Enrique I. Canela

Heterogeneous localization of some purine enzymes in subcellular fractions of rat brain and cerebellum.

The activity of guanine deaminase (GAH, E.C. 3.5.4.3) was lower in rat cerebellum soluble and microsomal fractions than in rat brain subfractions. Adenosine deaminase (ADA, E.C. 3.5.4.4) activity was released in higher proportion than guanine deaminase, purine nucleoside phosphorylase (PNP, E.C. 2.1.2.4), 5′-nucleotidase (5′N, E.C. 3.1.3.5), and lactate (LDH, E.C. 1.1.1.27) and malate (MDH, E.C. 1.1.1.37) dehydrogenase in press-juices of rat brain. Furthermore, nerve ending-derived fractions (synaptosomes and synaptic vesicles) showed an enrichment of adenosine deaminase and also of 5′-nucleotidase. The action of deoxycholate over the subfractions did not increase the activity of either enzyme. The contrary occurred with the remaining enzymes studied. Thus, it is possible that one set of enzymes are located on the surface of the particulate vesicles, whereas another set are located inside these vesicles, suggesting a compartmentation of purine catabolic enzymes in different areas of the central nervous system.

A free derivate program for non-linear regression analysis of enzyme kinetics to be used on small computers

A non-linear regression program, written in BASIC, is described. The program uses the Marquardt s algorithm as modified by Reich et al. (Eur. J. Biochem., 26 (1972) pp. 368-379). The user only supplies the expression to fit, since the program uses numerical differentiation. It is possible to fit models of 1 substrate, 2 substrates, 1 substrate and 1 inhibitor, and 2 substrates and 1 inhibitor. Likewise, several weighting patterns, as well as a simple or robust regression, can be selected.

Kinetics of the 5'-nucleotidase and the adenosine deaminase in subcellular fractions of rat brain

Suspensions of rat brain microsomes, synaptosomes, and synaptic vesicles were able to convert adenosine to inosine by means of adenosine deaminase. Isosbestic points of this transformation, at 222, 250 and 281 nm, remained unchanged with time-course. This fact suggests that adenosine deaminase (ADA, E.C. 3.5.4.4) is located on the surface of the vesicles whereas purine nucleoside phosphorylase (PNP, E.C. 2.1.2.4) is located inside the vesicles. Kinetic parameters of the particulate 5′-nucleotidase (5′N, E.C. 3.1.3.5) and adenosine deaminase were analogous to those of the cytosolic enzymes. These results suggest that soluble and particulate enzymes represent different pools of the same molecular species.

A program for deriving rate equations using small computers

Abstract A computer-assisted method to derive steady-state rate equations is described. It is based on the method previously published by Cornish-Bowden (1977). The program is written in BASIC , and the listing and its corresponding flow diagrams are given.

Simulation of the purine nucleotide cycle as an anaplerotic process in skeletal muscle

A computer model of purine nucleotide and citric acid cycles joined through fumarate is given. Steady-state equations corresponding to metabolic enzymes are written based on the information from the literature about their kinetic behavior. Numerical integration of this set of equations is performed and in order to maintain an overall stabilization between the two cycles, enzymatic activities, in the form of V, have been calculated. Sensitivity coefficients for enzymes indicate that the control is exerted, depending upon the intermediate concentrations, and furthermore, it is demonstrated that AMP concentration in muscle should be very low. From stabilization, simulation of exercise conditions has been performed by diminishing [ATP] and increasing accordingly [ADP] and [AMP]. In such conditions the operation of purine nucleotide cycle leads to a considerable increase in the level of citric acid cycle intermediates. Disruption of purine nucleotide cycle by altering some of the three enzymatic steps leads to a lesser increase of these intermediates. The set of results presented seems to confirm the hypothesis that purine nucleotide cycle acts as an anaplerotic process in muscle, as the experimental results of Aragon and Lowenstein (Aragon, J.J., and Lowenstein, J.M. (1980) Eur. J. Biochem. 110, 371-377) suggest.

A program for the numerical integration of enzyme kinetic equations using small computers

A simple program to integrate differential equations is given. The program is written in BASIC and has been devised to be used on small computers. The program has been used to integrate the differential equations corresponding to a single reaction-single enzyme, a bireaction-single enzyme and a four reaction-four substrate-single enzyme mechanism; it has been used also for studying the interconversions among various molecular forms of a single enzyme, viz. monomer - dimer - trimer - tetramer. The program has proven to be very effective in these models and its time consumption comparable with that of other longer programs. The BASIC version is shown, and the FORTRAN VS version is available upon request to the authors.

The distribution of A1 adenosine receptor and 5'-nucleotidase in pig brain cortex subcellular fractions.

Pig brain cerebral cortex was subfractionated by isopycnic centrifugation in sucrose gradients. In each subfraction the content of the agonist [3H]R-PIA binding, the activity of adenosine metabolizing enzymes (5′-nucleotidase and adenosine deaminase) and the activity of membrane marker enzymes were determined. The fractions were also examined by electron microscope. In general, the results suggest a widespread distribution of A1 adenosine receptors in membranes from different origins. Marker enzyme profile characterization indicated an enrichment of A1 adenosine receptor in pre-synaptic membranes isolated from the crude synaptosomal fraction (P2B subfraction) as well as in membranes of glial origin such as myelin. The receptor is also present in the endoplasmic reticulum and in membranes isolated from the microsomal fraction that seem to have a post-synaptic origin (P3B). In subfractions having a high content of adenosine receptor the equilibrium binding paramters were obtained as well as the proportion of high- to low-affinity sites. From the values of the equilibrium constants it was not possible to find differences between the receptor in the different subfractions. Analysis of the affinity state distribution showed a diminished percentage of high-affinity sites in fraction P3A, which can be accounted by the existence of myelin membranes; in contrast the percentage of high-affinity states was higher in P2 and P3B, indicating that in these fractions the receptor is present in synaptosomal membranes. The close correlation shown between the enzyme 5′-nucleotidase specific activity and the specific ligand binding distributions led us to postulate an important role for the enzyme in the regulation of adenosine action in pig brain cortex.

Enzymes of the purine metabolism in rat brain microsomes.

Abstract1)Rat brain microsomes, when they are suspended in moderate ionic strength medium, released enzyme activities of lactate dehydrogenase (LDH, E.C.1.1.1.27), malate dehydrogenase (MDH, E.C.1.1.1.37), adenosine deaminase (ADA, E.C.3.5.4.4), guanine deaminase (GAH, E.C.3.5.4.3), and purine nucleoside phosphorylase (PNP, E.C.2.1.2.4). The activities released decreased when the saline concentration of the medium was increased and the opposite occurred when 50 mM, pH 7.4 sodium phosphate medium was used.2)Rat brain microsomes that had been extracted previously by moderate ionic strength solutions still had activities of all the enzymes tested, and released these activities upon sonication or deoxycholate (DOC) treatment. The proportion of the activity released was similar for all the enzymes. DOC treatment released higher enzymic activities and a smaller amount of protein than sonication did. The proportion of activities released was similar to that found in the 105,000g supernatant.3)The suspension of microsomes still retained activities of the above-mentioned enzymes after consecutive extractions with increasing concentrations of detergent solutions (DOC and Triton X-100).4)The amount of enzymic activities released from the microsomes by sonication or DOC treatment did not depend on the protein composition of the homogenization medium. Thus, on increasing the enzyme concentration in the homogenization medium, the activities released did not increase in parallel.5)The set of results obtained showed that the microsomal fraction is as useful as the cytosolic one for studying purine catabolism in rat brain. Furthermore, the conditions in which purine enzymes are attached to the microsomal fraction are probably closer to “in vivo” conditions than those in which these enzymes are found in the soluble fraction.

Functional groups and quaternary structure of chicken liver xanthine dehydrogenase

Xanthine dehydrogenase from chicken liver is a dimeric enzyme, each hemimolecule containing one FAD and two Fe/S groups. Determination of sulfhydryl groups with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) andp-hydroxymercuribenzoic acid (PMB) showed a variable number of sulfhydryl groups depending onpH, ionic strength, and nature of the reaction medium and buffer. The number of disulfide bonds was determined with DTNB and reducing conditions. Amino groups were determined with 2,4,6,-trinitrobencensulfonic acid (TNBS). At constant temperature andpH the reaction of DTNB and TNBS with native xanthine dehydrogenase showed an exponential dependence on time. From the obtained parameters the number of available sulfhydryl and amino groups at infinite concentration of enzyme and the rate constant of the equation were determined. The absorption spectrum of the enzyme changed with time when a chaotropic agent (1 M sodium nitrate) was added to the medium. This difference was detected by measuring the absorbance in the range 450–550 nm. The absorption spectrum (between 350 and 600 nm) also changed when a denaturating agent (sodium dodecyl sulfate) was added. This modification increased with time and depended on the medium.

A microcomputer method for designing optimal experiments for estimating enzyme kinetic parameters

A computer program, written in BASIC, for designing optimal experiments with the aim of evaluating estimates of the parameters for any enzyme kinetic model is given. This computer program can be run on any microcomputer with less than 32 Kbytes of random access memory. The program uses the termed D-optimization design criterion, which minimizes the determinant of the variance-covariance matrix. The user only supplies the rate equation, the maximum and minimum concentrations of substrates and inhibitors, the weighting pattern, and the best possible values of the parameters. The computer supplies the optimal substrate and inhibitor concentrations (one for each parameter), for estimating the parameter values, and the determinant of the variance-covariance matrix. Likewise, the microcomputer supplies the eigenvalues and eigenvectors of information and redundancy matrices, the sensitivity and the global redundancy.