduction properties of the selenium moiety of the enzyme is suggested.     

Kinetic study of sinephrine oxidation by mushroom tyrosinase

Mushroom tyrosinase catalyzes the oxidation of sinephrine showing a marked lag period during appearance of adrenochrome and simultaneously adrenaline accumulation in the reaction medium can be detected. The adrenaline accumulation follows a sigmoidal curve until a constant level of adrenaline is reached when the system is in the steady-state. These experimental results agree with a model of enzymatic catalysis that includes the chemical evolution of adrenoquinone and permit us to explain these phenomenon as well as the influence that enzyme and sinephrine concentration present on the lag period and the level of adrenaline accumulated in the steady-state.     

Magnesium requirement for biological removal of phosphate by activated sludge

The effects of magnesium on excess uptake of phosphate in an aerobic-anaerobic activated sludge process were examined by the fill and draw procedure. The alternation of anaerobic and aerobic conditions in one cycle of fill and draw process was varied many ways.The presence of sufficient magnesium was necessary for uptake of excess phosphate. When sludge contained more phosphorus than the upper limit of phosphorus content in the usual aerobic activated sludge, 2.5% by weight, magnesium was also contained in more than an ordinary amount (0.5%). Their contents in the sludge at the end of each cycle of the process were correlated with each other by a linear equation with the correlation coefficient of 0.99. When magnesium concentration was insufficient for the uptake of excess phosphate, its concentration in the treated water was of the order of 0.1 mg/l.In the first anaerobic period both phosphate and magnesium were released, and in a successive aerobic period they were taken up again. The weight ratio of differential amounts of phosphorus and magnesium released or taken up changed with time in one cycle.Dynamic behaviors of phosphate and magnesium removal against the step change of feed magnesium concentration also showed a stoichiometric relationship supporting the correlation equation above mentioned.