Determination of enzymatic activities and metabolites in microliter sample size.

A new generation of spectrophotometers able to measure a wide range of absorbance in microliter aliquots is currently used for the determination of DNA, RNA, and proteins. The object of this article is to show that these instruments could be easily adapted for routine evaluation of enzymes and metabolites in 1-2-microl volumes of biological samples.

Synthesis of ATP derivatives of compounds of the mevalonate pathway (isopentenyl di- and triphosphate; geranyl di- and triphosphate, farnesyl di- and triphosphate, and dimethylallyl diphosphate) catalyzed by T4 RNA ligase, T4 DNA ligase and other ligases Potential relationship with the effect of bisphosphonates on osteoclasts.

Compounds of the mevalonate pathway containing a terminal di- or triphosphate (mev-PP or mev-PPP) were tested as substrates of several enzyme ligases (T4 RNA ligase, T4 DNA ligase, firefly luciferase and other ligases) for the synthesis of ATP derivatives of the mev-pppA or mev-ppppA type. T4 RNA ligase, in the presence of ATP and the substrates: geranyl, farnesyl or isopentenyl triphosphates, and geranyl, farnesyl, dimethylallyl or isopentenyl diphosphates, all at 0.3 mM concentration, catalyzed the synthesis of the corresponding ATP derivatives at a relative rate of activity of: 7.

Synthesis of bisphosphonate derivatives of ATP by T4 RNA ligase.

T4 RNA ligase catalyzes the synthesis of ATP beta,gamma-bisphosphonate analogues, using the following substrates with the relative velocity rates indicated between brackets: methylenebisphosphonate (pCH(2)p) (100), clodronate (pCCl(2)p) (52), and etidronate (pC(OH)(CH(3))p) (4). The presence of pyrophosphatase about doubled the rate of these syntheses. Pamidronate (pC(OH)(CH(2)-CH(2)-NH(2))p), and alendronate (pC(OH)(CH(2)-CH(2)-CH(2)-NH(2))p) were not substrates of the reaction.

Polyphosphates strongly inhibit the tRNA dependent synthesis of poly(A) catalyzed by poly(A) polymerase from Saccharomyces cerevisiae.

Polyphosphates of different chain lengths (P(3), P(4), P(15), P(35)), (1 microM) inhibited 10, 60, 90 and 100%, respectively, the primer (tRNA) dependent synthesis of poly(A) catalyzed poly(A) polymerase from Saccharomyces cerevisiae. The relative inhibition evoked by p(4)A and P(4) (1 microM) was 40 and 60%, respectively, whereas 1 microM Ap(4)A was not inhibitory. P(4) and P(15) were assayed as inhibitors of the enzyme in the presence of (a) saturating tRNA and variable concentrations of ATP and (b) saturating ATP and variable concentrations of tRNA.

Dinucleoside polyphosphates stimulate the primer independent synthesis of poly(A) catalyzed by yeast poly(A) polymerase.

Novel properties of the primer independent synthesis of poly(A), catalyzed by the yeast poly(A) polymerase are presented. The commercial enzyme from yeast, in contrast to the enzyme from Escherichia coli, is unable to adenylate the 3'-OH end of nucleosides, nucleotides or dinucleoside polyphosphates (NpnN). In the presence of 0.

Poly(A) polymerase from Escherichia coli adenylylates the 3'-hydroxyl residue of nucleosides, nucleoside 5'-phosphates and nucleoside(5')oligophospho(5')nucleosides (NpnN).

The capacity of Escherichia coli poly(A) polymerase to adenylylate the 3'-OH residue of a variety of nucleosides, nucleoside 5'-phosphates and dinucleotides of the type nucleoside(5')oligophospho(5')nucleoside is described here for the first time. Using micromolar concentrations of [alpha-32P]ATP, the following nucleosides/nucleotides were found to be substrates of the reaction: guanosine, AMP, CMP, GMP, IMP, GDP, CTP, dGTP, GTP, XTP, adenosine(5')diphospho(5')adenosine (Ap2A), adenosine (5')triphospho(5')adenosine (Ap3A), adenosine(5')tetraphospho(5')adenosine (Ap4A), adenosine(5')pentaphospho(5')adenosine (Ap5A), guanosine(5')diphospho(5') guanosine (Gp2G), guanosine(5')triphospho(5')guanosine (Gp3G), guanosine(5')tetraphospho(5')guanosine (Gp4G), and guanosine(5')pentaphospho(5')guanosine (Gp5G). The synthesized products were analysed by TLC or HPLC and characterized by their UV spectra, and by treatment with alkaline phosphatase and snake venom phosphodiesterase.

Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase and several ligases.

The findings presented here originally arose from the suggestion that the synthesis of dinucleoside polyphosphates (Np(n)N) may be a general process involving enzyme ligases catalyzing the transfer of a nucleotidyl moiety via nucleotidyl-containing intermediates, with release of pyrophosphate. Within this context, the characteristics of the following enzymes are presented. Firefly luciferase (EC 1.

IMP-GMP 5'-nucleotidase from rat brain: activation by polyphosphates.

IMP-GMP 5'-nucleotidase has been purified to homogeneity from total rat brain extracts. This preparation showed a unique band (Mr 54,000 +/- 1,509) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme presented the following properties: optimal pH value, 6.

Acyl coenzyme A synthetase from Pseudomonas fragi catalyzes the synthesis of adenosine 5'-polyphosphates and dinucleoside polyphosphates.

Acyl coenzyme A (CoA) synthetase (EC 6.2.1.

Synthesis of dehydroluciferin by firefly luciferase: effect of dehydroluciferin, coenzyme A and nucleoside triphosphates on the luminescent reaction.

The formation of dehydroluciferin (L) from luciferin (LH2) in the reaction catalyzed by firefly luciferase (EC 1.13.12.

2',3'-dideoxynucleoside triphosphates (ddNTP) and di-2',3'-dideoxynucleoside tetraphosphates (ddNp4ddN) behave differently to the corresponding NTP and Np4N counterparts as substrates of firefly luciferase, dinucleoside tetraphosphatase and phosphodiesterases.

2',3'-Dideoxynucleosides (ddN) and their derivatives are currently used as antiretroviral compounds. Their active agents are the corresponding 2',3'-dideoxynucleoside triphosphates (ddNTPs) generated inside the cell by host kinases. Dinucleoside tetraphosphates (Np4Ns) are molecules of interest in metabolic regulation; their synthesis in vitro can be catalyzed by firefly luciferase.

Risk factors of surgical wound infection in patients undergoing herniorrhaphy.

Objective: To study the causes of surgical wound infection in patients being operated on for abdominal hernias.

Design: Prospective cohort study.

Setting: A tertiary hospital, Spain.

Time course of luciferyl adenylate synthesis in the firefly luciferase reaction.

The time course of luciferyl adenylate formation in the reaction catalyzed by firefly luciferase (EC 1.13.12.

[Review: hybrid designs of cohort studies and case control studies].

Hybrid epidemiologic designs combining elements from case-control studies and cohort studies are reviewed. Firstly, the characteristics of selection of both index group and reference group are commented on. The selection of cases required a population-based disease registry.

Uric acid synthesis by rat liver supernatants from purine bases, nucleosides and nucleotides. Effect of allopurinol.

The synthesis of uric acid from purine bases, nucleosides and nucleotides has been measured in reaction mixtures containing rat liver supernatant and each one of the following compounds at 1 mM concentration (except xanthine, 0.5 mM and guanosine and guanine, 0.1 mM).

Validity and diagnostic bias in the clinical screening for congenital dysplasia of the hip.

To assess the validity of the clinical maneuvers and signs in the screening for congenital dysplasia of the hip and the presence of a diagnostic bias in this screening, a random sample of 261 newborns was studied at a tertiary hospital. All newborns were clinically and sonographically studied in the first 48 hours after birth. Hips were classified according to Graf's scheme.

Diadenosine 5',5"-P1,P4-tetraphosphate (Ap4A), ATP and catecholamine content in bovine adrenal medulla, chromaffin granules and chromaffin cells.

The level of diadenosine 5',5"-P1-P4-tetraphosphate (diadenosine tetraphosphate or Ap4A), catecholamines, ATP and other nucleotides has been investigated in perchloric acid extracts of bovine adrenal medulla, chromaffin granules and cultured chromaffin cells. As a control, the amount of Ap4A and ATP has also been measured in human blood platelets. The following values (nmol/mg protein) were found in adrenal medulla: Ap4A, 0.

Effect of diets containing adenosine, guanosine, inosine or xanthosine on the nucleotide content of Artemia. Influence of mycophenolic acid.

Artemia uses the stored diguanosine tetraphosphate as a source of adenine and guanine nucleotides during development from the encysted gastrula to the free swimming larva. Further development of the larvae depends on a dietary source of purine rings. We have investigated the growth of Artemia in axenic cultures supplemented with 0.

Diadenosine tetraphosphate is co-released with ATP and catecholamines from bovine adrenal medulla.

Secretion of adenosine(5')tetraphospho(5')adenosine (Ap4A) and ATP from perfused bovine adrenal glands stimulated with acetylcholine or elevated potassium levels was measured and compared with that of catecholamines. We have found a close correlation between the release of Ap4A and catecholamines elicited with all the secretagogues used in the presence of either Ca2+ or Ba2+, suggesting co-release of both constituents from the chromaffin granules. By contrast, ATP secretion, as measured with luciferase, showed a significantly different time course regardless of the secretagogue used.

Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase.

In the presence of ATP, luciferin (LH2), Mg2+ and pyrophosphatase, the firefly (Photinus pyralis) luciferase synthesizes diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) through formation of the E-LH2-AMP complex and transfer of AMP to ATP. The maximum rate of the synthesis is observed at pH 5.7.

Particulate diadenosine 5',5"'-P1,P3-triphosphate hydrolases in rat brain: two specific dinucleoside triphosphatases and two phosphodiesterase I-like hydrolases.

Rat liver and brain differ in the distribution pattern of the total hydrolytic activity on diadenosine 5',5"'-P1,P3-triphosphate (Ap3A) between the soluble and particulate fractions. The Ap3A-hydrolase activity in both the soluble and particulate liver fractions and in the brain soluble fraction had been previously studied in detail. We report now on the brain particulate fraction which, unlike liver, showed a low unspecific phosphodiesterase I-like (PDEaseI, EC 3.

Theoretical isoelectric points and electric charges of mutated human hemoglobin subunits.

Isoelectric point values have been determined from a theoretical standpoint for alpha, beta, gamma and delta human abnormal subunits bearing elimination or addition of 1-2 different aminoacids with acid-base residues. The charge distribution, in relation to pH values, of the four types of subunits are also reported. These values may be of interest both to predict the electrophoretic behaviour and to characterize human abnormal hemoglobins.

Firefly luciferase synthesizes P1,P4-bis(5'-adenosyl)tetraphosphate (Ap4A) and other dinucleoside polyphosphates.

The synthesis of P1,P4-bis(5'-adenosyl)tetraphosphate (Ap4A) has been considered, for a long time, to be catalyzed mainly by some aminoacyl-tRNA synthetases [Brevet et al. (1989) Proc. Natl.