Cu(II) catalysed hydrolysis of thiaminepyrophosphate: structure-reactivity relationship

The kinetics of the Cu(II) catalysed hydrolysis of thiamine-pyrophosphate (TPP) has been studied in aqueous solution at 56,64 and 78° over a pH fange of 3.0 to 7.0 at a constant ionic strength of 0.10 M (KNO₃). The pH rate profiles were analysed and the overall rate constants resolved into individual specific rate constants relating to various Cu:TPP chelate species in solution. Activation parameters ΔH², ΔS^° and ΔG² for the specific rate constants of various chelate species of TPP are reported. The possible mechanism of the Cu(II) promoted hydrolysis of TPP is discussed. The structure-reactivity relationship is also discussed.     

Mechanisms protecting plasma peptides from enzyme hydrolysis: a comparative study

1. The role of the enkephalin-protecting plasma substances in the protection of non-opioid peptides from enzyme hydrolysis has been studied in laboratory animals and in man. 2. The results obtained indicate that all the peptides hydrolyzed by the plasma enzymes are also protected from the hydrolysis by the enkephalin-protecting substances. 3. The protection is fairly uniform in all the species and for all the peptides examined. However, in the human species the protection of leucine enkephalin is considerably higher than the average. These results are discussed in terms of a possible differential inhibition of the different plasma aminopeptidases.     

Role of enzymes and inhibitors in leu-enkephalin metabolism in rabbit and human plasma

The hydrolysis of leucine enkephalin by the proteolytic enzymes present in human and rabbit plasma has been studied by kinetic and chromatographic techniques. Data obtained indicate the existence of noticeable intraspecific differences in the kinetics of leu-enkephalin degradation, and of formation of its hydrolysis by-products. The separation of the enzymes active on the substrate and of the inhibitors active on these enzymes evidences the existence of a species specific distribution of both groups of substances. Yet, the dissimilar kinetics of the substrate hydrolysis and of formation of its hydrolysis by-products appear to arise more from diversities in the competition between the enzymes present in plasma and in the role of inhibitors than from the differences in the enkephalin-degrading enzymes. It is suggested that differences observed may be related to the existence of species specific populations of the information-carrying plasma peptides.     

Kinetic studies of enzymatic hydrolysis of insoluble cellulose: analysis of the initial rates

A study was conducted on the kinetics of enzymatic hydrolysis of pure insoluble cellulose using unpurified culture filtrate Trichoderma reesei, with the emphasis on the initial reaction period. The initial hydrolysis rate and extent of enzyme (soluble protein)adsorption, either apparent or initial, were evaluated under various experimental conditions. It has been found that the various mass-transfer steps do not control the overall hydrolysis rate and that the hydrolysis rate is mainly controlled by the surface reaction step promoted by the adsorbed enzyme. It has also been found that the initial hydrolysis rate strongly depends on the initial extent of soluble protein adsorption and the effectiveness of the adsorbed soluble protein to promote the hydrolysis. The initial extent of soluble protein adsorption, in turn, is related to the initial cellulose concentration, enzyme concentration, and specific surface area of cellulose, whereas the effectiveness of the initially adsorbed soluble protein to promote the derived to interrelate these parameters without resorting to the Michaelis-Menten kinetics. The present result appear to imply that the role of enzyme-substrate complex formation should not be ignored in deriving a mechanistic kinetic model for enzymatic hydrolysis of cellulose.     

Loss of enzyme activity during turnover of the Bacillus cereus β-lactamase catalysed hydrolysis of β-lactams due to loss of zinc ion

Metallo-beta-lactamases are zinc-ion-dependent and are known to exist either as mononuclear or as dinuclear enzymes. The kinetics and mechanism of hydrolysis of the native zinc Bacillus cereus metallo-beta-lactamase (BcII) have been investigated under pre-steady-state conditions at different pHs and zinc-ion concentrations. Biphasic kinetics are observed for the hydrolysis of cefuroxime and benzylpenicillin with submicromolar concentrations of enzyme and zinc. The initial burst of product formation far exceeds the concentration of enzyme and the subsequent slower rate of hydrolysis is attributed to a branched kinetic pathway. The pH and metal-ion dependence of the microscopic rate constants of this branching were determined, from which it is concluded that two enzyme species with different metal-to-enzyme stoichiometries are formed during catalytic turnover. The dizinc enzyme is responsible for the fast route but during the catalytic cycle it slowly loses the less tightly bound zinc ion via the branching route to give an inactive monozinc enzyme; the latter is only catalytic following the uptake of a second zinc ion. The rate constant for product formation from the dinuclear enzyme and the branching rate constant show a sigmoidal dependence on pH indicative of important ionizing groups with pK (a)s of 9.0 +/- 0.1 and 8.2 +/- 0.1, respectively. The rate constant for the regeneration of enzyme activity depends on zinc-ion concentration. This unusual behaviour is attributed to an intrinsic property of metallo hydrolytic enzymes that depend on a metal bound water both as a ligand for the second metal ion and as the nucleophile which is consumed during hydrolysis of the substrate and so has to be replaced to maintain the catalytic cycle.     

Kinetics of the enzymatic hydrolysis of cellulose: 2. A mathematical model for the process in a plug-flow column reactor

The kinetics of enzymatic cellulose hydrolysis in a plug-flow column reactor catalysed by cellulases [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] from Trichoderma longibrachiatum adsorbed on cellulose surface have been studied. The maximum substrate conversion achieved was 90–94%. The possibility of enzyme recovery for a reactor of this type is discussed. A mathematical model for enzymatic cellulose hydrolysis in a plug-flow column reactor has been developed. The model allows for the component composition of the cellulase complex, adsorption of cellulases on the substrate surface, inhibition by reaction products, changes in cellulose reactivity and the inactivation of enzymes in the course of hydrolysis. The model affords a reliable prediction of the kinetics of d-glucose and cellobiose formation from cellulose in a column reactor as well as the degree of substrate conversion and reactor productivity with various amounts of adsorbed enzymes and at various flow rates.     

Kinetics of alpha-chymotrypsin catalyzed hydrolysis in equilibrium. II. Comparison of ester and amide substrates

Kinetic of the alpha-chymotrypsin catalyzed reversible hydrolytic reaction of methyl N-acetyl-L-phenylalaninate and N-acetyl-L-phenylalanylglycinamide at pH 5.5 and equilibrium conditions has been studied. The rates of the labeled reaction products incorporated into the substrate a different methanol concentrations shows that the reaction proceeds by a compulsory mechanism with the formation of N-acetyl-L-phenylalanine-alpha-chymotrypsin complex. For the amide substrate the data obtained are also in agreement with the compulsory mechanism of its hydrolysis. Equilibrium kinetics of ester and amide substrates hydrolysis has been compared.     

Urea hydrolysis by immobilized urease in a fixed-bed reactor: analysis and kinetic parameter estimation

Urea hydrolysis by urease immobilized onto ion exchange resins in a fixed-bed reactor has been studied. A modified Michaelis-Menten rate expression is used to describe the pH-dependent, substrate- and product-inhibited kinetics. Ionic equilibria of product and buffer species are included to account for pH changes generated by reaction. An isothermal, heterogeneous plug-flow reactor model has been developed. An effectiveness factor is used to describe the reaction-diffusion process within the particle phase. The procedure for covalent immobilization of urease onto macroporous cation exchangers is described. Urea conversion data are used to estimate kinetic parameters by a simplex optimization method. The best-fitted parameters are then used to predict the outlet conversions and pH values for systems with various inlet pH values, inlet urea and ammonia concentrations, buffers, particle sizes, and spacetimes. Very good agreement is obtained between experimental data and model predictions. This immobilized urease system exhibits quite different kinetic behavior from soluble urease because the pH near the enzyme active sites is different from that of the pore fluid. This effect results in a shift of the optimal pH value of the V(max) (pH) curve from 6.6 (soluble urease) to ca. 7.6 in dialysate solution, and ca. pH 8.0 in 20mM phosphate buffer. The reactor model is especially useful for estimating intrinsic kinetic parameters of immobilized enzymes and for designing urea removal columns.     

Studies of lipid turnover in cells with stable isotope and gas chromatograph-mass spectrometry

Phospholipids are major building blocks for biological membranes. In addition, metabolites derived from their degradation are important signals in major cellular events, such as proliferation and apoptosis. The concept of lipid signaling in cells is derived mainly from the measurement of change in the concentration of lipid molecules. However, these changes in concentration are only a small part of the underlying metabolic change induced by a perturbation in the cell. In contrast, metabolic kinetic studies documenting product-precursor relationships and turnover rates are useful in elucidating the responsible mechanisms. Historically, metabolic studies of phospholipids in cells have been carried out with pulse or pulse-chase methods using radioactive isotopes. While these studies provide valuable information, their scope is restricted by inherent limitations. In this paper we describe a method using [1,2,3,4-13C(4)]palmitate as the tracer for studying the metabolic kinetics of the molecular species of diacylglycerol, ceramide, phosphatidylcholine, and sphingomyelin. After growing cells in the presence of labeled palmitate complexed to serum albumin, the lipids are extracted and separated into lipid classes. After enzymatic hydrolysis, diacylglyerols and ceramides as bis-trimethylsilyl derivatives are determined quantitatively with capillary column gas chromatography. Internal standards for each lipid class are used in the procedure. In addition, the isotopic enrichments of the lipid molecular species are determined with gas chromatograph-mass spectrometry. We applied this method to the study of HL60 cells. Different turnover rates were found for various molecular species. In addition, the sn-1 and sn-2 acyl groups appear to be synthesized at different rates for different molecular species. Other information, such as chain elongation and desaturation, might also be derived through the use of this method.     

Carbachol stimulates a different phospholipid metabolism than nerve growth factor and basic fibroblast growth factor in PC12 cells.

We have examined 1,2-diglycerides (DGs) generated in PC12 cells in response to the muscarinic agonist carbachol and compared them with those generated in response to the differentiation factors nerve growth factor and basic fibroblast growth factor. Whereas carbachol stimulates a greater release of inositol phosphates, all three agonists generate similar levels of DGs. In this report, we have analyzed the molecular species of PC12 DGs generated in response to these three agonists. Additionally, we have analyzed the molecular species of PC12 phospholipids. The data indicate that 1) after 1 min of either nerve growth factor or basic fibroblast growth factor stimulation, DGs arise primarily from phosphoinositide hydrolysis; 2) in contrast, after 1 min of carbachol stimulation, DG are generated equally by both phosphoinositide and phosphatidylcholine hydrolysis; and 3) after 15 min of stimulation by any of these agonists, DGs are generated largely by phosphatidylcholine hydrolysis, with a smaller component arising from the phosphoinositides. These results suggest that at least part of the mechanism by which PC12 cells distinguish between different agonists is via alterations in phospholipid sources and kinetics of DG generation.     

Hydrolysis of oils by using immobilized lipase enzyme: A review

This review focuses on the use of immobilized lipase technology for the hydrolysis of oils. The importance of lipase catalyzed fat splitting process, the various immobilization procedures, kinetics, deactivation kinetics, New immobilized lipases for chiral resolution, reactor configurations, and process considerations are all reviewed and discussed.     

Cellulose hydrolysis in evolving substrate morphologies II: Numerical results and analysis

Numerical simulation results are presented for a cellulose hydrolysis model which incorporates both the enzymatic glucan chain fragmentation kinetics and the hydrolytic substrate morphology evolution within the general framework of our companion article I. To test the local Poisson (LP) approximation employed in the site number formalism of I, we numerically compare it to the corresponding exact chain number formalism of I. The LP results agree to very high accuracy with the exact chain number kinetics, assuming realistic parameters. From simulations of different types of random and non‐random model morphologies, we then show that the details of the random substrate morphology distribution, and its hydrolytic time evolution, profoundly affect the hydrolysis kinetics. Essential, likely very general, experimentally testable features of such morphology‐based hydrolysis models are (i) the existence of two distinct time scales, associated with the hydrolysis of the outermost surface‐exposed cellulose chains and, respectively, of the entire substrate; (ii) a strongly morphology‐dependent hydrolysis slow‐down effect, which has also been observed in previous experimental work. Our results also suggest that previously proposed non‐morphologic chain fragmentation models can only be applied to describe the hydrolytic short‐time behavior in the low enzyme limit. Further experiments to test our modeling framework and its potential applications to the optimization of the hydrolytic conversion process are discussed. Biotechnol. Bioeng. 2009; 104: 275–289 © 2009 Wiley Periodicals, Inc.     

黄河三角洲土壤运行多样性初步调查研究

1995－1996年对黄河三角洲9个代表性样区,进行了土壤运动定性,定量调查,并对古代,近代,现代黄河三角洲3个代表性样区开展了土壤动物周年调查,研究了土壤动物的种类组成,分布和节季变化,以及土壤动物多样性,调查研究结果,黄河三角洲共鉴定出土壤运动38种,土壤运动种类组成和数量分布因三个三角洲成土年龄不同而存在差异,土壤运动多样性以夏季为丰富,黄河三角洲土壤运动多样性]与均匀度表现为正相关关系,与单纯度呈负相关趋势。     

Kinetic and molecular species analyses of mitogen-induced increases in diglycerides: evidence for stimulated hydrolysis of phosphoinositides and phosphatidylcholine

A wide variety of agonist-induced events appear to be mediated through an increase in cellular diglyceride levels. With regard to the ability of diglycerides to mediate these events, three important parameters must be considered: a) the kinetics of diglyceride generation, b) the absolute mass levels, and c) their molecular species. While this increase is often due to a stimulated hydrolysis of phosphoinositides, there is increasing evidence that the stimulated hydrolysis of phosphatidylcholine also contributes to agonist-induced increases in diglyceride levels. The kinetics of mass increases in diglyceride levels stimulated in cultured fibroblasts are agonist-dependent. High concentrations of alpha-thrombin stimulate a biphasic increase in diglyceride levels with the first phase peaking at 15 s and the second phase peaking at 5 min. In contrast, stimulation with epidermal growth factor, or platelet-derived growth factor, results in a monophasic increase in cellular diglyceride levels. Furthermore, the molecular species and phospholipid source of the stimulated diglycerides are also agonist-dependent. While the hydrolysis of phosphoinositides is major source of diglycerides initially generated in response to some agonists (15 s with alpha-thrombin at 500 ng/ml), phosphatidylcholine is hydrolyzed as well. Following longer incubations, or at all times following stimulation by epidermal growth factor or platelet-derived growth factor, phosphatidylcholine hydrolysis is the principal source of the stimulated diglycerides.     

Cryptosporidium in wild placental mammals

Cryptosporidium species are common parasites of wild placental mammals. Recent parasitological studies combined with molecular genotyping techniques have been providing valuable new insight into the host specificity and potential transmission of various Cryptosporidium species/genotypes among animals and between these animals and humans. Although Cryptosporidium in wild animals may possess a potential public health problem due to oocyst contamination in the environment, studies at various regions of the world have indicated a strong host-adaptation by these parasites and a limited potential of cross-species transmission of cryptosporidiosis among placental mammals, suggesting that these animals are probably not a major reservoir for human infection. However, Cryptosporidium species/genotypes in placental animals have been reported occasionally in humans. Therefore, public health significance of some Cryptosporidium species in wild placental mammals, such as the cervine genotype, should not be overlooked and should be further studied.     

Bovine cardiac myosin subfragment 1. Transient kinetics of ATP hydrolysis

The kinetics of binding and hydrolysis of ATP by bovine cardiac myosin subfragment 1 has been reinvestigated. More than 90% of the total fluorescence amplitude associated with ATP hydrolysis occurs with an apparent second-order rate constant of 8.1 X 10(5) M-1 S-1 and a limiting rate constant of approximately 140 S-1 (100 mM KCl, 50 mM 1,3-bis-[tris(hydroxymethyl)methylamino]-propane, 10 mM MgCl2, pH 7.0, 20 degrees C); the remaining 10% occurs more slowly (approximately 1 S-1). The observed rate constants are independent of subfragment 1 concentration under pseudo first-order conditions for ATP with respect to protein. The fraction of protein which hydrolyzes ATP rapidly is not a function of the nucleotide or protein concentration and appears to be constant irrespective of ionic strength or temperature within the range studied (50-100 mM KCl, pH 7.0, 15-20 degrees C). These data are compared to that obtained previously using subfragment 1 prepared by a different method which showed ATP-dependent aggregation of two protein species.     

Thermodynamic arguments for temperature-induced cryptic conformational change of human plasma cholinesterase

The temperature and pressure dependence of the kinetics of the hydrolysis of o-nitrophenylbutyrate by human plasma tetrameric form cholinesterase (EC 3.1.1.8) was studied. The study was carried out on the one hand at atmospheric pressure by spectrophotometry at various temperatures ranging from 0 to 40 degrees C and, on the other hand by high-pressure stopped-flow spectrophotometry at 3.5, 25 and 35 degrees C in the pressure range 10(-3) to 2 kbar. The Arrhenius plot showed a break at 21 +/- 1 degrees C. Kinetic parameters, activation parameters and volume changes are reported. Discontinuities in the thermodynamic quantities obtained from temperature and pressure (up to 0.8 kbar) dependence of hydrolysis rates are discussed; they have been interpreted as the result of a temperature-induced cryptic conformational change of the enzyme at around 20 degrees C. Beyond 1 kbar the kinetics exhibited several complexities: curvature of the progress curves and high positive or negative activation volume changes depending on temperature and substrate concentration. These complex interacting effects between temperature, pressure and substrate concentration are discussed.     

Comparison of serum iron, total iron binding capacity, ferritin, and percent transferrin saturation in nine species of apparently healthy captive lemurs

Lemurs kept in captivity have been reported to be highly prone to accumulate excessive amounts of iron in tissues (hemosiderosis). Diagnosis of the condition is most commonly made during a postmortem examination because an antemortem diagnosis requires a liver biopsy, a procedure that may not be well tolerated by all animals. The lack of a noninvasive method to evaluate iron status in captive lemurs limits investigators' ability to effectively screen animals for the presence of hemosiderosis, and to detect the condition early when treatment protocols are most effective. This study was conducted in an effort to provide data regarding iron analyte values in healthy captive lemurs of multiple species. The relationship of various iron-related metabolites was evaluated in 177 clinically normal lemurs of nine different species. Serum iron (sI), total iron binding capacity (TIBC), and ferritin concentration were measured directly and the percent transferrin saturation (TS) was calculated. Significant differences in various iron metabolites were observed among several species, suggesting that normal reference values for iron metabolites in lemurs may need to be developed on a species by species basis.     

MEMBRANE-BOUND NEURAMINIDASE IN THE BRAIN OF DIFFERENT ANIMALS: BEHAVIOUR OF THE ENZYME ON ENDOGENOUS SIALO DERIVATIVES AND RATIONALE FOR ITS ASSAY

—The activity of brain membrane-bound neuraminidase on endogenous and exogenous substrates was comparatively studied in various animals (rat, chicken, rabbit, pig, calf and human). The maximum rate of hydrolysis of endogenous substrates by membrane-bound neuraminidase (using a crude preparation of the enzyme) was different in the various animals (from 0·05 to 0·73 units, referred to 1 mg protein) and was obtained under similar but not identical optimum conditions (pH from 4·1 to 5·1; requirement or not of Triton X-100). The maximum degree of hydrolysis of endogenous substates was also different (from 15 to 27 nmol released NeuNAc/mg protein) and was obtained within different incubation periods (from 2 to 18 h). It corresponded (in rabbit, calf, human brain only), or not, to the actual exhaustion of the endogenous substrates. The endogenous substrates were recognized as both gangliosides and sialoglycoproteins. The extent of hydrolysis of sialoglycoproteins varied from 1·5% in rabbit to 15·6% in chicken brain; the hydrolysis of gangliosides (ranging from 14·1% in pig to 53·7% in rabbit brain) reached only in some animals (rabbit, calf, human) the complete transformation of major oligosialogangliosides into the neuraminidase resistant monosialoganglioside GMI. Upon addition of exogenous substrates (sialyl-lactose, ganglioside GD1a, brain sialopeptides, ovine submaxillary mucin) the actual rate of liberation of total NeuNAc (from both endogenous and exogenous substrates) considerably exceeded, although at a different extent (depending on the animal and on the added substrate used) the rate of hydrolysis of sole endogenous substrates. The possibility of an accurate assay of brain membrane-bound neuraminidase in a crude enzyme preparation is evaluated and guidelines for the assay procedure suggested.     

Preparation, properties and biological applications of water soluble chitin oligosaccharides from marine organisms

Chitin oligosaccharides (COSs) can be isolated from various natural resources, which have widely been used in biological active supplements (BAS) for the benefit of humankind. Several technological approaches for the preparation of COSs such as enzymatic, chemical, acid-catalysts hydrolysis, microwave radiation, membrane bioreactor methods have been developed and among them, membrane bioreactor, bioconversion and continuous mass production technologies are reported to be excellent. Compounds isolated from natural products have made a drastic impact on the pharmaceutical industry and especially, water-soluble chitin oligosaccharides have shown greater clinical activity, which have been demonstrated in various cell lines of disease significance. The activities of these COSs were being investigated in different patients, animals and even plants as a broad phase clinical trial program. In the present article, we have discussed the COSs preparation by different methods through comprehensive diffraction procedures along with the merits and demerits given in detail. In addition, a summary of recent work describing the synthesis and biological activities of water-soluble COSs has been presented here.