Characterization of purified guanine aminohydrolase.

Guanine aminohydrolase (GAH) (E.C. 3.5.4.3) was purified by affinity chromatography on 9-(p-β-aminoethoxyphenyl)guanine-Sepharose to a specific activity of 35.5 units/mg. The molecular weight of the enzyme was estimated to be 110,000 by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) showed that the enzyme was composed of subunits with molecular weights of approximately 52,000. Data from SDS-gel electrophoresis in a discontinuous buffer system and from isoelectric focusing in the presence of 8-m urea indicated that more than one type of subunit were present. This was consistent with multiple forms of the native enzyme seen by electrophoresis and isoelectric focusing in polyacrylamide gels. The isoelectric points for the different forms of GAH were in the range of 4.65–4.85. Amino acid analyses showed cysteine to be the minimum amino acid and gave a calculated molecular weight for GAH of 53,016 when the assumption that there were four cysteines per subunit was made. Guanine, 8-azaguanine, and 6-thioguanine served as substrates for the enzyme but 3-deazaguanine, a potent competitive inhibitor of GAH, did not. Fluoride ion inhibited the enzyme in a noncompetitive manner, and this inhibition decreased as pH increased. Variation of the kinetic parameters with pH suggested that hydroxide ion might be the second substrate and that a functional group on the enzyme with a pK_a near 5.6 was involved in the reaction. The enzyme was inactivated by treatment with p-hydroxymercurobenzoate and by photooxidation in the presence of rose bengal. Two plausible mechanisms are proposed for the reaction catalyzed by GAH.     

Characterization of bovine liver guanine aminohydrolase

• 1.1. The isoelectric points of bovine liver guanine aminohydrolase and xanthine oxidase are 4.90 and 6.25, respectively.
• 2.2. The molecular weight of the guanine aminohydrolase is 95,000.
• 3.3. The guanine aminohydrolase is formed from two subunits of identical molecular weight.

     

A new fluorimetric assay for streptothricins

In strongly acidic medium (70% HClO4) streptothricins form a fluorophore (lambda ex = 312 nm; lambda em = 381 nm) with unknown structure. A fluorimetric determination of pure or crude products and cultures, respectively, was worked out based on this reaction. Concentrations for fluorescence measurements were in the range of 10(-8) - 2 X 10(-7) moles. Interferences of the assay are discussed, a statistical evaluation of results and a comparison between microbiological and fluorimetric findings are given.     

A sensitive fluorimetric assay for pyruvate

A sensitive and specific fluorimetric assay for the determination of pyruvate is reported here. This assay is based on the oxidation of pyruvate in the presence of pyruvate oxidase. Hydrogen peroxide generated by pyruvate oxidase reacts with nonfluorescent Amplex Red at a 1:1 stoichiometry to form the fluorescent product, resorufin. The assay is optimized with respect to pH of reaction buffer, enzyme concentration, dye concentration, and the time course. The usefulness of the assay is demonstrated by the accurate measurement of intracellular and extracellular pyruvate concentrations. The limit of detection of the assay is 5 nM.     

Purification of rabbit liver guanine aminohydrolase.

Rabbit liver guanine aminohydrolase has been purified 1250-fold by utilization of an affinity chromatographic separation on 9-(p-aminoethoxyphenyl) guanine-Sepharose with 50% recovery of activity. Polyacrylamide gel electrophoresis of the purified preparations revealed several protein bans which corresponded to regions of enzyme activity measured on gels which had been run under the same conditons. Gel concentration studies of the protein migration rate showed that the protein bans differed in molecular size. The minimum molecular weight was 100,000 from gel permeation chromatography studies. The pH optimum was near pH 8 and the Km, with guanine as substrate was 5.6 x 10-6 M. The latter values are in close agreement with partially purified preparations described in the literature.     

A fluorimetric assay for aminopeptidase W.

N-Methylformamide extracts of acid-treated precipitated VFe protein of the V-nitrogenase of Azotobacter chroococcum are yellow-brown in colour and contain vanadium, iron and acid-labile sulphur in the approximate proportions 1:6:5. E.p.r. spectra of the extracts exhibit a weak signal with g values near 4.5, 3.6 and 2.0 characteristic of an S = 3/2 metal-containing centre. The N-methylformamide extracts activated the MoFe protein polypeptides from mutants of nitrogen-fixing bacteria unable to synthesize FeMoco, the active centre of Mo-nitrogenase. The active hybrid protein exhibited the characteristic substrate-reducing phenotype associated with the VFe protein except that it could not reduce N2 to NH3. The above data are interpreted as demonstrating the existence of an iron- and vanadium-containing cofactor, FeVaco, within the VFe protein. It is suggested that nitrogen fixation requires specific interactions between FeVaco or FeMoco and their respective polypeptides. The biosynthesis of these cofactors is discussed.     

A simple fluorimetric assay for guanosine nucleotides

It is comparatively easy to assay adenosine 5′-triphosphate either in the presence or absence of other nucleoside triphosphates (1). An assay for UTP has been reported (2) based on its incorporation into RNA using RNA polymerase and a poly(dA-dT) template in the presence of excess [³H]-ATP; this method could be adapted to assay for GTP and CTP. It is known that the fluorescence of Tb³⁺ increases on binding to nucleic acids (3–5), and this enhancement has been attributed both to complex formation between Tb³⁺ and guanine residues in the nucleic acid (3) and also to binding of Tb³⁺ to thiouracil (4).     

A high-throughput fluorimetric assay for angiotensin I-converting enzyme

Angiotensin I-converting enzyme (ACE) assays are commonly used for measuring enzymatic activity in clinical and biological samples. The fluorimetric procedure described is sensitive, rapid and involves unsophisticated procedures and inexpensive reagents. It uses the substrate hippuryl-L-histidyl-L-leucine, and the fluorescent adduct of the enzyme-catalyzed product L-histidyl-L-leucine is quantified fluorimetrically. This assay has been adapted for a 96-well plate format that produces comparable data to previously described assays and has the advantage of greater efficiency with respect to both time and reagents. The protocol can be used for routine purposes or for more detailed kinetic analyses. The apparent Km and kcat values for purified testis ACE determined from a double reciprocal plot were 3.0 mM and 195.7 s(-1), respectively. The protocol can be completed within 4 h.     

A fluorimetric assay for acyl-CoA synthetase activities.

In this paper we describe a fluorimetric assay for the measurement of long-chain acyl-CoA synthetase activity in rat liver postnuclear supernatants. The method is based upon the use of acyl-CoA oxidase which catalyzes the dehydrogenation of acyl-CoA esters to yield enoyl-CoA esters and H2O2. H2O2 subsequently reacts with homovanillic acid in a horseradish peroxidase-catalyzed reaction to form a highly fluorescent dimer (see G. G. Guilbault, P. J. Brignac, and M. Zimmer (1968) Anal. Chem. 40, 190-196). The increase in fluorescence can be followed either continuously or discontinuously. The method described is able to detect acyl-CoA synthetase activities as low as 20 microU/ml which is almost as sensitive as the standard isotopic assay used in most laboratories. The method is applicable to measure the activation of a variety of fatty acids. Finally, the method provides a simple means of carrying out kinetic studies.     

A continuous fluorimetric assay for ATPase activity.

A new continuous coupled fluorimetric assay is described for ATPases in general. Thus phosphate released from ATP hydrolysis is coupled to the nucleoside phosphorylase reaction using 7-methylguanosine as a fluorescent substrate for the nucleoside phosphorylase reaction. The hydrolysis of 7-methylguanosine leads to 7-methylguanine, which has lower quantum yield and hence can be used to monitor ATP hydrolysis continuously. The method has the potential to be extended to GTPase and nucleotidyltransferase assays.     

A microplate fluorimetric assay for measuring dehalogenase activity

A fluorimetric assay was developed to measure halide release from halogenated compounds being degraded by microbes. The method relies on the property of halides to quench the fluorescence of 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) by collision quenching. The assay shows a wide response to halide concentration (1-500 mM) and tolerates a wide pH range. Furthermore, it is simple to use, has the potential for automation and uses an inexpensive non-toxic reagent.     

A fluorimetric Morgan-Elson assay method for hyaluronidase activity

Despite their physiological importance, hyaluronidases (HAases) have long been "neglected enzymes," due, presumably, in part to the lack of rapid, sensitive assays. Currently, the colorimetric Morgan-Elson assay method, which is based upon the generation of a new reducing N-acetyl-D-glucosamine terminus with each cleavage reaction, is most widely employed but is yet insensitive. We, therefore, reinvestigated the colorimetric method and established the fluorimetric Morgan-Elson assay for HAase activity, with the optimized tetraborate reagent. The fluorimetric assay, requiring neither specialized reagents nor a long time to perform, provided high sensitivity, nearly comparable to that of enzyme-linked immunosorbent assay (ELISA)-like assays, with a detection limit of 5 x 10(-3)NFU/ml of bovine testicular HAase after 1-h incubation. The increased sensitivity permitted rapid measurement of low HAase activity in biological samples such as human and rabbit serum HAases, the latter of which has not been detected either by an ELISA-like assay or by zymography. Human serum HAase was easily characterized it along with its optimum pH and kinetic parameters.     

A fluorimetric assay for available lysine in proteins

A two-dimensional fingerprinting gel system that provides sensitive analyses with high resolution of T1-resistant oligonucleotides of large RNA molecules is described. Unique oligonucleotides less than 30 bases in length are recovered quantitatively while longer oligonucleotides are recovered in very large (～90%) yields by active transfer of the fingerprint to DEAE paper. After elution of the oligonucleotides from DEAE paper, secondary analysis is performed by digestion of oligonucleotides with pancreatic RNase and separation of the products by high-voltage electrophoresis on polyethyleneimine cellulose. The complete analysis of up to 40 oligonucleotides can be accomplished within 4 days.     

A sensitive fluorimetric assay for gamma-glutamyl transferase.

A rapid, sensitive single-step assay for γ-glutamyl transferase is presented using l-γ-glutamyl-7-amino-4-methyl-coumarin as the donor substrate. Kinetic parameters and the relative activities with different acceptors were determined using this substrate. The results are compared with those obtained with an alternative substrate, l-γ-glutamyl-2-naphthyl-amide.     

Partial purification and characterization of guanine aminohydrolase fromtrypanosoma cruzi

Guanine deaminase (guanine aminohydrolase, EC 3.5.4.3) catalyzes the hydrolytic deamination of guanine to xanthine. A rapid procedure for the partial purification of guanine deaminase fromTrypanosoma cruzi using granulated bed electrofocusing was developed. Supernatants of cell sonicates (40,000 g) were subjected to electrofocusing with a broad range ampholyte (pH 4–9). Sections of the gel were eluted and assayed for xanthine production. Active fractions were pooled, concentrated, and again subjected to electrofocusing with a pH 5–7 range ampholyte. This procedure resulted in over 240-fold purification. The compounds 4-amino-5-imidazolecarboxamide andN ⁶-methyladenine were found to be potent competitive inhibitors of the enzyme. Their respective K_i values were 3.5×10^–6 M and 9.5×10^–6 M. Irreversible inactivation of the enzyme was observed upon incubation withp-chloromercurophenylsulfonic acid andN-ethyl-maleamide at 5.0×10^–4 M. The enzyme was labile to heat; a substantial loss of activity occurred upon incubation at 55°C for 5 min. A broad pH range of activity (pH 7.5–8.5) was observed in Tris, citrate, and phosphate buffers.     

Molecular forms of guanine aminohydrolase (author's transl)

Guanine aminohydrolase (E.C. 3.5.4.3) has been purified 11-fold from the supernatant fraction of guinea-pig liver homogenates in 0.25 M sucrose (centrifuged at 50,000 X g) through thermic denaturation at 60 degrees C and ammonium sulphate fractionation (30--60% saturation). The enzyme in the homogenates and purified preparations exhibited two Km values. In both preparations four enzymatic electrophoretic bands have been detected. Purified guanine aminohydrolase is chromatographically resolved on DEAE-sephadex in three components whose active forms appeared separately on their pherograms. The enzymatic form eluted at lower ionic strength has the least anodic mobility, is inhibited by guanine (4 X 10(-5) M) and presents only one Km value (1.5 X 10(-5) M). The enzymatic form eluted at greater ionic strength exhibits the highest anodic mobility, is also inhibited by guanine (7 X 10(-5) M) and its Km value seems to be 6.3 X 10(-6) M. Molecular weight of enzymatics forms determined by Sephadex G-200 chromatography, is 120,000 +/- 5,000. The preceding results, correlated with the chromatographic homogeneity of guanine aminohydrolase, purified in Sephadex G-100, suggests that the four molecular forms of the native enzyme may be considered as isozymes.     

A continuous fluorimetric assay for tumor necrosis factor-alpha converting enzyme

Fluorogenic peptide substrates with fluorophore/quencher-capped ends have found extensive use in monitoring protease activity in the screening of small-molecule libraries for protease inhibitors. We report here the identification and characterization of a fluorogenic substrate for tumor necrosis factor-alpha converting enzyme (TACE). This substrate is a 10-amino-acid peptide (LAQAVRSSSR) capped with an o-aminobenzoyl group on the N-terminal end and with a 3-(2,4-dinitrophenyl)-L-2,3-diaminopropionic amide group on the C-terminal end. Exhaustive enzymatic conversion of the substrate to products resulted in a fluorescence enhancement of -11-fold. A single cleavage occurred at the A-V scissile bond of the peptide. The validity of this fluorimetric assay for TACE was corroborated by an independent HPLC method. Interestingly, the hydrolysis of the substrate displayed positive cooperativity with a Hill coefficient of 1.5, while the hydrolysis of the corresponding uncapped peptide displayed Michaelis-Menten kinetics. A k(cat) value of 21.6 s(-1) and an S(0.5) value of 342 microM were obtained for the fluorogenic substrate. The addition of the two capping groups on the two ends of the peptide enhanced the k(cat) value by 64-fold. Nine additional decapeptides that contained the same capping groups on the two ends and substitutions at the P1 and P1' sites were also tested. TACE appears to slightly prefer the A-V scissile bond. The enzyme also cleaves scissile bonds such as F-V, A-I, and A-L efficiently.     

A fluorimetric assay for human reticulocyte 15-lipoxygenase-1 activity

A rapid and sensitive fluorescence-based assay for the determination of human 15-lipoxygenase-1 (15-LOX-1) activity is described in this article. The assay utilizes the ability of 15-LOX-1-generated lipid hydroperoxides to oxidize nonfluorescent dihydrorhodamine 123, producing the highly fluorescent dye rhodamine 123. Formation of rhodamine 123 can be monitored through fluorescence spectroscopy using Ex/Em of 500 nm/536 nm. The IC(50) values of three well-known 15-LOX-1 inhibitors, nordihydroguaiaretic acid, quercetin, and fisetin, were evaluated in 96- and 384-well formats, and they conform to previously reported data. We believe this assay can be broadly used for the discovery of novel lipoxygenase inhibitors.