4-alkylidene oxazolones as substrates and inhibitors for sensitive assays of the normality of active sites and the catalytic properties of hydrolases.

The suitability of eleven 4-alkylidene oxazolones for the determination of four hydrolases, alpha-chymotrypsin, trypsin, carboxylesterase and leucine aminopeptidase, was tested. The specific activities were in general low compared with those obtained with the classical substrates but the Kmapp values were also small. Hence, the kcat/Kmapp ratios of the oxazolones, the optimal indicator of activity, remained in the usual range. The high differential absorption coefficients of the oxazolones in the UV range render these substrates very suitable for the determination of active site normalities if the solubilities of the acyl enzymes and the magnitudes of the rapid bursts are sufficient. Since some of the oxazolones fluoresce, the sensitivity of the method may be increased up to 1000 x by fluorescence detection. The titrations may be carried out in organic solvents, e.g. dimethyl sulphoxide, which greatly stabilize the hydrolases. The high specificity of the oxazolones permits active site titrations in the presence of other hydrolases.

Isolation and characterization of the human liver ethanolamine kinase.

Ethanolamine kinase was purified from human liver to apparent homogeneity and its properties were studied. The minimum molecular weight of the purified enzyme was estimated by SDS-PAGE to 42,000 +/- 2000 Da. The molecular weight of the native enzyme determined by Superose 12 (FPLC) gel filtration was 87,000 +/- 4000 Da, suggesting that ethanolamine kinase in human liver consists of two chains of the same or almost the same molecular weight. The titration of the active site normality showed 1.03 +/- 0.05 active sites/chain. The Km value for ethanolamine was 0.25 +/- 0.01 mM.

Evaluation of the kinetic parameters of the activation of trypsinogen by trypsin.

Kinetic analysis of the mechanism of trypsinogen activation by trypsin under rapid equilibrium conditions and certain relationships between the rate constants are presented. The kinetic equations are valid from the beginning of the reaction. In addition, we suggest a procedure, based on the above equations, for the evaluation of the kinetic parameters of the reaction. This procedure is applied to a set of experimental data collected during the activation of bovine trypsinogen by trypsin at 30 degrees C (pH 8.1) in 0.01 M CaCl2. In this system, the amount of active enzyme increases exponentially, as expected from an autocatalytic process. The apparent rate constant, delta, governing this increase would vary linearly with the trypsinogen concentration, [Z]0, if no Michaelis complex was detectable. However, the increase in delta with [Z]0 is clearly non-linear and fits a hyperbola (delta = k2[Z]0/(Kz + [Z]0)) well.

Elucidation of a new biological function of an old protein: unique structure of the cobra serum albumin controls its specific toxin binding activity.

Although few proteins have been studies as thoroughly as serum albumin, a new biological property of this evolutionary ancient protein was recently discovered: The ability of cobra serum albumin (CSA) to specifically sequester lethal endogenous toxins. A study of the structural basis of this property is reported in this contribution. Two independent approaches were used to alter the structure of the CSA at defined positions: Directed mutagenesis and limited proteolysis. The conserved pattern of the disulfide linkages in the primary structure of the serum albumins showed in the case of the cobra snake (Naja naja kaouthia) an anomaly at C11 and C502, which suggested the existence of a unique spatial structure in this protein. The two cysteine residues were singly replaced with the consensus residue, i.e. C11-->F and C502-->T. The former substitution increased the specific neurotoxin binding capacity of the CSA by the factor 1.7 +/- 0.2, whereas the latter replacement reduced it to (25 +/- 2)%. The limited proteolysis yielded the large tryptic peptides T60, T40, T30 and T18, which after isolation by PAGE followed by HPLC had retained a strong toxin affinity. The location of these peptides in the amino-acid sequence was identified by Edman degradation and suggested the order of their release. On the basis of these data, a model of the unfolding and of the activity changes of the CSA caused by the structural perturbations was composed and the kinetic parameters associated with the process were evaluated. The results support the hypothesis of the existence of a structure of multiple homologous domains with a disulfide linkage between C11 and C502 in the native CSA that joins the chain ends to form a dense conformation.

Kinetic analysis of enzyme systems with suicide substrate in the presence of a reversible competitive inhibitor, tested by simulated progress curves.

The use of suicide substrates remains a very important and useful method in enzymology for studying enzyme mechanisms and designing potential drugs. Suicide substrates act as modified substrates for the target enzymes and bind to the active site. Therefore the presence of a competitive reversible inhibitor decreases the rate of substrate-induced inactivation and protects the enzyme from this inactivation. This lowering on the inactivation rate has evident physiological advantages, since it allows the easy acquisition of experimental data and facilitates kinetic data analysis by providing another variable (inhibitor concentration). However despite the importance of the simultaneous action of a suicide substrate and a competitive reversible inhibition, to date no corresponding kinetic analysis has been carried out. Therefore we present a general kinetic analysis of a Michaelis-Menten reaction mechanism with double inhibition caused by both, a suicide substrate and a competitive reversible inhibitor. We assume rapid equilibrium of the reversible reaction steps involved, while the time course equations for the reaction product have been derived with the assumption of a limiting enzyme. The goodness of the analytical solutions has been tested by comparison with the simulated curves obtained by numerical integration. A kinetic data analysis to determine the corresponding kinetic parameters from the time progress curve of the product is suggested. In conclusion, we present a complete kinetic analysis of an enzyme reaction mechanism as described above in an attempt to fill a gap in the theoretical treatment of this type of system.

Flavonoids, a class of natural products of high pharmacological potency.

A review has been presented of the biochemistry and pharmacology of a class of natural products, the flavonoids. These substances which are widely distributed in the plant kingdom and present in considerable quantities in common food products, spices and beverages have in a concentrated form (Propolis) been used since ancient times by physicians and laymen to treat a great variety of human diseases but they have yet to pass the tests of modern, controlled, clinical experimentation. An attempt has been made to present the fundamental evidence from the basic biological sciences which is required to stimulate the interest of the clinicians in this new field. The few existing reports on the careful pharmacodynamic, pharmacokinetic and clinical studies which have been made have been summarized to provide a basis for a full-scale investigation of the therapeutic potential of flavonoids.

Potential linkage disequilibrium between schizophrenia and locus D22S278 on the long arm of chromosome 22.

Locus D22S278 at 22q12 has been implicated in schizophrenia by sib-pair analysis. In order to replicate these results, we performed the transmission test for linkage disequilibrium (TDT) in 113 unrelated schizophrenic patients and their 226 parents. Evidence for potential linkage disequilibrium was obtained between schizophrenia and allele 243 of the marker AFM 182xd12 at the locus D22S278 (P = 0.02). The results of our study suggest a detectable oligogenic gene in a multigene system for schizophrenia closely linked to D22S278 on the long arm of chromosome 22. If confirmed by others, this finding could lead to the identification of a schizophrenia susceptibility gene.

Biochemical analysis of normal human tissues and those of lymphoma patients by high-resolution protein mapping.

The technique of high-resolution, two-dimensional electrophoresis was applied to crude membrane protein fractions of various normal human tissue samples, and those, mainly spleens and lymph nodes, of patients with lymphomas of different types. The obtained protein maps show a high reproducibility, tissue specificity, and sensitivity to specific oncologic changes. The relevance of the latter aspect to tumor characterization is illustrated. Quantitatively, minor proteins were detected by a sensitive silver stain. The feasibility of objective, computerized quantification and evaluation of the protein maps is described.

Molecular weight analysis of Fc gamma-binding proteins of lymphoid leukemia, myeloid leukemia, and hairy-cell leukemia.

The molecular weights of EDTA-mercaptoethanol-soluble Fc gamma-affined proteins isolated from chronic lymphocytic leukemia of the B type, prolymphocytic leukemia of the B type, chronic myeloid leukemia and hairy-cell leukemia were compared. SDS polyacrylamide gel electrophoresis of the Fc gamma-binding material obtained from all six cases of B type leukemia revealed a single peak with an apparent molecular weight of 28,000. The Fc gamma-affined material isolated from the cells of two cases of chronic myeloid leukemia showed two peaks, one with an apparent molecular weight of 42,600 and one with an apparent molecular weight of 18,800. The Fc gamma-affined material isolated from the cells of two cases of hairy-cell leukemia electrophoresed in the form of a closely spaced double peak. One component of the double peak had an apparent molecular weight of 28,000 and thus corresponds to the Fc gamma-binding material of leukemic B cells. The second component had a slightly lower molecular weight. The latter component is not present on either leukemic B cells or myeloid cells. The results indicate that the EDTA-mercaptoethanol-soluble Fc gamma-affined proteins of different types of cells differ in molecular weight, and thus in molecular structure.

Linear free energy relationship for osmotic water flow through a membrane.

A linear free-energy relationship has been found for the osmotic water flux through membranes in a broad variety of systems including electrolytes, organic compounds, intact biological cells and industrial scale filtration. In all cases, broad concentration ranges were found in which the equation 1n v = alpha 1n m + beta (v, flux (in kg cm-2 min-1; m, molarity] was valid. The parameters alpha and beta were interpreted in terms of molecular weight, mean ionic radius, enthalpy of solvation, electronic structure and H-bonding propensity. The equation is independent of the membrane material and of the presence of other solutes and precipitates, as long as the latter are incompressible. Its parameters are only slightly dependent upon the temperature. The contributions from different solutes to the osmotic flux are at appreciable concentrations even additive. The relationship permits the prediction of the osmotic water flux and of the rate of filtration of systems of known composition. For simple systems it permits determination of the molecular weight, mean ionic radius, degree of hydration and enthalpy of solvation. It is suggested that osmosis is primarily due to the shift of hydration equilibria and that guanidine hydrochloride, in a realistic concentration range, forms practically infinite clathrates with water. The properties of the urea and Gdn X HCl systems indicate that these solutes either reversibly change the membrane structure and/or display intrinsic hysteresis.

General linear compartment model with zero input: II. The computerized derivation of the kinetic equations.

The final equations obtained in the first article of this series describing the concentrations of substances in any of the compartments of a model are here cast in an easily programmable form. A computer program with an easy input method and the ability to expand all of the coefficients in the kinetic equations in terms of the model parameters has been developed. The program has been written in the BASIC language.

General linear compartment model with zero input: I. Kinetic equations.

The derivation of kinetic equations is described for n-compartment linear models, in which the substance may be simultaneously introduced into one or more compartments at t = 0 and eliminated from any compartment. For a given zero-input, general formulas are derived which describe the amount of tracer in any of the compartments as a function of time and the model parameters. New algorithms have been developed which allow the expression of the kinetic equations.

An analysis of the kinetics of enzymatic systems with unstable species.

We present a general kinetic analysis of the Michaelis-Menten mechanism for the case in which the substrate, the enzyme-substrate complex, and the product are unstable. The equations for the rapid equilibrium conditions are obtained as a particular case of the general equations of the transient-phase. The kinetic data analysis which we suggest is based on the time progress curve of the product of the enzymatic reaction, or on the progress curve of the species into which the immediate product is transformed. This analysis allows the determination of the rate and equilibrium constants if adequate experimental results are available. It assumes, in contrast to most previous treatments of enzyme kinetics, that the concentration of the enzyme is much higher than that of the substrate. Since this condition often is satisfied inside cells, it can be more relevant to physiological problems than the classical assumption, [E] << [S], which sooner pertains to the situation in the laboratory.

The kinetic effect of product instability in a Michaelis-Menten mechanism with competitive inhibition.

In most kinetic studies it is assumed that both the reactant and the products are stable. However, under certain conditions spontaneous decomposition or deterioration caused by one of the participating species occurs. Studies, in which a species (the free enzyme, the enzyme-substrate complex, an inhibitor or the product of the reaction) is unstable, have appeared in the literature. However, to our knowledge, the enzymatic systems, in which a competitive inhibition and a decomposition or transformation of the products take place simultaneously, have not been studied so far. In this paper, we present a kinetic analysis of an enzyme reaction that follows a Michaelis-Menten mechanism, in which the free enzyme suffers a competitive inhibition simultaneously with the decomposition of the immediate product. In this study, we have linearised the differential equations that describe the kinetics of the process. Under the assumption of limiting concentration of enzyme, we have obtained and tested the explicit equation describing the time dependence of the product concentration using numerical calculus. With this equation and the experimental progress curve of the product, we constructed an easy procedure for the evaluation of the principal kinetic parameters of the process.

Transient phase of enzyme reactions. Time course equations of the strict and the rapid equilibrium conditions and their computerized derivation.

In this contribution, we present the derivation, from the strict transient phase equations of enzyme reactions, of the transient phase equations under the usual assumptions that one or more of the reversible steps involved in the mechanism of the enzyme reaction are assumed to be in rapid equilibrium. Moreover, we present the transient phase equations of all of the species in a general enzyme system model, valid for the partial or total rapid equilibrium conditions, as well as the particular case of the strict transient phase equations. In the case of the rapid equilibrium assumptions, the equations may be given either as functions of the individual rate constants in the reversible steps assumed in rapid equilibrium or as functions of the corresponding equilibrium constants. The steady state equations are easily obtained from the transient phase equations by setting the time --> infinity. We have implemented a computer program, easy to use and with a user-friendly format for the input of data and output of results, which allows the user to derive the symbolic strict transient phase equations and/or those corresponding to the assumption that one or more of the reversible reaction steps are in rapid equilibrium.

Kinetics of enzyme systems with unstable suicide substrates.

This paper deals with kinetic studies of enzyme reaction mechanisms with enzyme inactivation induced by an unstable suicide substrate. An initial steady-state of the catalytic route is assumed and the time course equations for the total active enzyme forms and the reaction product have been derived. The goodness of the analytical solutions has been tested by comparison with the simulated curves obtained by numerical integration. A kinetic data analysis to determine the corresponding kinetic parameters is suggested and the time course equations of an important reaction mechanisms involving a stable suicide substrate and which can be regarded as particular case of that under study has also been derived from the corresponding equations. The simplicity of our method allows its systematic application to more complex mechanisms.

Time course equations of the amount of substance in a linear compartmental system and their computerized derivation.

In this contribution, we present the symbolic time course equations corresponding to a general model of a linear compartmental system, closed or open, with or without traps and with zero input. The steady state equations are obtained easily from the transient phase equations by setting the time --> infinity. Special attention has been given to the open systems, for which an exhaustive kinetic analysis has been developed to obtain important properties. Besides, the results have been particularized to open systems without traps and an alternative expression for the distribution function of exit times has been provided. We have implemented a versatile computer program, that is easy to use and with a user-friendly format of the input of data and the output of results. This computer program allows the user to obtain all the information necessary to derive the symbolic time course equations for closed or open systems as well as for the derivation of the distribution function of exit times.

[The molecular cloning and sequencing of the cobra serum antitoxic protein gene].

In the previous study the authors found at least one peptide of 39 amino acid residues of several purified tryptic-fragments of the cobra serum antitoxic protein (CSAP), whose sequence had shown certain correlation with the rat serum albumin. For further clarification of the toxin-binding activity of CSAP in relation with its corresponding structure, the cDNA gene library of cobra liver cells was established and DNA primers were designed according to the known amino acid sequences of the tryptic peptides of CSAP. The specific probes were prepared and the specific clone was screened out from the library by PCR. Finally, the CSAP cDNA was sequenced from its sub-clones. Then the complete amino acid sequence of CSAP was elucidated. It is a polypeptide chain of 614 amino acid residues with a molecular size of 69.8 KDa. In comparing the whole sequence of CSAP, especially its signal peptide, with mammalian serum albumins, the authors have come to realize that CSAP is just the cobra serum albumin (CSA).