Assay for the enzymic hydrolysis of penicillin and acylhydroxamates

A rapid and easily reproducible assay for penicillin acylase and aliphatic amidase activity is described. The method, which is based on the colorimetric estimation of proton release, is very sensitive and rates as low as 0.5 nmoles/minute can be measured readily. Determination of the kinetic constants of the enzymes gave results as good as or better than standard procedures.     

Monooxygenase activities of dioxygenases. Benzphetamine demethylation and aniline hydroxylation reactions catalyzed by indoleamine 2,3-dioxygenase

Benzphetamine demethylase and aniline hydroxylase activities were determined with various hemoproteins including indoleamine 2,3-dioxygenase in a cytochrome P-450-like reconstituted system containing NADPH, NADPH-cytochrome P-450 reductase, and O2. The highest specific activities, almost comparable to those of liver microsomal cytochrome P-450, were detected with indoleamine 2,3-dioxygenase from the rabbit intestine. The indoleamine 2,3-dioxygenase-catalyzed benzphetamine demethylation reaction was inhibited by catalase but not by superoxide dismutase. Exogenous H2O2 or organic hydroperoxides was able to replace the reducing system and O2. The stoichiometry of H2O2 added to the product formed was essentially unity. These results indicate that the dioxygenase catalyzes the demethylation reaction by the so-called "peroxygenation" mechanism using H2O2 generated in the reconstituted system. On the other hand, the dioxygenase-catalyzed aniline hydroxylation reaction was not only completely inhibited by catalase but also suppressed by superoxide dismutase by about 60%. Although the O2- and H2O2-generating system (e.g. hypoxanthine-xanthine oxidase) was also active as the reducing system, neither exogenous H2O2 nor the generation of O2- in the presence of catalase supported the hydroxylation reaction, indicating that both H2O2 and O2- were essential for the hydroxylation reaction. However, typical scavengers for hydroxyl radical and singlet oxygen were not inhibitory. These results suggest that a unique, as yet unidentified active oxygen species generated by H2O2 and O2- participates in the dioxygenase-mediated aniline hydroxylation reaction.     

Determination of initial velocities of enzymic reactions from progress curves.

The present communication describes a novel method for estimating initial velocities (v) of enzyme-catalysed reactions. It is based on an approximation of experimental data obtained by the cubic spline function. The initial velocity of a reaction is calculated as a derivative of the approximating function at a time value equal to zero. The proposed method is usable on a computer with a FORTRAN IV program. The method can be successfully used in such cases as substantial extents of substrate conversion, the inactivation of an enzyme in the course of a reaction, the existence of large experimental error or when the reaction mechanism is unknown.     

Determination of diamine oxidase in lentil seedlings by enzymic activity and immunoreactivity

A competitive radioimmunoassay for the quantitation of diamine oxidase (EC 1.4.3.6) from Lens culinaris is reported. Specific antibodies raised in rabbits immunized with a homogeneous preparation of the enzyme were incubated with purified ¹²⁵I-enzyme and with either unlabeled diamine oxidase or plant material. Antigen-antibody complexes were isolated from the mixture by incubation with Staphylococcus protein A. The sensitivity of the test was about 5 nanograms in terms of enzyme protein. This assay was applied to the determination of the enzyme in extracts from lentil shoots grown either in the dark or in the light. Diamine oxidase activity and enzyme protein (as determined by radioimmunoassay) were measured during 7 days after germination. Both enzymic activity and enzyme protein declined slowly in the dark and rapidly in the light. These results indicate that fluctuation of the enzymic activity in this organ, both in the light and in the dark, are mediated via changes in the amount of the enzyme protein and not via the action of an inhibitor.     

The enzymic preparation of diphosphoinositides.

A procedure for the preparation of diphosphoinositides is described. Triphosphoinositides isolated from bovine brain are hydrolysed by the triphosphoinositide phosphatase (EC 3.1.3.36) from Crithidia fasciculata in the presence of MgC12 and cetyltrimethyl-ammonium bromide. The diphosphoinositides produced are not degraded further and can be recovered from the reaction mixture in greater than 80% yield. The product is chromatographically pure and has the same structure (1-phosphatidylinositol 4-phosphate) as naturally occurring diphosphoinositides.     

Assay of nicotinamide deamidase. Determination of ammonia by the indophenol reaction

The indophenol reaction for the determination of ammonia has been adopted for the assay of nicotinamide deamidase. Nicotinamide interferes with the development of the blue color characteristic of indophenol by competing with ammonia for the assay reagents. This difficulty is circumvented by varying the concentration of nitroprusside used in the determination of ammonia with the nicotinamide contents of individual samples. It is not possible to adopt a unique concentration of nitroprusside for all nicotinamide concentrations because both insufficient and excessive amounts of the reagent lead to low color yields. The method permits the determination of 0.02–1.4 μmoles ammonia and as little as 1 μg rabbit liver nicotinamide deamidase. The reduction of the scale of the procedure to one-tenth is possible and would permit a ten-fold increase in sensitivity. The inhibition of the indophenol reaction by amino acids has also been found to be reversed by increased nitroprusside levels in assay mixtures.Since a number of substances can interfere with the indophenol method, attention must be given to the control of their effects on the color reaction. Ammonia and other nitrogenous compounds may have to be removed if their concentrations in assay samples are high. The effects of low levels of such compounds ordinarily can be controlled through the appropriate use of reagent blanks, internal standards, and adjustment of the nitroprusside concentration employed in the ammonia assay mixtures.     

Mammalian protein methylesterase. Physical and enzymic properties.

Protein methylesterase (PME) amino acid composition and substrate specificity towards methylated normal and deamidated protein substrates were investigated. The enzyme contained 23% acidic and 5% basic residues. These values are consistent with a pI of 4.45. The product formed from methylated protein by PME was confirmed as methanol by h.p.l.c. The kcat. and Km values for several methylated protein substrates ranged from 20 x 10(-6) to 560 x 10(-6) s-1 and from 0.5 to 64 microM respectively. However, the kcat./Km ratios ranged within one order of magnitude from 11 to 52 M-1.s-1. Results with the irreversible cysteine-proteinase inhibitor E-64 suggested that these low values were in part due to the fact that only one out of 25 molecules in the PME preparations was enzymically active. When PME was incubated with methylated normal and deamidated calmodulin, the enzyme hydrolysed the latter substrate at a higher rate. The Km and kcat. for methylated normal calmodulin were 0.9 microM and 31 x 10(-6) s-1, whereas for methylated deamidated calmodulin values of 1.6 microM and 188 x 10(-6) s-1 were obtained. The kcat./Km ratios for methylated normal and deamidated calmodulin were 34 and 118 M-1.s-1 respectively. By contrast, results with methylated adrenocorticotropic hormone (ACTH) substrates indicated that the main difference between native and deamidated substrates resides in the Km rather than the kcat. The Km for methylated deamidated ACTH was 5-fold lower than that for methylated native ACTH. The kcat./Km ratios for methylated normal and deamidated ACTH were 43 and 185 M-1.s-1 respectively. These results indicate that PME recognizes native and deamidated methylated substrates as two different entities. This suggests that the methyl groups on native calmodulin and ACTH substrates may not be on the same amino acid residues as those on deamidated calmodulin and ACTH substrates.     

Accleration of autooxidation of human oxyhemoglobin by aniline and its relation to hemoglobin-catalyzed aniline hydroxylation.

Changes in the ultraviolet/visible spectrum of human oxyferrohemoglobin upon addition of aniline were indicative of a concentration-dependent interaction of aniline with hemoglobin, resulting in accelerated autooxidation of the hemoprotein. Oxygen was found to markedly inhibit this interaction of aniline with oxyhemoglobin. The dependence of the rate of autooxidation on aniline concentration followed saturation kinetics and showed a half-maximal response at 8 mM aniline. This value is equal to the value of Km for aniline as substrate for the O2-dependent, hemoglobin-catalyzed hydroxylation reaction which yields p-aminophenol (Mieyal, J. J., Ackerman, R.S., Blumer, J.L., and Freeman, L.S. (1976) J. Biol. Chem. 241, 3436-3441). Thus, an aniline-oxyhemoglobin complex is implicated in the overall catalytic reaction. No detectable p-aminophenol was formed when aniline was combined with oxyhemoglobin in the absence of an electron donor, but hydroxylation of aniline does occur when NADPH, NADPH plus P-450 reductase, or Na2S2O4 are also added.     

Complete enzymic digestion of acidic proteins.

Acidic proteins are usually resistant to complete enzymic hydrolysis. The increasing number of "unusual" amino acids, which are unstable to acid hydrolysis, makes it necessary to have a method of enzymic hydrolysis applicable to all proteins. The complete hydrolysis of four acidic proteins by subtilisin plus leucine amino-peptidase plus prolidase followed by carboxypeptidase C, is described. Recoveries of amino acids were in excellent agreement with the expected content from the known sequences.