Fitting progress curves in assays of slow-binding enzyme inhibitors.

Practical aspects of fitting progress curves deriving from assays of slow-binding enzyme inhibitors in order to extract characteristic parameters is briefly discussed. An approach little-used, but which may provide more accurate estimates, is the simultaneous analysis of two assays differing from one another in the order of addition of reactants.     

Structure and possible catalytic residues of Taka-amylase A

A complete molecular model of Taka-amylase A consisting of 478 amino acid residues was built with the aid of amino acid sequence data. Some typical structural features of the molecule are described. A model fitting of an amylose chain in the catalytic site of the enzyme showed a possible productive binding mode between substrate and enzyme. On the basis of the difference Fourier analysis and the model fitting study, glutamic acid (Glu230) and aspartic acid (Asp297), which are located at the bottom of the cleft, were concluded to be the catalytic residues, serving as the general acid and base, respectively.     

Analysis of progress curves for a highly concentrated Michaelian enzyme in the presence or absence of product inhibition.

Methods are given for analysing the time course of an enzyme-catalysed reaction when the concentration of the enzyme itself is high, a situation which is often found in vivo. (1) The integrated form of the kinetic equation for a concentrated Michaelian enzyme in absence of product inhibition is given. Parameters are shown to be calculated easily using non-linear fitting procedures. (2) A general algorithm to analyse progress-curve data in more complex cases (i.e. when the analytical form of the integrated rate equation is not known or is exceedingly complex) is proposed. This algorithm may be used for any enzyme mechanism for which the differential form of the kinetic equation may be written analytically. We show that the method allows differentiation between the main types of product inhibition which may occur in the case of a highly concentrated Michaelian enzyme.     

Using the beta-lactamase protein-fragment complementation assay to probe dynamic protein-protein interactions

We have developed a general experimental strategy that enables the quantitative detection of dynamic protein-protein interactions in intact living cells, based on protein-fragment complementation assays (PCAs). In this method, protein interactions are coupled to refolding of enzymes from cognate fragments where reconstitution of enzyme activity acts as the detector of a protein interaction. We have described a number of assays with different reporter readouts, but of particular value to studies of protein interaction dynamics are assays based on enzyme reporters that catalyze the creation of products, thus taking advantage of the amplification of signal afforded. Here we describe protocols for one such PCA based on the enzyme TEM beta-lactamase as a reporter in mammalian cells. The beta-lactamase PCA consists of fusing complementary fragments of beta-lactamase to two proteins of interest. If the proteins interact, the fragments are brought together and fold into active beta-lactamase. Here we describe a protocol for this PCA that can be completed in a few hours, using two different substrates that are converted to fluorescent or colored products by beta-lactamase.     

A highly sensitive protein-protein interaction assay based on Gaussia luciferase

Protein-fragment complementation assays (PCAs) provide a general strategy to study the dynamics of protein-protein interactions in vivo and in vitro. The full potential of PCA requires assays that are fully reversible and sensitive at subendogenous protein expression levels. We describe a new assay that meets these criteria, based on the Gaussia princeps luciferase enzyme, demonstrating chemical reversal, and induction and inhibition of a key interaction linking insulin and TGFbeta signaling.     

Two sensitive, convenient, and widely applicable assays for marker enzyme activities specific to endoplasmic reticulum

Two assay protocols are described for enzyme activities known to reside in the endoplasmic reticulum of a wide variety of species and tissue types, with the intent that they be used as marker enzyme assays in subcellular fractionations. The enzyme activities assayed are choline phosphotransferase and dolichol-P-mannosyl synthase, both of which result in synthesis of lipid products. The assays are constructed to make them easy to perform and sensitive enough to detect enzyme activity even using microgram quantities of cell protein. The assay methodologies are effective not only in vertebrate cells, but in insect cells and yeast cells as well. This implies that these assays should be useful as marker enzyme assays for a wide variety of eukaryotic cells.     

Modeling substrate inhibition of microbial growth

This article presents a general equation for substrate inhibition of microbial growth using a statistical thermodynamic approach. Existing empirical models adapted from enzyme kinetics, for example, the Haldane-Andrews equation, often criticized for not being physically based for microbial growth, are shown to derive from the general equation in this article, and their empirical parameters are shown to be well defined physically. Three sets of experimental data from the literature are used to test the modeling abilities of the general equation to represent experimental data. The results are compared with those obtained by fitting the same data set to a widely used empirical model existing in the literature. The general equation is found to represent all three experimental data sets better than the alternative model tested. In addition, a graphical method existing in enzyme kinetics is successfully adapted and further developed to determine the number of inhibition sites of a basic functional unit of a bacterial cell. (c) 1996 John Wiley & Sons, Inc.     

Histochemical technique : a general method for quantitative enzyme assays of single cell ;extracts' with a time resolution of seconds and a reading precision of femtomoles

Biochemists who study single cells have been constrained by the lack of a general methodology of high time resolution and high measurement sensitivity for quantitatively assaying enzyme activities using natural substrates in solution. The methods we describe will remove this limitation. In brief, nanogram tissue samples are dissected from frozen-dried tissue. The samples are `extracted' in microdroplets of assay cocktail. The enzyme activity, indicated fluorometrically by the oxidation/reduction of NAD(P), is followed in real time on a computer display. In the development of this method, we evaluated several parameters required for optimization; the most important of these evaluations, including numerous empirically derived relationships, are reported here and in supplemental material provided with reprints.

With these methods, assays of pyruvate orthophosphate dikinase on samples enriched in bundlesheath cells and mesophyll cells of Flaveria brownii yielded the predictable results. Assays of this enzyme in guard cells dissected from Vicia faba leaflets gave results like those recently reported by another laboratory for protoplasts derived from these cells. The results of assays by this method and by enzymic cycling for NAD(P)triose-P dehydrogenase were comparable. Phosphoenolpyruvate carboxylase, the most extensively studied enzyme activity, was present at high levels in guard cells, which has been demonstrated previously in other reports based on diverse assay approaches.

     

Modelling of different enzyme productions by solid-state fermentation on several agro-industrial residues

A simple kinetic model, with only three fitting parameters, for several enzyme productions in Petri dishes by solid-state fermentation is proposed in this paper, which may be a valuable tool for simulation of this type of processes. Basically, the model is able to predict temporal fungal enzyme production by solid-state fermentation on complex substrates, maximum enzyme activity expected and time at which these maxima are reached. In this work, several fermentations in solid state were performed in Petri dishes, using four filamentous fungi grown on different agro-industrial residues, measuring xylanase, exo-polygalacturonase, cellulose and laccase activities over time. Regression coefficients after fitting experimental data to the proposed model turned out to be quite high in all cases. In fact, these results are very interesting considering, on the one hand, the simplicity of the model and, on the other hand, that enzyme activities correspond to different enzymes, produced by different fungi on different substrates.     

Computerized analysis of enzyme cascade reactions using continuous rate data obtained with an ELISA reader

Two programs have been written which permit analysis of multiple continuous-rate enzyme-cascade assays conducted with the use of an ELISA spectrophotometer and a synthetic chromogenic substrate. Because the product of the first reaction functions as the enzyme in the second reaction, production of chromophore continuously accelerates and it is the rate of acceleration which serves to measure the rate of the initial reaction in the system. The first program determines the rate of acceleration using linear regression to analyze the reaction curves as a function of the square of time. The second program, using a Simplex algorithm, determines the parameters which establish the assay standard curve by fitting the rate data to the Hill equation. Used together, these programs facilitate the analysis of many kinetic experiments conducted simultaneously.     

Subnanoliter enzymatic assays on microarrays

Many areas of research today are based on enzymatic assays most of which are still performed as enzyme-linked immunosorbent assays in microtiter plates. The demand for highly parallel screening of thousands of samples eventually led to a miniaturization and automation of these assays. However, the final transfer of enzymatic assays from a microtiter-based technology to microarrays has proven to be difficult for various reasons, such as the inability to maintain unbound reaction products on the spot of reaction or the missing capability of multiplexing. Here, we have conducted multiplex enzymatic assays in subnanoliter volumes on a single microarray using the multiple spotting technology. We were able to measure enzymatic activity with a sensitivity down to 35 enzyme molecules, applying only conventional flat microarray surfaces and standard microarray hardware. We have performed assays of inhibition and applied this format for the detection of prognostic markers, such as cathepsin D. The new approach allows the rapid and multiplex screening of thousands of samples on a single microarray with applications in drug screening, metagenomics, and high-throughput enzyme assays.     

Functional heterogeneity of monoclonal antibodies obtained using different screening assays

Two alternate screening methods have enabled the detection of monoclonal antibodies with different specificities toward the lysosomal enzyme alpha-mannosidase of Dictyostelium discoideum. Spleen/myeloma hybrid cell cultures were screened for antibody production by separate assays: an indirect enzyme-linked immunoadsorbent assay (ELISA) based on the antibody binding to enzyme adsorbed on plastic, and a direct assay of the antibodies' ability to precipitate enzyme activity with fixed Staphylococcus aureus cells (Pansorbin). Fourteen stable antibody-producing cell lines resulted from a single fusion; these fell into three distinct classes based on their screening characteristics. A group of eight were positive in both assays, and these immunoprecipitated a 140,000 Mr precursor form of alpha-mannosidase in addition to the 58,000 and 60,000 Mr mature enzyme subunits from [35S]methionine-labeled total secreted protein preparations. Two of the antibodies were positive only in the immunoprecipitation assay; these failed to precipitate the 140,000 Mr precursor. The third class consisted of four antibodies that were positive only in the ELISA method. These exclusively recognized an altered conformation of the enzyme (precursor and mature forms) that was immobilized either on plastic or on nitrocellulose paper. In addition, only members of this class were able to bind to immobilized fragments of protease-treated enzyme. The implications of these findings for the general design of monoclonal antibody screenings and for the alternative structures of this enzyme are discussed.     

Application of a metabolic balancing technique to the analysis of microbial fermentation data

A general method for the development of fermentation models, based on elemental and metabolic balances, is illustrated with three examples from the literature. Physiological parameters such as the (maximal) yield on ATP, the energetic maintenance coefficient, the P/O ratio and others are estimated by fitting model equations to experimental data. Further, phenomenological relations concerning kinetics of product formation and limiting enzyme activities are assessed. The results are compared with the conclusions of the original articles, and differences due to the application of improved models are discussed.     

Steady state kinetics of consecutive enzyme catalysed reactions involving single substrates: procedures for the interpretation of coupled assays

Sets of differential rate equations are written describing a linear sequence of reactions occurring in solution each catalysed by a control enzyme or one of the Michaelis-Menten type. It is shown that the solutions of these equations may be formulated as a set of Maclaurin polynomials, expressing the concentration of each reactant and of final product as a function of time. From arrays of such polynomials, general expressions are induced for the first non-zero term of the series. These are used to formulate a procedure (illustrated with an example simulated by numerical integration) by which results of coupled enzymic assays may be analysed in terms of maximal velocities and apparent Michaelis constants: correlation is made with other established methods for conducting coupled assays. The present procedure assumes a steady state of enzyme-substrate complexes but not of intermediate reactants.     

Mlab--a mathematical modeling tool

An interactive interpreter called Mlab is described. One uses Mlab by typing commands. In this sense, Mlab is a programming language. It has various mathematical and graphical facilities which make it a useful tool for mathematical modeling. The curve fitting capabilities of Mlab are augmented with differential-equation-handling and matrix-manipulation capabilities which provide a powerful and civilized facility for curve fitting. Many people are engaged in this activity, and, in general, they use programs which are neither sufficiently general nor easy to use. (Some conventional programming is usually required, for example.) Mlab purports to be easier than alternate approaches. The nature of Mlab is discussed with accompanying examples. The main example is the use of curve fitting to determine molecular weight from ultracentrifuge data. This example was chosen because it exhibits a special feature of Mlab, namely the root operator, which appears in the definition of the model function.     

Application of protein-fragment complementation assays in cell biology

We have developed a general experimental strategy that enables the quantitative detection of dynamic protein-protein interactions in intact living cells, based on protein-fragment complementation assays (PCAs). In this method, protein-protein interactions are coupled to refolding of enzymes from cognate fragments where reconstitution of enzyme activity acts as the detector of a protein interaction. Here we discuss the application of PCA to different aspects of cell biology.     

Tubulin carboxypeptidase assay based on the action of the enzyme on [14C]tyrosinated tubulin bound to nitrocellulose membrane

We have developed a method for the determination of tubulin carboxypeptidase activity which is based on the action of the enzyme on the substrate, [14C]tyrosinated tubulin, previously adsorbed on nitrocellulose membrane. In addition to being two to three times more sensitive than previous carboxypeptidase assays, this method allows the determination of dilute enzyme preparations even containing high salt (inhibitory) concentrations. This is a valuable property specially under circumstances in which numerous high salt-containing fractions with scarce activity should be analyzed (for example after certain chromatographic stages during enzyme purification). Our method is simpler, less time-consuming, and suitable for multiple, simultaneous determinations and the substrate bound to nitrocellulose can be stored for several months without significant alteration of its properties. Peptidases other than tubulin carboxypeptidase can act on [14C]tyrosinated tubulin bound to nitrocellulose, solubilizing radioactive compounds, suggesting the eventual applicability of this method to assay proteases in general. Other features and advantages of the assay as well as its limitations are discussed.     

The role of lysine 532 in the catalytic mechanism of human topoisomerase I

Based on co-crystal structures of human topoisomerase I with bound DNA, Lys(532) makes a minor groove contact with the strongly preferred thymidine residue at the site of covalent attachment (-1 position). Replacement of Lys(532) with either arginine or alanine has essentially no effect on the sequence preference of the enzyme, indicating that this interaction is not required for the preference for a T at the -1 position. Although both the cleavage and religation activities of the K532R mutant enzyme are reduced, cleavage is reduced to a greater extent than religation. The reverse is true for the K532A mutant enzyme with religation so impaired that the nicked intermediate accumulates during plasmid relaxation assays. Consistent with the shift in the cleavage religation equilibrium toward cleavage for the K532A mutant enzyme, expression of the mutant enzyme in Saccharomyces cerevisiae is cytotoxic, and thus this mutant enzyme mimics the effects of the anticancer drug camptothecin. Cleavage assays with the mutant enzymes using an oligonucleotide containing a 5'-bridging phosphorothiolate indicate that Lys(532) functions as a general acid during cleavage to protonate the leaving 5'-oxygen. It is possible that the contact with the -1 base is important during catalysis to provide positional rigidity to the active site. The corresponding residues in the vaccinia virus topoisomerase and the tyrosine recombinases may have similar critical roles in catalysis.     

Exact and user-friendly kinetic analysis of the two-step rapid equilibrium Michaelis-Menten mechanism

Most enzyme kinetic experiments are carried out under pseudo-first-order conditions, that is, when one of the reactant species (the enzyme or the substrate) is in a large excess of the other species. More accurate kinetic information about the system can be gained without the restrictions of the pseudo-first-order conditions. We present a practical and general method of analysis of the common two-step rapid equilibrium Michaelis-Menten mechanism. The formalism is exact in that it does not involve any other approximations such as the steady-state, limitations on the reactant concentrations or on reaction times. We apply this method to the global analysis of kinetic progress curves for bovine alkaline phosphatase assays carried out under both pseudo-first-order and pseudo-second-order conditions.     

A cDNA library functional screening strategy based on fluorescent protein complementation assays to identify novel components of signaling pathways

Progress towards a deeper understanding of cellular biochemical networks demands the development of methods to both identify and validate component proteins of these networks. Here, we describe a cDNA library screening strategy that achieves these aims, based on a protein-fragment complementation assay (PCA) using green fluorescent protein (GFP) as a reporter. The strategy combines a simple cell-based cDNA-screening approach (interactions of a "bait" protein of interest with "prey" cDNA products) with specific functional assays that use the same system and provide initial validation of the cDNA products as being biologically relevant. We applied this strategy to identify novel interacting partners of the protein kinase PKB/Akt. This method provides very general means of identifying and validating genes involved in any cellular process and is particularly designed for identifying enzyme substrates or regulatory proteins for which the enzyme specificity can only be defined by their interactions with other proteins in cells in which the proteins are normally expressed.