Gallotannin biosynthesis: beta-glucogallin: hexagalloyl 3-O-galloyltransferase from Rhus typhina leaves.

Leaves of staghorn sumac (Rhus typhina) contain several galloyltransferases that catalyze the beta-glucogallin dependent transformation of 1,2,3,4,6-pentagalloylglucose to gallotannins. Among these, an enzyme has been isolated that preferentially acylates the 3-position of the hexagalloylglucose, 3-O-digalloyl-1,2,3,4,6-tetra-O-galloylglucose, to afford the corresponding heptagalloylglucose being characterized by a 3-O-meta-trigalloyl side-chain. The enzyme, for which a M(r) of ca. 260,000 was determined, was purified to apparent homogeneity. SDS-PAGE suggested an alpha4beta4-conformation of the native enzyme. It had a pH-optimum and an isolelectric point at pH 5.6, was most stable at pH 4.0-4.3, and displayed excellent heat-stability and in particular an extreme cold-tolerance. We propose the systematic name "beta-glucogallin: hexagalloylglucose 3-O-galloyltransferase" for this new enzyme.     

A macrophage-nanozyme delivery system for Parkinson's disease

Selective delivery of antioxidants to the substantia nigra pars compacta (SNpc) during Parkinson's disease (PD) can potentially attenuate oxidative stress and as such increase survival of dopaminergic neurons. To this end, we developed a bone-marrow-derived macrophage (BMM) system to deliver catalase to PD-affected brain regions in an animal model of human disease. To preclude BMM-mediated enzyme degradation, catalase was packaged into a block ionomer complex with a cationic block copolymer, polyethyleneimine-poly(ethylene glycol) (PEI-PEG). The self-assembled catalase/PEI-PEG complexes, "nanozymes", were ca. 60 to 100 nm in size, stable in pH and ionic strength, and retained antioxidant activities. Cytotoxicity was negligible over a range of physiologic nanozyme concentrations. Nanozyme particles were rapidly, 40-60 min, taken up by BMM, retained catalytic activity, and released in active form for greater than 24 h. In contrast, "naked" catalase was rapidly degraded. The released enzyme decomposed microglial hydrogen peroxide following nitrated alpha-synuclein or tumor necrosis factor alpha activation. Following adoptive transfer of nanozyme-loaded BMM to 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine-intoxicated mice, ca. 0.6% of the injected dose were found in brain. We conclude that cell-mediated delivery of nanozymes can reduce oxidative stress in laboratory and animal models of PD.     

Biosynthesis and subcellular distribution of hydrolyzable tannins.

Pathways to complex gallotannins have been elucidated by enzyme studies, indicating that beta-glucogallin is required as principal acyl donor. Evidence for the in vitro oxidation of pentagalloylglucose, the pivotal metabolite in this sequence, to ellagitannins, is presented. Immunohistochemical studies with antibodies raised against pentagalloylglucose and the galloyltransferase catalyzing the formation of this ester revealed that leaf mesophyll cell walls were a typical site of origin and deposition of hydrolyzable tannins. Seasonal changes of these compounds were studied with extracts from cell walls and intracellular space of oak leaves.     

Enzymology of gallotannin and ellagitannin biosynthesis

Gallotannins and ellagitannins, the two subclasses of hydrolyzable tannins, are derivatives of 1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranose. Enzyme studies with extracts from oak leaves (Quercus robur, syn. Quercus pedunculata; Quercus rubra) and from staghorn sumac (Rhus typhina) revealed that this pivotal intermediate is synthesized from beta-glucogallin (1-O-galloyl-beta-D-glucopyranose) by a series of strictly position-specific galloylation steps, affording so-called 'simple' gallotannins, i.e., mono- to pentagallyoylglucose esters. Besides its role as starter molecule, beta-glucogallin was also recognized as the principal energy-rich acyl donor required in these transformations. Subsequent pathways to 'complex' gallotannins have recently been elucidated by the isolation of five different enzymes from sumac leaves that were purified to apparent homogeneity. They catalyzed the beta-glucogallin-dependent galloylation of pentagallyoylglucose to a variety of hexa- and heptagalloylglucoses, plus several not yet characterized higher substituted analogous galloylglucoses. With respect to the biosynthesis of ellagitannins, postulates that had been formulated already decades ago were proven by the purification of a new laccase-like phenol oxidase from leaves of fringe cups (Tellima grandiflora) that regio- and stereospecifically oxidized pentagallyoylglucose to the monomeric ellagitannin, tellimagrandin II. This compound was further oxidized by a similar but different laccase-like oxidase to yield a dimeric ellagitannin, cornusiin E.     

Unique phenolic carboxylic acids from Sanguisorba minor

The unique phenolic carboxylic acids, 4,8-dimethoxy-7-hydroxy-2-oxo-2H-1-benzopyran-5,6-dicarboxylic acid and 2-(4-carboxy-3-methoxystyryl)-2-methoxysuccinic acid were isolated and identified from the whole Sanguisorba minor plant. The known phenolics, gallic acid; ellagic acid; quercetin-3-O-(6"-galloylglucose); beta-glucogallin; 2,3-hexahydroxydiphenoyl-(alpha/beta)-glucose; 1-galloyl-2,3-hexahydroxydiphenoyl-alpha-glucose together with its beta-isomer were also characterized. Structures were established by conventional methods of analysis and confirmed by NMR and ESI-MS spectral analysis.     

12a-羟基鱼藤酮对斜纹夜蛾生殖力的影响及其作用机理

为探讨12a-羟基鱼藤酮对斜纹夜蛾Prodenia litura (Fabricius)生殖力的影响及其作用机理, 本文测定了该虫取食12a-羟基鱼藤酮后的产卵量。在用高效液相色谱仪确定其卵巢组织中存在12a-羟基鱼藤酮后, 通过MTT法测定该化合物对斜纹夜蛾卵巢细胞（PL细胞）的细胞毒性,并利用流式细胞仪检测该化合物对PL细胞细胞周期、膜电位、胞内钙离子浓度、线粒体膜电位的影响。结果表明:斜纹夜蛾幼虫取食12a-羟基鱼藤酮后产卵量下降80％以上, 并从卵巢组织中检测出该化合物。12a-羟基鱼藤酮对PL细胞增殖具有抑制活性, 其IC₅₀为6.6 mg/L。该化合物将PL细胞周期阻滞于S期, 导致PL细胞膜电位、线粒体膜电位和细胞内游离钙离子浓度均显著升高。该化合物导致斜纹夜蛾繁殖力降低。12a-羟基鱼藤酮将PL细胞增殖阻滞于细胞周期的S期。同时, 该化合物对卵巢细胞具有较弱的毒杀活性, 导致一些卵巢细胞死亡。由于上述原因, 卵巢细胞数量逐渐减少而导致卵巢管萎缩。卵巢管的萎缩使卵巢管内的卵母细胞不能发育成卵细胞, 故其生殖力下降。     

Alkaline-active xylanase produced by an alkaliphilicBacillus sp isolated from kraft pulp

ABacillus sp (V1-4) was isolated from hardwood kraft pulp. It was capable of growing in diluted kraft black liquor at pH 11.5 and produced 49 IU (mol xylose min^–1 ml^–1) of xylanase when cultivated in alkaline medium at pH 9. Maximal enzyme activity was obtained by cultivation in a defined alkaline medium with 2% birchwood xylan and 1% corn steep liquor at pH 9, but high enzyme production was also obtained on wheat bran. The apparent pH optimum of the enzyme varied with the pH used for cultivation and the buffer system employed for enzyme assay. With cultivation at pH 10 and assays performed in glycine buffer, maximal activity was observed at pH 8.5; with phosphate buffer, maximal activity was between pH 6 and 7. The xylanase temperature optimum (at pH 7.0) was 55°C. In the absence of substrate, at pH 9.0, the enzyme was stable at 50°C for at least 30 min. Elecrophoretic analysis of the crude preparation showed one predominant xylanase with an alkaline pl. Biobleaching studies showed that the enzyme would brighten both hardwood and softwood kraft pulp and release chromophores at pH 7 and 9. Because kraft pulps are alkaline, this enzyme could be used for prebleaching with minimal pH adjustment.     

The presence of thiolated compounds allows the immobilization of enzymes on glyoxyl agarose at mild pH values: New strategies of stabilization by multipoint covalent attachment

Highly activated glyoxyl-supports rapidly immobilize proteins at pH 10 (where the -amino groups of the Lys groups of the protein surface are very reactive), and stabilize them by multipoint covalent attachment. However, they do not immobilize proteins at pH 8. This paper shows that the enzyme immobilization at this mild pH value is possible by incubation of the enzymes in the presence of different thiolated compounds (dithiothreitol, DTT; was selected as optimal reagent). The thiolated compounds (even the not reducing ones) stabilized the imino bonds formed at pH 8 between the aldehydes in the support and the amino groups of the protein. However, thiolated compounds are unable to reduce the imino bonds or the aldehyde groups and a final reduction step (e.g., using sodium borohydride) was always necessary. After enzyme immobilization through the most reactive amino group of the protein, the further incubation of this immobilized enzyme at pH 10 would improve the reactivity of the -amino groups of the Lys residues of the protein surface. Then, an intense multipoint covalent reaction of the enzyme with the dense layer of glyoxyl groups in the support could be obtained, increasing the stability of the immobilized enzyme. Using three different industrially relevant enzymes (penicillin G acylase from Escherichia coli (PGA), lipase from Bacillus thermocatenulatus (BTL2) and glutaryl acylase from Pseudomonas sp. (GA)), new immobilized-stabilized biocatalysts of the enzymes were produced. After reduction, the preparations incubated at pH 10 were more stable than those that were only immobilized and reduced at pH 8. In the case of the PGA, this preparation was even 4–5-fold more stable than those obtained by direct immobilization at pH 10 (around 40,000–50,000-fold more stable than the soluble enzyme).     

Purification and Properties of NADP-Dependent Maltose Dehydrogenase Produced by Alkalophilic Corynebacterium sp. No. 93–1

NADP-dependent maltose dehydrogenase (NADP-MalDH) was completely purified from the cell free extract of alkalophilic Corynebacterium sp. No. 93–1. The molecular weight of the enzyme was estimated as 45,000~48,000. The enzyme did not have a subunit structure. The isoelectric point of the enzyme was estimated as pH 4.48. The pH optimum of the enzyme activity was pH 10.2, and it was stable at pH 6 to 8. The temperature optimum was 40°C, and the enzyme was slightly protected from heat inactivation by 1 mm NADP. The enzyme oxidized d-xylose, maltose and maltotriose, and the Km values for these substrates were 150mm, 250 mm and 270 mm, respectively. Maltotetraose and maltopentaose were suitable substrates. The Km value for NADP was 1.5 mm with 100mm maltose as substrate. The primary product of this reaction from maltose was estimated as maltono-δ-lactone, and it was hydrolyzed non-enzymatically to maltobionic acid. The enzyme was inhibited completely by PCMB, Ag⁺ and Hg²⁺.     

Stabilization and functional properties of Escherichia coli penicillin G acylase by covalent conjugation of anionic polysaccharide carboxymethylcellulose

The stabilization of Escherichia coli penicillin G acylase (PGA) conjugated with carboxymethylcellulose (CMC) against temperature and pH was studied. The 2,3-dialdehyde derivative of CMC obtained by periodate oxidation was covalently conjugated to PGA via Schiff's base formation. The inactivation mechanism of both native and CMC-conjugated PGA appeared to obey first order inactivation kinetics during prolonged incubations at 40–60 °C and in the pH range 4–9. Inactivation rate constants of conjugated enzyme were always lower, and half-life times were always higher than that of native PGA. The activation free energy of inactivation (G _i values) of CMC-conjugated enzyme were found to be always higher than that of native PGA at all temperatures and pH values studied as another indicator of enzyme stabilization. Highest stability of CMC-conjugated enzyme was observed as nearly four-fold at 40 °C and pH 8.0. No changes were observed on the temperature and pH profiles of PGA after CMC conjugation. Lower K _m and higher k _cat values of PGA obtained after CMC conjugation indicates the improved effect of conjugation on the substrate affinity and catalytic performance of the enzyme.     

Purification and Characterization of DNA Polymerase Obtained from the Membrane Fraction of E. coli

Purification studies were conducted on DNA polymerase bound to the membrane fraction of E. coli HF 4704. Purified enzyme (Fraction V) required Mg²⁺ and showed an optimun pH of 7.2. Various kinds of salt indicated a stimulative effect at concentrations lower than 0.1 m. Fraction V was unstable at an acidic condition (pH 5.0) but was rather stable at an alkaline condition (pH 9.0). The enzyme activity was lost by incubation at 45°C for 30min but was stabilized by the addition of DNA. The enzyme contained exonuclease activity but no endonuclease activity. The enzyme produced only light density DNA of various sizes. The function of this enzyme as considered to fill single stranded region of the double stranded primer DNA.     

Variation in pH Optima of Hydrolytic Enzyme Activities in Tropical Rain Forest Soils

Extracellular enzymes synthesized by soil microbes play a central role in the biogeochemical cycling of nutrients in the environment. The pH optima of eight hydrolytic enzymes involved in the cycles of carbon, nitrogen, phosphorus, and sulfur, were assessed in a series of tropical forest soils of contrasting pH values from the Republic of Panama. Assays were conducted using 4-methylumbelliferone-linked fluorogenic substrates in modified universal buffer. Optimum pH values differed markedly among enzymes and soils. Enzymes were grouped into three classes based on their pH optima: (i) enzymes with acidic pH optima that were consistent among soils (cellobiohydrolase, β-xylanase, and arylsulfatase), (ii) enzymes with acidic pH optima that varied systematically with soil pH, with the most acidic pH optima in the most acidic soils (α-glucosidase, β-glucosidase, and N-acetyl-β-glucosaminidase), and (iii) enzymes with an optimum pH in either the acid range or the alkaline range depending on soil pH (phosphomonoesterase and phosphodiesterase). The optimum pH values of phosphomonoesterase were consistent among soils, being 4 to 5 for acid phosphomonoesterase and 10 to 11 for alkaline phosphomonoesterase. In contrast, the optimum pH for phosphodiesterase activity varied systematically with soil pH, with the most acidic pH optima (3.0) in the most acidic soils and the most alkaline pH optima (pH 10) in near-neutral soils. Arylsulfatase activity had a very acidic optimum pH in all soils (pH ≤3.0) irrespective of soil pH. The differences in pH optima may be linked to the origins of the enzymes and/or the degree of stabilization on solid surfaces. The results have important implications for the interpretation of hydrolytic enzyme assays using fluorogenic substrates.Measurements of the activities of extracellular enzymes involved in the turnover of nutrients from organic compounds provide important information on biogeochemical cycles in tropical soils (13, 59, 63). In particular, they are the primary mechanism by which microbes decompose organic matter and can provide key information on the nutrient status of the ecosystem (45, 60). For example, changes in the activities of phosphatase and N-acetyl-β-glucosaminidase in soil chronosequences reflect long-term changes in nitrogen and phosphorus availability during pedogenesis in both tropical and temperate rain forests (3, 34).The pH of the soil solution exerts a strong control on enzyme activity, because it influences the conformation of the enzyme, its adsorption on solid surfaces, and the ionization and solubility of substrates and cofactors (38, 51). Although some studies have determined enzyme activity at the soil pH (e.g., references 19 and 63), assays are usually conducted at the optimum pH for the enzyme, which yields a measure of its maximum potential activity at a given temperature (7, 27). For example, the assay of acid phosphomonoesterase activity using the chromogenic substrate para-nitrophenyl phosphate is typically performed at pH 6.5 (51), based on the determination of the pH optima of this enzyme in a series of temperate agricultural soils (15, 21, 50).Once the optimum pH for a given enzyme has been determined, it is usually assumed that this will apply broadly to other soils, allowing the recommendation of a single buffer pH in standardized procedures (44, 51). However, the pH optima of some enzymes can vary markedly among soils. For example, Niemi and Vepsäläinen (33) reported soil-specific pH optima for three hydrolytic enzymes (acid phosphomonoesterase, phosphodiesterase, and N-acetyl-β-glucosaminidase) in six soils under contrasting land uses in Finland. Such soil-specific pH optima led Malcolm (27) to recommend, in a critique of soil enzyme assays, that the pH optimum of the enzyme under study should be determined for each soil.Differences among soils in relation to the pH optima of an individual enzyme might be due to a variety of factors, including the composition of the soil microbial community (i.e., if isoenzymes originating from different organisms have different pH optima) and the location of the enzyme in the soil matrix (e.g., intracellular, free in solution, or adsorbed on solid surfaces, etc.) (4). For example, the sorption of an enzyme on a clay surface can increase its optimum pH by one or two pH units relative to that of the same enzyme in solution (31, 40). This is due to “unfolding” of enzymes on solid surfaces, which is most likely to occur at soil pH values below the isoelectric point of the enzyme (26, 38).Information on the optimum pH of enzyme activity is of particular importance for studies that use fluorogenic substrates in multiwell plates to assay several enzymes simultaneously. Such studies are usually simplified by assaying all enzymes in a single buffer, such as acetate at pH 5.5 (57) or 2-(N-morpholino)ethanesulfonic acid (MES) at pH 6.1 (30). However, this may not coincide with the pH optima of all the enzymes involved, especially if the optimum pH for a given enzyme varies among soils. Further, most studies of the pH optima of hydrolytic enzymes have been conducted using chromogenic substrates linked to para-nitrophenol, and it is not clear whether the values correspond to the pH optima for fluorogenic substrates linked to 4-methylumbelliferone.Here I report the pH optima for eight hydrolytic enzymes involved in the cycles of carbon, nitrogen, phosphorus, and sulfur in a series of soils under a lowland tropical rain forest in the Republic of Panama. The aim was to determine the extent to which the pH optima of activity varied among enzymes and soils, in order to develop a method suitable for the measurement of enzyme activities in a broad range of tropical rain forest soils.     

Regulation of horse-liver glutathione reductase

• 1.1. The enzyme was rapidly inactivated by NAD(P)H, GSH, dithionite or borohydride, while activity increased in the presence of NAD(P)+ or GSSG. NADH was more efficient for inactivation than NADPH. Redox inactivation required neutral or alkaline pH, was maximal at pH 8.5, and depended on the presence of metal cations.
• 2.2. GSSG and dithiothreitol fully protected the enzyme from inactivation at concentrations stoichiometric with NAD(P)H. Ten-fold higher ferricyanide or GSH concentrations were required to obtain partial protection. NAD+ or NADP+ were quite ineffective.
• 3.3. GSSG fully reactivated the inactive enzyme at 38°C and neutral to acidic pH (5.5–7.5). Reactivation by dithiothreitol was accomplished in short periods of time at pH 8.5 although the activity was progressively lost afterwards. Ferricyanide and GSH also reactivated the enzyme to different extents.

     

Studies on Isomerization of Sugars by Bacteria

An enzyme, which catalyzes the isomerization of d-glucose to d-fructose, has been found in a newly isolated bacterium which tentatively identified as Pacacolobacterum aerogenoides. The enzyme converts not only d-glucose but also d-mannose to d-fructose, and NAD and Mg⁺⁺ are required as cofactor for this isomerization. The properties of this enzyme were summarized as follows: (1) As a cofactor for the isomerization by this enzyme, NAD was absolutely necessary, whereas NADP, FMN and FAD were not. (2) The optimum pH was found to be at 7.5 and optinum temperature was at about 40°C. (3) The enzyme activity was markedly reduced by EDTA treatment and the reduced activity by EDTA was restored by the addition of Mg⁺⁺, Mn⁺⁺ or Co⁺⁺. (4) The enzyme activity was strongly inhibited by monoiodoacetate, p-chloromercuribenzoate, and Cu⁺⁺, however, the activity was recovered by adding cysteine or glutathione.     

Penicillinase (β-lactamase) formation by blue-green algae

-Lactamase (penicillinase) activity was found in a number of strains of blue-green algae. In some cases, this enzyme permitted algae to overcome the inhibitory effects of penicillin. Production and localization of -lactamase were studied in a unicellular species, Coccochloris elabens (strain 7003), and in a filamentous, nitrogen-fixing Anabaena species (strain 7120). When cells were grown in a neutral medium with NaNO₃ as N source, the pH rose during growth; at a pH of about 10, most of the enzyme was extracellular and all the cell-bound enzyme was expressed equally well in intact or disrupted cells. If the pH was kept near neutrality during growth by gassing with CO₂ in N₂ or by growth under conditions of N₂ fixation, the enzyme remained cell-bound and cryptic for most of the growth phase, being measurable only after cells were disrupted. The enzymes from strains 7003 and 7120 had greater activity on benzyl penicillin and other penicillins than on cephalo-sporins. Some differences were observed in the substrate profiles of penicillinases from the two strains against different penicillins.A preliminary account of this work was presented at the 1974 meetings of the American Society for Microbiology in Chicago (Abstracts of Meetings, M37)     

Purification and properties of protein methylase II

Protein methylase II (S-adenosyl-methionine:protein-carboxyl methyltransferase) from calf thymus was purified approximately 2400-fold with a yield of 7% by incorporating the pH 5.1 treatment and QAE (triethylaminoethyl)-Sephadex column chromatography to the published purification steps (Kim and Paik (1970) J. Biol. Chem., 245, 1806). The enzyme is found stable at pH 10.2, but loses 50% of its activity in 60 min at pH 5. The enzyme activity disappeared in 8 m urea 2.5 m guanidine hydrochloride at pH 8.0. However, about 80% of the activity returned upon dialysis of the mixture. The highly purified enzyme is stable for at least 2 yr in the presence of 50% glycerol at pH 8.0 or in the form of lyophilized powder. Protein methylase II from different tissues exhibits different pI values, determined by isoelectrofocusing; 4.85 with the enzyme preparation isolated from calf thymus, 5.8 from calf spleen, and 5.08 from rat testis. Reinvestigation of the methanol-forming enzyme system from calf posterior pituitary gland by Axelrod and Daly [Science 150, 892 (1965)] indicated that this enzyme is identical with protein methylase II.     

Immobilization of Bacillus licheniformis L-Arabinose Isomerase for Semi-Continuous L-Ribulose Production

Bacillus licheniformis L-arabinose isomerase (BLAI) with a broad pH range, high substrate specificity, and high catalytic efficiency for L-arabinose was immobilized on various supports. Eupergit C, activated-carboxymethylcellulose, CNBr-activated agarose, chitosan, and alginate were tested as supports, and Eupergit C was selected as the most effective. After determination of the optimum enzyme concentration, the effects of pH and temperature were investigated using a response surface methodology. The immobilized BLAI enzyme retained 86.4% of the activity of the free enzyme. The optimal pH for the immobilized BLAI was 8.0, and immobilization improved the optimal temperature from 50 °C (free enzyme) to a range between 55 and 65 °C. The half life improved from 2 at 50 °C to 212 h at 55 °C following immobilization. The immobilized BLAI was used for semi-continuous production of L-ribulose. After 8 batch cycles, 95.1% of the BLAI activity was retained. This simple immobilization procedure and the high stability of the final immobilized BLAI on Eupergit C provide a promising solution for large-scale production of L-ribulose from an inexpensive L-arabinose precursor.     

Kinetic and allosteric properties of highly-purified, biosynthetic L-threonine dehydrogenase from brewer's yeast Saccharomyces carlsbergensis

Kinetic and allosteric propeties of highly purified "biosynthetic" L-threonine dehydratase from brewer's yeast S. carlbergensis were studied at three pH values, using L-threonine and L-serine as substrates. It was shown that the plot of the initial reaction rate (v) versus initial substrate concentrations ([S]0 pH 6.5 is hyperbolic (Km=5.0.10-2M), while these at pH 7.8 and 9.5 have a faintly pronounced sigmoidal shape with fast occurring saturation plateaus ([S]0.5= 1.0.10-2 and 0.9.10-2M, respectively). the ratios between L-threonine and L-serine dehydratation rates depend on pH. The kinetic properties and the dependence of substrate specificity on pH suggest that the enzyme molecule undergoes pH-induced (at pH 7.0) conformational changes. The determination of pK values of the enzyme functional groups involved in L-threonine binding demonstrated that these groups have pK is approximately equal to 7.5 and 9.5. The latter group was hypothetically identified as a epsilon-NH2-group of the lysine residue. High concentrations of the allosteric inhibitor (L-isoleucine) decrease the rates of L-threonine and L-serine dehydratation and induce the appearance (at pH 6.5) or increase (at pH 7.9 and 9.5) of homotropic cooperative interactions between the active sites in the course of L-threonine dehydratation. The enzyme inhibition by L-isoleucine increases with a decrease of L-threonine concentrations. Low L-isoleucine concentrations, as well as the enzyme activator (L-valine) stimulate the enzyme at non-saturating substrate concentrations (when L-threonine or L-serine are used as substrates) without normalization of (v) versus [S]0 plots. The maximal activation of the enzyme is observed at pHG 8.5--9.0. It is assumed that the molecule of "biosynthetic" L-threonine dehydratase from brewer's yeast contains two types of sites responsible for the effector binding, i.e., "activatory" and "inhibitory" ones.     

Kinetic and Structural Properties of NADP-Malic Enzyme from Sugarcane Leaves

Oligomeric structure and kinetic properties of NADP-malic enzyme, purified from sugarcane (Saccharam officinarum L.) leaves, were determined at either pH 7.0 and 8.0. Size exclusion chromatography showed the existence of an equilibrium between the dimeric and the tetrameric forms. At pH 7.0 the enzyme was found preferentially as a 125 kilodalton homodimer, whereas the tetramer was the major form found at pH 8.0. Although free forms of l-malate, NADP⁺, and Mg²⁺ were determined as the true substrates and cofactors for the enzyme at the two conditions, the kinetic properties of the malic enzyme were quite different depending on pH. Higher affinity for l-malate (K_m = 58 micromolar), but also inhibition by high substrate (K_i = 4.95 millimolar) were observed at pH 7.0. l-Malate saturation isotherms at pH 8.0 followed hyperbolic kinetics (K_m = 120 micromolar). At both pH conditions, activity response to NADP⁺ exhibited Michaelis-Menten behavior with K_m values of 7.1 and 4.6 micromolar at pH 7.0 and 8.0, respectively. Negative cooperativity detected in the binding of Mg²⁺ suggested the presence of at least two Mg²⁺ - binding sites with different affinity. The K_a values for Mg²⁺ obtained at pH 7.0 (9 and 750 micromolar) were significantly higher than those calculated at pH 8.0 (1 and 84 micromolar). The results suggest that changes in pH and Mg²⁺ levels could be important for the physiological regulation of NADP-malic enzyme.     

Isolation and characterization of a trypsin fraction from the pyloric ceca of chinook salmon (Oncorhynchus tshawytscha)

A trypsin fraction was isolated from the pyloric ceca of New Zealand farmed chinook salmon (Oncorhynchus tshawytscha) by ammonium sulfate fractionation, acetone precipitation and affinity chromatography. The chinook salmon enzyme hydrolyzed the trypsin-specific synthetic substrate benzoyl-dl-arginine-p-nitroanilide (dl-BAPNA), and was inhibited by the general serine protease inhibitor phenyl methyl sulfonyl fluoride (PMSF), and also by the specific trypsin inhibitors — soybean trypsin inhibitor (SBTI) and benzamidine. The enzyme was active over a broad pH range (from 7.5 to at least pH 10.0) at 25 °C and was stable from pH 4.0 to pH 10.0 when incubated at 20 °C, with a maximum at pH 8.0. The optimum temperature for the hydrolysis of dl-BAPNA by the chinook salmon enzyme was 60 °C, however, the enzyme was unstable at temperatures above 40 °C. The molecular mass of the chinook salmon trypsin was estimated as 28 kDa by SDS–PAGE.