An enzyme kinetics study of the pH dependence of chloride activation of oxygen evolution in photosystem II

Oxygen evolution by photosystem II (PSII) involves activation by Cl^? ion, which is regulated by extrinsic subunits PsbQ and PsbP. In this study, the kinetics of chloride activation of oxygen evolution was studied in preparations of PSII depleted of the PsbQ and PsbP subunits (NaCl-washed and Na₂SO₄/pH 7.5-treated) over a pH range from 5.3 to 8.0. At low pH, activation by chloride was followed by inhibition at chloride concentrations >100 mM, whereas at high pH activation continued as the chloride concentration increased above 100 mM. Both activation and inhibition were more pronounced at lower pH, indicating that Cl^? binding depended on protonation events in each case. The simplest kinetic model that could account for the complete data set included binding of Cl^? at two sites, one for activation and one for inhibition, and four protonation steps. The intrinsic (pH-independent) dissociation constant for Cl^? activation, K _S, was found to be 0.9 ± 0.2 mM for both preparations, and three of the four pK _as were determined, with the fourth falling below the pH range studied. The intrinsic inhibition constant, K _I, was found to be 64 ± 2 and 103 ± 7 mM for the NaCl-washed and Na₂SO₄/pH7.5-treated preparations, respectively, and is considered in terms of the conditions likely to be present in the thylakoid lumen. This enzyme kinetics analysis provides a more complete characterization of chloride and pH dependence of O₂ evolution activity than has been previously presented.     

Phosphatidohydrolase activity in a solubilized preparation from rat brain particulate fraction.

Phospholipase D activity (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) was demonstrated in vitro in a solubilized preparation from rat brain particulate fraction which also possessed the transphosphatidylation activity. The preparation attacked a phosphatidylcholine microdispersion and cleaved the terminal phosphate diester bond of this phospholipid resulting in the formation of phosphatidic acid. The pH optimum for the phosphatidohydrolase activity was broad with an apparent peak aroung 6.0 whereas the transphosphatidylation showed a sharp pH optimum at 7.2 Ca²⁺ was not essential for the hydrolysis, but merely stimulated slightly with an optimum about 5 mm, however, it could be replaced by Mg²⁺. The hydrolysis of phosphatidylcholine by the enzyme was almost completely inhibited in the presence of either diethyl ether (20% by volume) or p-chloromercuriophenyl sulfonate (6 × 10 ^?5m). The latter inhibition was reduced by the addition of dithiothreitol (6 × 10^?4m). The result suggests an essential role of sulfhydryl group in the formation of the enzyme-substrate complex.The apparent K_m for phsophatidylcholine for the phosphatidohydrolase activity was about 8.3 × 10 ^?4m.     

Kinetic properties of pig pyruvate kinases type A from kidney and type M from muscle.

Kinetic properties of homogeneous preparations of pig kidney and pig muscle pyruvate kinases (EC 2.7.1.40) were studied. Both isozymes showed a hyperbolic relationship to ADP with an apparent K_m of 0.3 mm. K⁺ and Mg²⁺ were necessary for the activity of both isozymes, and their dependences on these cations were similar. The muscle isozyme expressed Michaelis-Menten type of kinetics with respect to phosphoenolpyruvate, and the apparent K_m was the same (0.03 mm) from pH 5.5 to pH 8.0. In contrast, the dependence on phosphoenolpyruvate changed with pH for the kidney isozyme. It showed similar properties to the muscle isozyme at pH 5.5–7.0 (apparent K_m of 0.08 mm), while two apparent K_m values for this substrate were present at pH 7.5–8.0, one low (0.1 mm) and one high (0.3–0.6 mm). At pH 7.5, fructose 1,6-bisphosphate converted the kidney isozyme to a kinetical form where only the lower apparent K_m for phosphoenolpyruvate was detected. On the other hand, in the presence of alanine or phenylalanine the kidney pyruvate kinase showed only the higher K_m for this substrate. At low phosphoenolpyruvate levels both isozymes were inhibited by phenylalanine, and half-maximal inhibition was found at 0.3 and 2.2 mm for the kidney and muscle isozymes, respectively. At a 5 mm concentration of the substrate only the kidney isozyme was inhibited, the apparent K_i being the same. Alanine inhibited the kidney isozyme (apparent K_i at 0.3 mm, irrespective of substrate concentration). No effect was seen on the muscle isozyme. Fructose 1,6-bisphosphate was an activator of the kidney isozyme at phosphoenolpyruvate concentrations below 1.0 mm It also counteracted the inhibition by alanine or phenylalanine of this isozyme. ATP inhibited both isozymes, and this inhibition was not counteracted by fructose 1,6-bisphosphate. The kidney isozyme showed both a high and a low apparent K_m for phosphoenolpyruvate in the presence of ATP. The influence of the effectors on the activity of both isozymes varied markedly with pH, except for the action of ATP. At low substrate concentrations, however, the inhibitor action of ATP on the muscle enzyme was diminished around pH 7.5, in contrast to higher or lower pH values. Alanine or phenylalanine were more effective as inhibitors at higher pH values, and fructose 1,6-bisphosphate stimulated the kidney isozyme only at pH levels above pH 6.5. The influence of activators and inhibitors on the regulation of the kidney and muscle pyruvate kinases is discussed.     

Purification and properties of enolase from swine kidney

The first, enolase (2-phospho-d-glycerate hydrolyase, EC 4.2.1.11) to be isolated from a gluconeogenic tissue, swine kidney, was purified more than 600-fold to near homogeneity, as estimated from sedimentation equilibrium and velocity measurements and from disc electrophoresis patterns. The physical properties of the enzyme were examined. Purified kidney enolase has a s^0.87%_20,w = 5.87 S, M_r = 90,000 ± 4,500, e^0.1%_280,1cm = 1.07/mg/ml, a Stokes radius of 37.0 Å, and an apparent subunit molecular weight of 52,000.The amino acid composition was determined and compared with those of mammalian muscle enolases. The partial specific volume calculated from the amino acid composition was found to be 0.728 cc/g. Swine kidney enolase had 12 cysteines per mole; in the native enzyme, two reacted with DTNB.The enzyme was stabilized by magnesium, sucrose, or glycerol; activity lost, on prolonged storage could be completely recovered by treatment with mercaptoethanol and EDTA at 37 °C. Some evidence was obtained for the existence of active monomers of this enzyme. This form of swine kidney enolase was quite unstable, however. The pH optimum was at 6.8. The Michaelis constants for 2-phospho-d-glycerate and phosphoenolpyruvate were 5.10^?5m and 10^?4m; that for magnesium was 4.10^?4m. Substrate inhibition was found for 2-phosphoglycerate but not for phosphoenolpyruvate. No inhibition is seen under comparable conditions with mammalian enolases from glycolytic tissues. This finding is discussed.     

Subcellular localization,metal ion requirement and kinetic properties of arginase from the gill tissue of the bivalve Semele solida

Arginase activity (3.1 ± 0.5 units/g (wet wt) of tissue) was found associated to the cytosolic fraction of the gill cells of the bivalve Semele solida. The enzyme, with a molecular weight of 120,000 ± 3000, was partially purified, and some of the enzymic properties were were examined. The activation of the enzyme by Mn²⁺ followed hyperbolic kinetics with a K_Mn value of 0.10 ± 0.02 μM. In addition to Mn²⁺, the metal ion requirement of the enzyme was satisfied by Ni²⁺, Cd²⁺ and Co²⁺; Zn²⁺ was inhibitory to ail the Values of K_m for arginine and K_i for lysine inhibition, were the same, regardless of the metal ion used to activate the enzyme; K_m values were 20 mM at pH 7.5 and 12 mM at the optimum pH of 9.5. Competitive inhibition was caused by ornithine, lysine and proline, whereas branched chain amino acids were non competitive inhibitors of the enzyme.     

Plant pyruvate dehydrogenase complex: analysis of the kinetic properties and metabolite regulation

The pyruvate dehydrogenase complex was isolated from the mitochondria of broccoli florets and shown to be similar in its reaction mechanism to the complexes from other sources. Three families of parallel lines were obtained for the initial velocity patterns, indicating a multisite ping-pong mechanism. The apparent K_m values obtained were 321 ± 18, 148 ± 13, and 7.2 ± 0.51 μm for pyruvate, NAD⁺, and CoA, respectively. Product inhibition studies using acetyl-CoA and NADH yielded results which were in agreement with those predicted by the multisite ping-pong mechanism. Acetyl-CoA and NADH were found to be competitive inhibitors versus CoA and NAD⁺, respectively. All other substrate-product combinations showed uncompetitive inhibition patterns, except for acetyl-CoA versus NAD⁺. Among various metabolites tested, only hydroxypyruvate (K_i = 0.11 mM) and glyoxylate (K_i = 3.27 mM) were found to be capable of inhibiting the broccoli enzyme to a significant degree. Initial velocity patterns using Mg²⁺? or Ca²⁺-thiamine pyrophosphate and pyruvate as the variable substrate were found to be consistent with an equilibrium ordered mechanism where Mg? or Ca-thiamine pyrophosphate bind first, with dissociation constants of 33.8 and 3 μm, respectively. The Mg- or Ca-thiamine pyrophosphate complexes also dissociated rapidly from the enzyme complex.     

Lunularic acid decarboxylase from the liverwort Conocephalum conicum

Some properties of a preparation of an enzyme, lunularic acid decarboxylase, from the liverwort Conocephalum conicum are described. The enzyme is normally bound and could be solubilized with Triton X-100; at least some of the bound decarboxylase activity appears to be associated with chloroplasts. For lunularic acid the enzyme has K_m 8.7 × 10^?5 M (pH 7.8 and 30°). Some substrate analogues have been tested but no other substrate was found. Pinosylvic acid is a competitive inhibitor for the enzyme, K_i 1.2 × 10^?4 M (pH 7.8 and 30°). No product inhibition was observed. Lunularic acid decarboxylase activity has also been observed with a cell-free system from Lunularia cruciata.     

Pituitary corticotropin-inhibiting peptide: Properties and use in study of corticotropin action

The biological properties of the naturally occurring pituitary peptide α_h^7–38-adrenocorticotropin (ACTH) have been investigated. α_h^7–38-ACTH is devoid of steroidogenic activity but inhibits competitively ACTH-induced steroidogenesis in vitro as well as in vivo. The long-term actions of ACTH on normal and tumor adrenal cells in culture are also antagonized by α_h^7–38-ACTH. The apparent K_i for the inhibition of cyclic AMP production by α_h^7–38-ACTH (301 ± 62 nm) was significantly higher than the apparent K_i for the inhibition of corticosterone synthesis (21.6 ± 6.8 nm). Analysis of the inhibition of ACTH-induced steroidogenesis and cyclic AMP production in normal rat adrenocortical cells indicates that two separate receptors may be involved in mediating these responses.     

Role of electrolytes and starvation in altering apparent radiosensitivity of baker's yeast

1. Respiration and fermentation of yeast receiving 90,000 r of 250 kv. x-rays were studied under a variety of conditions. This dose will nearly completely inhibit growth or colony formation. 2. The apparent effects of irradiation are quite dependent on the K⁺ and H⁺ of the suspending medium. At pH 4.5 stimulatory effects were observed in KH₂PO₂ buffer and inhibition in potassium-free (T-S-T) buffer. At pH 6.5 the situation was reversed and the effects were very small (about 10 per cent). Addition of K⁺ to irradiated yeast in T-S-T buffer at pH 4.5 can completely reverse the inhibition seen. 3. Starving increases the apparent radiosensitivity of respiration and fermentation, probably by depletion of metabolite and/or electrolyte reserves. 4. Treatment with a cation exchange resin (dowex 50) results in marked inhibition of these processes in irradiated yeast, either fresh or starved. This was most effective if given after irradiation. Almost complete inhibition of anaerobic CO₂ production occurs with starvation, irradiation, and dowex treatment combined. 5. The effects of starvation and cation exchange resin treatment can be reversed, though not completely, by adding K⁺ to the medium.     

Preparation,characterization and laboratory-scale application of an immobilized glucoamylase

Glucoamylase (1,4-α-d-glucan glucohydrolase, EC 3.2.1.3) has been covalently immobilized on a polyacrylamide-type support containing carboxylic groups activated by water-soluble carbodiimide. The activity was 5.5– 6.0 units g^?1solid. The optimum pH for catalytic activity was pH 3.8. The apparent optimum temperature was found at 60°C. With soluble starch as substrate the K_m value was 14 mg ml^?1. The pH for maximum stability was pH 4.0–4.5. In the presence of 8 m urea the immobilized glucoamylase retained most of its catalytic activity but it was more susceptible to guanidinium hydrochloride than the soluble enzyme. The practical applicability of immobilized glucoamylase was tested in batch process and continuous operation.     

Kinetics of cyanide binding to chloroperoxidase in the presence of nitrate: detection of the influence of a heme-linked acid group by shift in the appa

The kinetics of the binding of cyanide to ferric chloroperoxidase have been studied at 25°C and ionic strength 0.11 M using a stopped-flow apparatus. The dissociation constant (K_CN) of the peroxidase-cyanide complex and both forward (k₊) and reverse (k_?) rate constants are independent of the H⁺ concentration over the pH range 2.7 to 7.1. The values obtained are k_cn = (9.5 ± 1.0) × 10^-5 M, k₊. = (5.2 ± 0.5) × 10⁴ M^?1 sec^?1 and k_- = (5.0± 1.4) sec^-1. In the presence of 0 06 M potassium nitrate the affinity of cyanide for chloroperoxidase decreases due to the inhibition of the forward reaction. The dissociation rate is not affected. The nitrate anion exerts its influence by binding to a protonated form of the enzyme, whereas the cyanide binds to the unprotonated form. Binding of nitrate results in an apparent shift towards higher pK_a values of the ionization of a crucial heme-linked acid group. Hence the influence of this group can be detected in the accessible pH range. Extrapolation to zero nitrate concentration yields a value of 3.1±0.3 for the pK_a of the heme-linked acid group.     

d-Xylose,an anomer-specific inhibitor of α-galactosidase

d-xylose is a highly anomer-specific, powerful and competitive inhibitor of plant α-galactosidases. The apparent inhibition constant (K_i) for the interaction of d-xylose with chick pea (Cicer arietinum) α-galactosidase and the apparent number of inhibitor molecules (n) bound per enzyme molecule, using p-nitrophenyl-α-d-galactopyranoside as substrate, were found to be 0.4 × 10^?2M and 0.8, respectively.     

Phosphatidohydrolase and base-exchange activity of commercial phospholipase D

Base-exchange activity was contrasted to the usual phosphatidohydrolase activity of commercial phospholipase D preparation from cabbage. The former activity was assayed by measuring the incorporation of labeled ethanolamine and choline into phospholipids. The latter activity was assayed by measuring the formation of phosphatidic acid with radioactive phosphatidylcholine microdispersion as substrate. The pH optimum for the base-exchange activity was about 9.0, whereas the phosphatidohydrolase activity had a pH optimum around 5.6. The incorporation of ethanolamine and choline into phospholipid was dependent upon the amount of acceptor asolectin microdispersion present. The optimum concentration of Ca²⁺ in the base-exchange reaction was about 4 mm, whereas the optimum concentration for the phosphatidohydrolase activity was greater than 28 mm. The incorporation of ethanolamine into phospholipid was decreased 50% by heating the enzyme preparation at 50°C for about 10 min, whereas the choline incorporation decreased approximately 20% and the phosphatidohydrolase activity decreased by about 10% under these conditions.Hemicholinium-3 was found to be a noncompetitive inhibitor for the incorporation of both ethanolamine and choline into phospholipid with respective K_i, values of 1.25 × 10^?3 and 2.50 × 10^?3m. The K_m values for ethanolamine and choline in the base-exchange reaction were 1.25 × 10^?3 and 2.50 × 10^?3m, respectively. The apparent K_m for phosphatidylcholine for the phosphatidohydrolase activity was about 1.5 × 10^?3m, and there was no inhibition by hemicholinium-3.     

Transport of vitamin B12 into mouse leukemia cells.

Transport of [⁵⁷Co]cyanocobalamin (vitamin B₁₂) into L1210 murine leukemia cells, mediated by transcobalamin-II, is a biphasic process. The primary step, which occurs very rapidly (within the first minute), appears to involve binding of the B₁₂-transcobalamin complex to the external membrane of the cells; at 37 °C and pH 7.3, apparent K_m and V values are 180 pM and 5.9 pmol/min/10⁹ cells, respectively. This step is relatively insensitive to temperature, has an apparent pK_a of 6.0, and is inhibited by EDTA; Ca²⁺ or Mg²⁺ can reverse the inhibition. The slower secondary step appears to involve translocation of the vitamin into the cell; V for this step is 0.4 pmol/min/10⁹ cells. Temperature and pH optima are 30 ° and 6.5?7.0, respectively. This step is stimulated by glucose and inhibited by cyanide, arsenite, arsenate, p-chloromercuriphenylsulfonate and dinitrophenol. Labeled B₁₂ in an amount corresponding to that taken up in the initial step can be released by: (a) resuspending the cells in a B₁₂-free medium; (b) addition of unlabeled B₁₂-transcobalamin-II; and (c) addition of EDTA. The material released is chromatographically and functionally identical with B₁₂-transcobalamin-II.     

Purification and characterization of selenium-glutathione peroxidase from hamster liver

Hamster liver glutathione peroxidase was purified to homogeneity in three chromatographic steps and with 30% yield. The purified enzyme had a specific activity of approximately 500 μmol cumene hydroperoxide reduced/min/mg of protein at 37 °C, pH 7.6, and 0.25 mm GSH. The enzyme was shown to be a tetramer of indistinguishable subunits, the molecular weight of which was approximately 23,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single isoelectric point of 5.0 was attributed to the active enzyme. Amino acid analysis determined that selenocysteine, identified as its carboxymethyl derivative, was the only form of selenium. One residue of cysteine was found to be present in each glutathione peroxidase subunit. The presence of tryptophan was colorimetrically determined. Pseudo-first-order kinetics of inactivation of the enzyme by iodoacetate was observed at neutral pH with GSH as the only reducing agent. An optimal pH of 8.0 at 37 °C and an activation energy of 3 kcal/mol at pH 7.6 were found. A ter-uni-ping-pong mechanism was shown by the use of an integrated-rate equation. At pH 7.6, the apparent second-order rate constants for reaction of glutathione peroxidase with hydroperoxides were as follows: k₁ (t-butyl hydroperoxide), 7.06 × 10⁵ mm min^?1; k₁ (cumene hydroperoxide), 1.04 × 10⁶ mm^?1 min^?1; k₁ (p-menthane hydroperoxide), 1.2 × 10⁶ mm^?1 min^?1; k₁ (diisopropylbenzene hydroperoxide), 1.7 × 10⁶ mm^?1 min^?1; k₁ (linoleic acid hydroperoxide), 2.36 × 10⁶ mm^?1 min^?1; k₁ (ethyl hydroperoxide), 2.5 × 10⁶ mm^?1 min^?1; and k₁ (hydrogen peroxide), 2.98 × 10⁶ mm^?1 min^?1. It is concluded that for bulky hydroperoxides, the more hydrophobic the substrate, the faster its reduction by glutathione peroxidase.     

The effect of pH on the inorganic carbon source for photosynthesis in the seagrass Zostera muelleri irmisch ex aschers

The ability of the seagrass Zostera muelleri Irmisch ex Aschers. to use HCO₃⁻ as well as CO₂ for photosynthesis was investigated by measuring photosynthetic O₂ evolution over a range of pH values. It was found that the apparent K_m CO₂ fell from 0.128 mM at pH 7.9 to 0.016 mM at pH 9.1 indicating that HCO₃⁻ as well as CO₂ may act as a substrate for photosynthesis.The true K_m CO₂ could not be determined due to inhibition of photosynthesis at pHs less than 7.8 K_m CO₂ must be at least 0.128 mM, the apparent K_m at pH 7.9, and is probably of the order of 0.200 mM CO₂, the same as that reported for other marine plants. K_m HCO₃⁻¹ is about 20 mM when CO₂-dependent photosynthesis is minimal. Such a high K_m HCO₃⁻ resembles values reported for freshwater, rather than marine plants.Photosynthetic O₂ evolution is not saturated with respect to total inorganic carbon in natural seawater (pH 8.2). It is suggested that the distinctive shoulder from pH 8.1 to 8.5 in the pH profile of photosynthetic O₂ evolution at a constant concentration of inorganic carbon is caused by an effect of pH on HCO₃⁻ uptake. The effect of pH on HCO₃⁻ uptake was determined by constructing a pH profile of photosynthesis at constant HCO₃⁻ concentration, and subtracting the estimated contribution of CO₂ to photosynthesis from this rate. The resultant curve has a maximum at pH 8.4 and declines sharply at pHs less than 8.     

Fructokinase (Fraction III) of Pea Seeds

A second fructokinase (EC 2.7.1.4) was obtained from pea seed (Pisum sativum L. var. Progress No. 9) extracts. The enzyme, termed fructokinase (fraction III), was specific for fructose and had little activity with glucose. With fructose concentrations above 0.25 millimolar, there was strong substrate inhibition at the optimum pH (8.0) and also at pH 6.6. The apparent K_m values at pH 8.0 for fructose and glucose were 0.06 millimolar and 0.14 millimolar, respectively. The apparent K_m for Mg adenosine 5′-triphosphate (MgATP) was 0.06 millimolar and excess MgATP was inhibitory. Mg²⁺ was essential for activity but the enzyme was inhibited by excess Mg²⁺ or ATP. Mg adenosine 5′-pyrophosphate was also inhibitory. Activity was stimulated by the addition of monovalent cations: of those tested K⁺, Rb⁺, and NH₄⁺ were the most effective. The possible role of fructokinase (fraction III) is discussed.     

Myoinositol hexaphosphate as a potential inhibitor of α-amylases

Myoinositol hexaphosphate (MHP) strongly inhibited α-amylases of different origins. The inhibition of wheat α-amylase is noncompetitive with an apparent K_i value of 1 mM, pH dependent and markedly increased by the preincubation of enzyme with MHP before the addition of substrate. Addition of Ca²⁺ did not reverse the inhibition of α-amylase indicating that its inhibition was not due to the binding of Ca²⁺ by MHP.     

Purification and biochemical characterization of a new alkali-stable laccase from Trametes sp. isolated in Tunisia: role of the enzyme in olive mill waste water treatment

A white-rot basidiomycete, isolated from decayed acacia wood (from Northwest of Tunisia) and identified as Trametes sp, was selected in a broad plate screening because of its ability to decolorize and dephenolize olive oil mill wastewater (OMW) efficiently. The major laccase was purified and characterized as a monomeric protein with apparent molecular mass of 61 kDa (SDS-PAGE). It exhibits high enzyme activity over broad pH and temperature ranges with optimum activity at pH 4.0 and a temperature of 60 °C. The purified laccase is stable at alkaline pH values. The enzyme retained 50 % of its activity after 90 min of incubation at 55 °C. Using ABTS, this laccase presented K _m and V _max values of 0.05 mM and 212.73 μmoL min^?1 mg^?1, respectively. It has shown a degrading activity towards a variety of phenolic compounds. The purified laccase was partially inhibited by Fe²⁺, Zn²⁺, Cd²⁺ and Mn²⁺, while Cu²⁺ acted as inducer. EDTA (10 mM) and NaN₃ (10 mM) were found to completely inhibit its activity. 73 % OMW was dephenolized after 315 min incubation at 30 °C with 2 U mL^?1 of laccase and 2 mM HBT.     

Inhibition of Escherichia coli glutamine synthetase by phosphinothricin

The inhibition of Escherichia coli glutamine synthetase by phosphinothricin [2-amino-4-(methylphosphinyl)butanoic acid] has been studied. This amino acid was observed to function as an active site directed inhibitor exhibiting time-dependent inhibition of glutamine synthetase in the presence of ATP or adenylylimidodiphosphate (AMPPNP) but not adenylyl(β,γ-methylene) diphosphonate (AMPPCP). The inactivation was observed to be pseudo-first order. Phosphinothricin was also found to inhibit the enzyme reversibly under initial rate conditions and was competitive with respect to glutamate with K_1S = 18 ± 3 μm. The inactive enzyme inhibitor complex was found to contain approximately 11 molecules of ADP and of ³²P per dodecamer using [γ-³²P]ATP. Reactivation of the inactive enzyme complex was achieved by incubating the enzyme complex in 50 mm acetate (pH 4.4), 1 m KCl, and 0.40 m (NH₄)₂SO₄. ADP, phosphinothricin, and P_i were released upon reactivation.