Diadenosine 5′,5‴-P1,P3-triphosphate in eukaryotic cells: Identification and quantitation

A highly sensitive enzymatic assay for diadenosine 5′,5?-P¹,P³-triphosphate (Ap₃A) has been established on the basis of the coupled luminescence assay for diadenosine 5′,5?-P¹,P³-tetraphosphate (A. Ogilvie (1981)Anal. Biochem.115, 302–307). Snake venom phosphodiesterase splits Ap₃A into AMP plus ADP which can be measured in a luminescence reaction containing pyruvate kinase, phosphoenolpyruvate and luciferin-luciferase. The procedure is linear with Ap₃A levels ranging from 0.1 to 2 pmol. The assay has been used to measure Ap₃A in various eukaryotic cells after ion-exchange chromatography and high-performance liquid chromatography of acidic extracts of the cells. The level of diadenosine triphosphate was higher in all instances than the level of diadenosine tetraphosphate. When growing in the abdominal cavity of mice, Ehrlich ascites tumor cells contained high amounts of Ap₃A ($\text{0.1}\text{nmol}\text{10}{}^6\text{cells}$), allowing direct optical determination in the HPLC chromatography. The quantitative measurement of Ap₃A with the luminescence assay gave identical results. Ap₃A extracted from Ehrlich cells was also chromatographed with authentic nucleotide in two thin-layer systems providing additional proof for the existence of Ap₃A in biological material.     

Detection of chemiluminescence in lipid peroxidation of biological systems and its application to HPLC

A combined system of chemiluminescence detection and high performance liquid chromatography (CL–HPLC) was developed to determine primary peroxidation products in biological tissues, such as phosphatidylcholine hydroperoxide (PCOOH). The CL–HPLC assay consists of separation of lipid classes with HPLC and detection of hydroperoxide-specific chemiluminescence. Hydroperoxides react with heme compounds to produce oxidants as suggested by our early studies on tissue low-level chemiluminescence in which singlet molecular oxygen is generated as one of the excited species in several biological systems involving free radical events. In the CL–HPLC method, a cytochrome c–luminol mixture was used as a hydroperoxide-specific luminescent reagent, and the quantification of hydroperoxide was performed by detecting chemiluminescence due to the luminol oxidation caused by the oxidant produced during the lipid hydroperoxides with heme. The detection limit of PCOOH was 10 pmole hydroperoxide–O₂. PCOOH in normal human blood was found to be 10–500 pmol/ml plasma and significantly higher levels of PCOOH were observed in some hospitalized patients.     

Alteration of opioid receptors in seizure-susceptible El mouse brain

The distribution density of opioid receptors in the brain of El mice (seizure-susceptible strain) was examined to determine the relation between seizures and the opioid system. Saturation curves and Scatchard plots of [³H]2-d-alamine-5-d-leucine enkephalin binding revealed that the opioid delta receptor density in adult El mice during interictal periods was significantly increased in the cerebral cortex, hippocampus, and septal area. It was further shown that the concentration of such receptors in 25-day-old El mice that had no seizures was also significantly increased in the hippocampus and septal area, with no changes in apparent affinities, as compared with in the corresponding regions in ddY mice (seizure-nonsusceptible strain; the mother strain of El). Such up-regulation of opioid receptors in the El mouse brain could result from deficits in endogenous opioid peptides, which could be associated with the pathogenesis of seizure diathesis in the El mouse.     

Halcyon days with Bill Arnold

The circumstances that led to the discovery that plants luminesce after they are illuminated are described, as are other discoveries that would not have been possible were it not for the fortuitous association I had with my dear and most admirable friend, W.A. Arnold, to whom this special issue is dedicated.     

Isolation and promoter characterization of barley geneItr1 encoding trypsin inhibitor BTI-CMe: differential activity in wild-type and mutantlys3a endosperm

The geneItr1, encoding trypsin inhibitor BTI-CMe, has been obtained from a genomic library ofHordeum vulgare L. The gene has no introns and presents in its 5-upstream region 605 bp that are homologous to the long terminal repeats (LTR) of the copia-like retro-transposon Bare-1. Functional analysis of theItr1 promoter by transient expression in protoplasts derived from different barley tissues, has shown that in this system theItr1 promoter retains its endosperm specifity and thetrans-regulation mediated by theLys3a gene. The proximal promoter extending 343 bp upstream of the translation initiation ATG codon is sufficient to confer fullGUS expression and for endosperm specifity. In protoplasts derived from thelys3a mutant, Risø 1508,GUS activity was less than 5% of that obtained with the same constructs in the protoplasts of wild-type Bomi from which it derives. Gel retardation experiments, after incubation with proteins obtained from both types of endosperm nuclei, also show differential patterns. Possible reasons for these differences are discussed.Equal authours     

Peroxyoxalate chemiluminescent assay for oxidase activities based on detecting enzymatically formed hydrogen peroxide

A sensitive peroxyoxalate chemiluminescent (PO-CL) assay for activities of oxidases (uricase, choline oxidase, cholesterol oxidase and xanthine oxidase) which catalyse a formation of hydrogen peroxide was developed using 4,4′-oxalyl-bis[(trifluoromethylsulphonyl)imino]trimethylene-bis(4-methylmorpholinium)trifluoromethanesulphonate as a chemiluminogenic reagent and 2,4,6,8-tetramorpholinopyrimido[5,4-d]pyrimidine as a fluorophore. The standard curve for hydrogen peroxide was linear over the range 1 × 10^?7-1 × 10^?4 mol/L. Relative standard deviations for oxidase assays were 5.1–12.7% (n = 10). Detection limits were 1 × 10^?3 U/mL for uricase, 5 × 10^?4 U/mL for choline oxidase, 5 × 10^?3 U/mL for cholesterol oxidase and 5 × 10^?4 U/mL xanthine oxidase (sample to blank ratio, 3).     

A novel bioluminogenic assay for α-chymotrypsin

The use of 6-(N-acetyl-L -phenylalanyl)-aminoluciferin as a novel substrate for α-chymotrypsin has been demonstrated. The kinetic parameters determined are K_M = 0.38mmol/L, k_cat = 6.5 s^?1 and k_cat/k_M = 17,100 (L/mols). The test principle of the coupled assay is the release of aminoluciferin by enzymatic cleavage of 6-(N-acetyl-L -phenylalanyl)-aminoluciferin. Aminoluciferin is oxidized, with light emission, by firefly luciferase (Photinus pyralis) and can be quantified in a luminometric assay. The detection limit for chymotrypsin was found to be 0.3 ng per assay. 6-(N-acetyl-L -phenylalanyl)-aminoluciferin has been synthesized as an example for a new class of highly sensitive substrates. By modification of the peptide residue these new substrates may be suitable for ultrasensitive detection of different proteinases.     

Ultrafast protein determinations using microwave enhancement

We present here microwave-based modifications of standard protein assays that dramatically reduce the time required to determine protein concentrations. Typical protein determinations involve incubation times ranging from 15–60 min. Microwave irradiation of specimens reduces this time requirement to 10–20 s without compromising accuracy or reliability. The remarkable speed with which protein determinations may be carried out using microwave enhancement greatly simplifies general laboratory procedures that depend on the estimation of protein concentrations. An erratum to this article is available at .     

The extracellular nuclease of Serratia marcescens: studies on the activity in vitro and effect on transforming DNA in a groundwater aquifer microcosm

A quantitative endonuclease assay, which relies on the introduction of single and double strand breaks into supercoiled plasmid DNA, was used to study the activity of the extracellular nuclease of Serratia marcescens SM6 in buffer and in groundwater. The parallel enzyme concentration-dependent production of relaxed and linear plasmid molecules suggests that the nuclease produces single and double strand breaks in duplex DNA. Bovine serum albumin stimulated the nuclease activity towards DNA and RNA and increased the stability of the enzyme against thermal inactivation. The DNase activity at 4 °C and 50 °C was almost half of that at the optimum temperature (37 °C). The nuclease was active in groundwater, although the specific activity was lower than in buffer. In a groundwater aquifer microcosm, mineral-adsorbed transforming DNA was substantially less accessible to the nuclease than was dissolved DNA. The data suggest that the extracellular nuclease of Serratia marcescens may contribute to DNA turnover in the environment and that adsorption of DNA to minerals provides protection against the nuclease.Abbreviations GW groundwater GWA groundwater aquifer