Relative Concentration Of Enzyme Research Articles

Coupled chemistry kinetics demonstrate the utility of functionalized Sup35 amyloid nanofibrils in biocatalytic cascades

Concerns over the environment are a central driver for designing cell-free enzymatic cascade reactions that synthesize non–petrol-based commodity compounds. An often-suggested strategy that would demonstrate the economic competitiveness of this technology is recycling of valuable enzymes through their immobilization. For this purpose, amyloid nanofibrils are an ideal scaffold to realize chemistry-free covalent enzyme immobilization on a material that offers a large surface area. However, in most instances, only single enzyme–functionalized amyloid fibrils have so far been studied. To embark on the next stage, here we displayed xylanase A, β-xylosidase, and an aldose sugar dehydrogenase on Sup35(1–61) nanofibrils to convert beechwood xylan to xylonolactone. We characterized this enzymatic cascade by measuring the time-dependent accumulation of xylose, xylooligomers, and xylonolactone. Furthermore, we studied the effects of relative enzyme concentrations, pH, temperature, and agitation on product formation. Our investigations revealed that a modular cascade with a mixture of xylanase and β-xylosidase, followed by product removal and separate oxidation of xylose with the aldose sugar dehydrogenase, is more productive than an enzyme mix containing all of these enzymes together. Moreover, we found that the nanofibril-coupled enzymes do not lose activity compared with their native state. These findings provide proof of concept of the feasibility of functionalized Sup35(1–61) fibrils as a molecular scaffold for biocatalytic cascades consisting of reusable enzymes that can be used in biotechnology.

Degradation of proteoglycan 4/lubricin by cathepsin S: Potential mechanism for diminished ocular surface lubrication in Sjögren's syndrome

Sjögren's syndrome (SS) is an autoimmune disease affecting the lacrimal and salivary glands with hallmark clinical symptoms of dry eye and dry mouth. Recently, markedly increased cathepsin S (CTSS) activity has been observed in the tears of SS patients. Proteoglycan 4 (PRG4), also known as lubricin, is an effective boundary lubricant that is naturally present on the ocular surface. While PRG4 is susceptible to proteolytic digestion, the potential effect of CTSS on PRG4 remains unknown. The objective of this study was to assess the ability of CTSS to enzymatically degrade purified PRG4, and PRG4 naturally present in human tears, and alter ocular surface boundary lubricating properties. To assess the potential time course and dose-dependency of PRG4 digestion by CTSS, full-length recombinant human PRG4 (rhPRG4) was incubated at 37 °C with or without CTSS in an enzymatic digestion buffer. Digestion of PRG4 by CTSS was also examined within normal human tear samples, both with and without supplementation by rhPRG4. Finally, digestion of endogenous PRG4 by CTSS, and the effect of a CTSS inhibitor, was examined in SS tears on Schirmer strips. Digestion products were separated on 3–8% SDS-PAGE and visualized by protein staining and western blotting. The boundary lubricating ability of rhPRG4 samples was assessed using an in vitro human eyelid-cornea friction test. Finally, SDS-PAGE protein stain bands resulting from rhPRG4 digestion were submitted for tandem mass spectrometry analysis to confirm their identity as PRG4 and identify non-tryptic cleavage sites. CTSS digested rhPRG4 in a time and dose dependent manner. CTSS digestion of rhPRG4 at 1% (where % is the mass ratio of CTSS to rhPRG4) resulted in a time dependent decrease in the full-length, ∼460 kDa, monomeric rhPRG4 band, and an appearance of lower MW fragments. After 20 h, no full-length rhPRG4 was observed. Furthermore, with an increased relative enzyme concentration of 3%, no protein bands were observed after 2 h, indicating complete digestion of rhPRG4. Western blotting demonstrated PRG4 is present in normal human tears, and that rhPRG4, tears, and tears supplemented with rhPRG4 incubated with 3–9% CTSS demonstrated decreased intensity of high MW PRG4 bands, indicative of partial degradation by CTSS. Similarly, western blotting of PRG4 in SS tears incubated with CTSS demonstrated decreased intensity of high MW PRG4 bands, which was reversed in the presence of the CTSS inhibitor. CTSS treatment of rhPRG4 resulted in an increased friction coefficient, compared to untreated controls. Lastly, the lower MW bands were confirmed to be PRG4 fragments by tandem mass spectrometry, and 6 non-tryptic cleavage sites were identified. rhPRG4 is susceptible to proteolytic digestion by CTSS, both alone and in human tears, which results in diminished ocular surface boundary lubricating ability. Moreover, endogenous PRG4 is susceptible to proteolytic digestion by CTSS, both in normal and SS tears. Given the elevated activity of CTSS in SS tears, and the role intact PRG4 plays in ocular surface health and lubrication, degradation of PRG4 by CTSS is a potential mechanism for diminished ocular surface lubrication in SS. Collectively these results suggest that tear supplementation of PRG4 may be beneficial for SS patients.

Amino Groups of Chitosan Are Crucial for Binding to a Family 32 Carbohydrate Binding Module of a Chitosanase from Paenibacillus elgii

We report here the role and mechanism of specificity of a family 32 carbohydrate binding module (CBM32) of a glycoside hydrolase family 8 chitosanase from Paenibacillus elgii (PeCsn). Both the activity and mode of action of PeCsn toward soluble chitosan polymers were not different with/without the CBM32 domain of P. elgii (PeCBM32). The decreased activity of PeCsn without PeCBM32 on chitosan powder suggested that PeCBM32 increases the relative concentration of enzyme on the substrate and thereby enhanced enzymatic activity. PeCBM32 specifically bound to polymeric and oligomeric chitosan and showed very weak binding to chitin and cellulose. In isothermal titration calorimetry, the binding stoichiometry of 2 and 1 for glucosamine monosaccharide (GlcN) and disaccharide (GlcN)2, respectively, was indicative of two binding sites in PeCBM32. A three-dimensional model-guided site-directed mutagenesis and the use of defined disaccharides varying in the pattern of acetylation suggested that the amino groups of chitosan and the polar residues Glu-16 and Glu-38 of PeCBM32 play a crucial role for the observed binding. The specificity of CBM32 has been further elucidated by a generated fusion protein PeCBM32-eGFP that binds to the chitosan exposing endophytic infection structures of Puccinia graminis f. sp. tritici Phylogenetic analysis showed that CBM32s appended to chitosanases are highly conserved across different chitosanase families suggesting their role in chitosan recognition and degradation. We have identified and characterized a chitosan-specific CBM32 useful for in situ staining of chitosans in the fungal cell wall during plant-fungus interaction.

Lipopolysaccharide O antigen size distribution is determined by a chain extension complex of variable stoichiometry in Escherichia coli O9a.

The lengths of bacterial polysaccharides can be critical for their biological function. Unlike DNA or protein synthesis, where polymer length is implicit in the nucleic acid template, the molecular mechanisms for regulating polysaccharide length are poorly understood. Two models are commonly cited: a "molecular clock" regulates length by controlling the duration of the polymer extension process, whereas a "molecular ruler" determines length by measurement against a physical structure in the biosynthetic complex. Escherichia coli O9a is a prototype for the biosynthesis of O polysaccharides by ATP-binding cassette transporter-dependent processes. The length of the O9a polysaccharide is determined by two proteins: an extension enzyme, WbdA, and a termination enzyme, WbdD. WbdD is known to self-oligomerize and also to interact with WbdA. Changing either enzyme's concentration can alter the polysaccharide length. We quantified the O9a polysaccharide length distribution and the enzyme concentration dependence in vivo, then made mathematical models to predict the polymer length distributions resulting from hypothetical length-regulation mechanisms. Our data show qualitative features that cannot be explained by either a molecular clock or a molecular ruler model. Therefore, we propose a "variable geometry" model, in which a postulated biosynthetic WbdA-WbdD complex assembles with variable stoichiometry dependent on relative enzyme concentration. Each stoichiometry produces polymers with a distinct, geometrically determined, modal length. This model reproduces the enzyme concentration dependence and modality of the observed polysaccharide length distributions. Our work highlights limitations of previous models and provides new insight into the mechanisms of length control in polysaccharide biosynthesis.

Intermediate instability at high temperature leads to low pathway efficiency for anin vitroreconstituted system of gluconeogenesis inSulfolobus solfataricus

Four enzymes of the gluconeogenic pathway in Sulfolobus solfataricus were purified and kinetically characterized. The enzymes were reconstituted in vitro to quantify the contribution of temperature instability of the pathway intermediates to carbon loss from the system. The reconstituted system, consisting of phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, triose phosphate isomerase and the fructose 1,6-bisphosphate aldolase/phosphatase, maintained a constant consumption rate of 3-phosphoglycerate and production of fructose 6-phosphate over a 1-h period. Cofactors ATP and NADPH were regenerated via pyruvate kinase and glucose dehydrogenase. A mathematical model was constructed on the basis of the kinetics of the purified enzymes and the measured half-life times of the pathway intermediates. The model quantitatively predicted the system fluxes and metabolite concentrations. Relative enzyme concentrations were chosen such that half the carbon in the system was lost due to degradation of the thermolabile intermediates dihydroxyacetone phosphate, glyceraldehyde 3-phosphate and 1,3-bisphosphoglycerate, indicating that intermediate instability at high temperature can significantly affect pathway efficiency.

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Starting from zero-, first-, and second-order integrated laws for chemical kinetics, some cases are shown which produce fractional orders. Taking the Michaelis-Menten mechanism as a first example, it is shown that substrate order can go from 1 to zero, depending on relative concentration of enzyme and substrate. Using other examples which show fractional orders higher than one and even negative (inhibition), it is shown that the presence of an equilibrium before or parallel to the rate determining step can be the reason for fractional orders, which is an indication of a more complex mechanism.

Preparation of W/O/W microcapsule containing enzyme without alcohol

We proposed a novel method for the preparation of W/O/W type microcapsules containing enzymes. In this method, a gas phase was used in place of alcohol that has been conventionally used in solvent change processes for the preparation of W/O/W type microcapsules. As a model system for this study, we prepared glucose-oxidase encapsulated poly( l-lysine-alt-terephthalic acid) microcapsules whose volume can be decreased in the presence of glucose and evaluate the effect of glucose on the shrinking process. To estimate the relative concentration of enzyme in microcapsule, a simple model was proposed on assumption of linear dependence of the rate of change in the size and in the charge density of the microcapsule membrane. The diameters of the shrunk microcapsule observed were consistent with the theoretical value. By a fitting of the observed time course of the microcapsule size to the theoretical equations, two time constants were obtained, both related inversely to the relative enzyme concentration in the microcapsules. From the comparison of their constants for microcapsules prepared by the novel and conventional method, it became apparent that the glucose-oxidase containing microcapsule prepared by the novel method exhibited about 1.5 times higher enzyme activity of the case for the conventional method using ethanol in the solvent change processes.

Versatile regulation of multisite protein phosphorylation by the order of phosphate processing and protein–protein interactions

Multisite protein phosphorylation is a common regulatory mechanism in cell signaling, and dramatically increases the possibilities for protein-protein interactions, conformational regulation, and phosphorylation pathways. However, there is at present no comprehensive picture of how these factors shape the response of a protein's phosphorylation state to changes in kinase and phosphatase activities. Here we provide a mathematical theory for the regulation of multisite protein phosphorylation based on the mechanistic description of elementary binding and catalytic steps. Explicit solutions for the steady-state response curves and characteristic (de)phosphorylation times have been obtained in special cases. The order of phosphate processing and the characteristics of protein-protein interactions turn out to be of overriding importance for both sensitivity and speed of response. Random phosphate processing gives rise to shallow response curves, favoring intermediate phosphorylation states of the target, and rapid kinetics. Sequential processing is characterized by steeper response curves and slower kinetics. We show systematically how qualitative differences in target phosphorylation--including graded, switch-like and bistable responses--are determined by the relative concentrations of enzyme and target as well as the enzyme-target affinities. In addition to collective effects of several phosphorylation sites, our analysis predicts that distinct phosphorylation patterns can be finely tuned by a single kinase. Taken together, this study suggests a versatile regulation of protein activation by the combined effect of structural, kinetic and thermodynamic aspects of multisite phosphorylation.

Heterogeneous kinetics of the enzymatic degradation of poly(β-hydroxyalkanoates)

The kinetics of the enzymatic degradation reactions of the naturally produced polyesters poly(β-hydroxybutyrate) (PHB) and poly(β-hydroxybutyrate- co-β-hydroxyvalerate) (PHBV) by appropriate depolymerases were studied. Specifically, the interaction between the extracellular PHB depolymerase of Pseudomonas lemoignei and both PHB powder and solution-cast films of PHBV was investigated. Kinetic analysis of titristatically and turbidimetrically determined rates of degradation has revealed that the observed degradation behaviour was inconsistent with classical Michaelis-Menten enzymatic kinetics. Depending upon the relative concentrations of enzyme and substrate, each exhibited a range of kinetic dependencies from zero order to first order. At high relative concentrations of enzyme, the reaction rate became inversely proportional to enzyme concentration. A new kinetic treatment was derived which predicted the observed variations in kinetic dependence on both enzyme and substrate, and it successfully described previously published kinetic data for this and other PHB depolymerases. The degradation of PHB by the PHB depolymerase of Aspergillus fumigatus M2A was investigated for comparison. While the specific rate constants of this enzyme differed from that of P. lemoignei, the general kinetic model was still applicable.

Monoamine oxidase-B in astrocytes.

In the present report we describe the astrocytic localization and content of monoamine oxidase-B (MAO-B) by means of a 3H-L-deprenyl emulsion autoradiography in primary cultures of rat astrocytes, in cryosectioned astrocytoma surgical specimen, and in cryosections of human spinal cords from patients dying in amyotrophic lateral sclerosis (ALS) and controls. The occurrence of MAO-B enzyme protein depends on the degree of cellular differentiation as demonstrated by studies on astrocytes in primary cultures analyzed at two different stages of maturation. Highly differentiated cells exhibited high relative enzyme concentration whereas glioblasts lacked or showed very low contents of MAO-B enzyme. This was further substantiated by studies performed on human astrocytoma tissue using 3H-L-deprenyl emulsion autoradiography in combination with immunohistochemical detection of glial fibrillary acidic protein (GFAP). Regional increases of MAO-B concentration were found in ALS lumbar sections with quantitative 3H-L-deprenyl autoradiography. On the basis of results obtained from double staining for GFAP and MAO-B, the increase in MAO-B seemed to be due to an increased number of astrocytes as well as an increased content of MAO-B in reactive species of astrocytes. A cell culture model has been used that produces cells with morphology and GFAP-content similar to reactive cells. These astrocytes exhibited high relative content of the MAO-B enzyme protein. In the light of the presented data, taking into account the finding that a subpopulation of reactive cells contained low levels of MAO-B, a heterogeneity among reactive astrocytes was observed.

Multipoint kinetic method for simultaneously measuring the combined concentrations of acetoacetate-beta-hydroxybutyrate and lactate-pyruvate.

This is a multipoint kinetic method for simultaneously determining acetoacetate (AcAc) plus beta-hydroxybutyrate and lactate plus pyruvate in a single cuvette of the Multistat III centrifugal analyzer. In the first step, AcAc and pyruvate are completely reduced, using 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) and lactate dehydrogenase (EC 1.1.1.27) in the presence of excess NADH at pH 7.5, to beta-hydroxybutyrate and lactate, respectively. After dilution, the endogenous beta-hydroxybutyrate and lactate and that resulting from reduction are simultaneously oxidized by their respective dehydrogenases in the presence of excess NAD+ at pH 9.0. Adjustment of the relative enzyme concentrations allows simultaneous estimation of AcAc plus beta-hydroxybutyrate and lactate plus pyruvate by analyzing multipoint absorbance data, collected during the oxidation reaction, with use of a two-component linear-regression model. Total run-to-run CVs were 6.4% and 6.1% at 5 mmol/L beta-hydroxybutyrate and 5 mmol/L lactate, respectively. The method was designed to be useful for identifying the cause of an increased anion gap in serum.

Development and evaluation of an enzyme inhibition assay for methotrexate.

A method for the measurement of methotrexate based on its inhibition of dihydrofolate reductase (EC 1.5.1.3) is described. The performance of the method is evaluated and a brief assessment of its clinical usefulness made. Reaction time, pH, and the relative concentration of enzyme and inhibitor all have a significant effect on the shape of the standard curve. Pre-incubation of enzyme with methotrexate resulted in increased inhibition, but did not improve the sensitivity or linearity of the assay. Data are presented on the imprecision, specificity and accuracy of the method. The method is simple to perform, rapid and inexpensive. The sensitivity and specificity of the method are such as to allow for effective monitoring of patients on methotrexate therapy.

Detection of femtomole quantities of proteases by high-performance liquid chromatography

Small amounts (femtomoles) of proteases, as might be present in cell extracts or secretions, were detected using reverse-phase high-performance liquid chromatography. Carboxymethylated lysozyme and cytochrome c were incubated with trypsin and chymotrypsin. Peptide peaks were present in the column elution profiles (as detected by absorbance, 206 nm) from incubations with as little as 0.1 fmol of chymotrypsin and 5 fmol of trypsin. In addition, the disappearance of the substrate peak or the increase in peptide peaks could be quantitated by integrating the areas under the peaks. In this way estimates of relative enzyme concentrations or duration of incubation can be determined. However, when [ 14C]lysozyme was used as a substrate and the radioactivity of collected peaks was measured, the assay was less sensitive than that using uv absorbance. This finding probably is related to the selective radiolabeling of the substrate, in contrast to uv detection, which should detect all the peptides. The technique reported in this paper should prove to be a sensitive indicator of proteolytic activity in cell or tissue preparations where the use of synthetic ester or amide substrates might lead to erroneous conclusions regarding the nature of the enzymatic activity present. Furthermore, by the collection of the peptides generated, one would have the ability to determine amino acid compositions or sequences and thus ascertain the specificity of enzymatic cleavage.

The inhibition by clorgyline of 5-hydroxytryptamine deamination by the rat liver.

The inhibition of the monoamine oxidase activity in the rat liver by the substrate selective inhibitor clorgyline has been investigated with 5-hydroxytryptamine as substrate. The results obtained are consistent with a theoretical model whereby the inhibition of enzyme activity by clorgyline follows a reversible association phase leading to an irreversible 'suicide' reaction. The relative concentrations of enzyme and inhibitor are of the same order, and can account both for the failure of the reaction to go to completion, and for the differences in the apparent sensitivity of enzyme preparations to inhibition by clorgyline. The possible value of this type of inhibition as a means of assay for monoamine oxidase active centres is discussed.

Graphical Determination of Relative Concentrations of Enzyme and Endogenous Inhibitor by Dilution

Graphical Determination of Relative Concentrations of Enzyme and Endogenous Inhibitor by Dilution

Quantitative distribution of lactic dehydrogenase in the regenerating liver lobule of the rat.

Regeneration of rat liver is maximal during the first day following partial hepatectomy. In order to obtain quantitative data on lactic dehydrogenase during this period of increased energy utilization, microdissection of lobules at 0, 15 and 30 hr after partial hepatectomy was carried out. Isolated samples from periportal, midzone and pericentral areas weighing 0.05-0.2 µg, dry weight, were analyzed for LDH and DNA. Sections were also stained for glycogen, fat and LDH. Qualitative results revealed depletion of glycogen from portal and midzonal regions, accumulation of fat in periportal zones, and heavier concentrations of LDH in peripheral areas of the regenerating lobule. Quantitative analysis showed LDH activity to be 40-50% higher in peripheral compared to central zones. Similar levels and relative concentrations of enzyme persisted throughout the 30-hr regenerative period investigated. DNA content increased by about 30% in the peripheral zone during the same period. The possible meaning of these findings is discussed.

The Cholinesterase of Erythrocytes in Anemias

Abstract1. Activity-pS curves were determined experimentally for the cholinesterase of the erythrocytes in various human anemias.2. The data were analyzed to give experimental values for the apparent dissociation constants, K8 of the active enzyme-substrate complex (ES) and K2 of the inactive complex (ES2), and the theoretic limiting velocity V.3. The analysis showed that the enzyme in the cells of anemia is the same as that in normal cells. The abnormally high enzymic activity (initial velocity at a single substrate concentration) known to occur in various anemias and the change from low activity in pernicious anemia in relapse to high activity during treatment are attributable to changes in concentration of the enzyme.4. The enzyme in the red cells of pernicious anemia in relapse was not activated by folic acid or liver extract.5. It was shown experimentally that the reticulocytes and young cells of normal blood and of pernicious anemia during treatment contain much higher concentrations of enzyme than old cells. The increased concentration persists for some time after the reticulum has disappeared.6. The relative concentration of enzyme in the red cells is a more sensitive indicator of hyperactive hematopoiesis than the reticulocyte count.7. Evidence is presented that failure of elevation of enzymic concentration in severe anemia is associated specifically with suppression or malfunction of the hematopoietic system.8. It is believed that the relative concentration of enzyme can be used as a test of bone marrow function in severe anemias. It is hoped that further investigation may result in a useful test of wider application.