Fluorescent modification of adenosine-containing coenzymes. Biological activities and spectroscopic properties

Secrist, John A.; Barrio, Jorge R.; Leonard, Nelson J.; Weber, Gregorio

doi:10.1021/bi00769a001

Cited by 517 publications

(314 citation statements)

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“…The latter were subsequently eluted with 0.5 ml of 0.4 mM ammonium phosphate, pH 5.1, containing 50% methanol . Samples were then dried using a Speed-Vac concentrator (Savant, Farmingdale, NY) and adenine nucleosides were converted to fluorescent etheno-(&-)derivatives by the chloroacetaldehyde reaction (Secrist et al, 1972). The etheno-derivatives of adenosine and 2'-deoxyadenosine were resolved by HPLC using an isocratic mobile phase consisting of 20 mM ammonium phosphate, pH 5.1, containing 9% methanol.…”

Section: Adenosine Extraction and Analysismentioning

confidence: 99%

Adenosine levels in the postimplantation mouse uterus: Quantitation by HPLC‐fluorometric detection and spatiotemporal regulation by 5′‐nucleotidase and adenosine deaminase

Blackburn

Gao

Airhart

et al. 1992

Developmental Dynamics

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Extracellular adenosine has the potential to influence many aspects of target cell metabolism. The present study has determined the endogenous levels of adenosine in the pregnant mouse uterus and developing embryodecidual unit with respect to the expression of two key enzymes of adenosine metabolism, 5'-nucleotidase (5'-NT; EC 3.1.3.5) and adenosine deaminase (ADA; EC 3.5.4.4). To measure adenosine levels, nucleoside extracts were etheno-derivatized and quantitated by high-performance liquid chromatography-fluorescence detection (0.03 pmollmg protein sensitivity). Adenosine levels were determined to be 0.18 nmol/mg protein in the nonpregnant uterus; however, two statistically significant changes were identified in the pregnant uterus: (1) a periimplantation surge between day 3 (0.24 nmoVmg protein) and day 5 (0.59 nmoYmg protein) of gestation (plug day 0; implantation day 4); and (2) an early postimplantation decline between day 6 (0.54 nmollmg protein) and day 7 (0.10 nmollmg protein). The periimplantation adenosine surge coincided with uterine expression of 5'-NT, an enzyme which catalyzes the irreversible dephosphorylation of 5'-AMP to adenosine. 5'-NT expression was shown by Northern blot analysis to peak in the embryo-decidual unit on day 5 of gestation and then to decline through day 9; transcripts remained elevated in the placenta between day 9 and day 13 (the latest day examined in this study). By use of specific enzyme histochemistry, most 5'-NT activity was localized to the primary decidual :zone on day 5. This expression subsequently declined during regression of the primary decidua; however, 5'-NT appeared on giant trophoblast ((days 7-13) and the metrial gland (days 11-13).Other purine catabolic enzymes degrading AMP (adenylate deaminase) or generating adenosine (S-adenosylhomocysteine hydrolase) were not detected in the embryo-decidual unit suggesting that the net flux of utero-placental AMP catabolism proceeds with adenosine as an intermediate, this being the major pathway of adenosine formation.The sharp drop in adenosine levels between day 6 and day 7 coincided with a rise in the activity and mRNA expression of ADA, an enzyme which catalyzes the irreversible deamination of adenosine to inosine. ADA was previously localized to the secondary decidual zone (days 6 1 l), secondary giant cells (days 7-13), and spongiotrophoblasts (days 8-13) in the mouse (Knudsen et al., 1991). Results of developmental Northern blot analysis demonstrated a direct correlation of relative 5'-NT/ADA mRNA band intensity to adenosine content between day 4 and day 9 of gestation, suggesting that the local availability of adenosine in the antimesometrium is dependent upon the distribution of these enzymatic activities. Purine nucleoside phosphorylase and xanthine oxidase, which are two catabolic enzymes acting subsequent to 5'-NT and ADA in the sequential degradation of AMP to xanthine, remained low and constant in the tissues examined suggesting that the catabolic pathway is geared toward regulation of adenosine le...

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Section: Adenosine Extraction and Analysismentioning

confidence: 99%

Adenosine levels in the postimplantation mouse uterus: Quantitation by HPLC‐fluorometric detection and spatiotemporal regulation by 5′‐nucleotidase and adenosine deaminase

Blackburn

Gao

Airhart

et al. 1992

Developmental Dynamics

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“…Solutions of these compounds were prepared and assayed as previously described [4]. e-ADP and e-NAD' were prepared according to [2,3] and purified to homogeneity by chromatography on Dowex X 1. Their concentrations were measured photometrically using the absorption coefficients e26s = 5.7 cm-' mM-' for e-ADP and e26s = lO.OCITl- ' mM_r for e-NAD' [2,3 1. e-NADH was prepared by enzymatic reduction of e-NAD' with liver alcohol dehydrogenase and 3% cyclohexanol at pH 9.4 as described for NADH [7].…”

Section: Mannermentioning

confidence: 99%

“…The 1 ,@ -ethenoadenine analogue (e-NAD") described by Leonard and coworkers [2,3] allows fluorescence studies in the oxidized state of the coenzyme. Provided the analogue is active as a coenzyme in the dehydrogenase reaction, these studies should give useful information about coenzyme binding and the influence of effecters or substrates on enzymecoenzyme complexes.…”

Section: Introductionmentioning

confidence: 99%

Nicotinamide 1, N⁶‐ethenoadenine dinucleotide, a coenzyme for glutamate dehydrogenase

1974

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“…Studies of the nucleotide cleft in actin were facilitated by the development of the fluorescent etheno-labeled nucleotide analogs, €-ATP and €-ADP (Secrist et al, 1972;Leonard, 1984). Early work utilizing etheno-nucleotides led to a controversy over whether G-and F-actin showed different fluorescent lifetimes for bound €-ATP (Mihashi & Wahl, 1975;Harvey et al, 1977).…”

mentioning

confidence: 99%

“…A sharp increase in critical concentration and polymerization halftimes occurs between pH 6.6 and pH 7.4 (Zimmerle & Frieden, 1988a,b;Wang et al, 1989), which suggests that an increase in the positive charge of some region on actin is important for its polymerization. Interestingly, the pKO2 of ATP and ADP is in the range of 6.5-6.7 (Secrist et al, 1972). Possibly, the charge on the terminal phosphate of the nucleotide may affect the polymerization of actin.…”

mentioning

confidence: 99%

The accessibility of etheno‐nucleotides to collisional quenchers and the nucleotide cleft in G‐ and F‐actin

Root

Reisler

1992

Protein Science

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Recent publication of the atomic structure of G-actin (Kabsch, W., Mannherz, H.G., Suck, D., Pai, E.F., & Holmes, K.C., 1990, Nature 347, 37-44) raises questions about how the conformation of actin changes upon its polymerization. In this work, the effects of various quenchers of etheno-nucleotides bound to G-and F-actin were examined in order to assess polymerization-related changes in the nucleotide phosphate site. The Mg2+-induced polymerization of actin quenched the fluorescence of the etheno-nucleotides by approximately 20% simultaneously with the increase in light scattering by actin. A conformational change at the nucleotide binding site was also indicated by greater accessibility of F-actin than G-actin to positively, negatively, and neutrally charged collisional quenchers. The difference in accessibility between G-and F-actin was greatest for I-, indicating that the environment of the etheno group is more positively charged in the polymerized form of actin. Based on calculations of the change in electric potential of the environment of the etheno group, specific polymerization-related movements of charged residues in the atomic structure of G-actin are suggested. The binding of S-1 to €-ATP-G-actin increased the accessibility of the etheno group to I-even over that in Mg2+-polymerized actin. The quenching of the etheno group by nitromethane was, however, unaffected by the binding of S-1 to actin. Thus, the binding of S-1 induces conformational changes in the cleft region of actin that are different from those caused by Mg2+ polymerization of actin. The pH dependence of collisional quenching shows that the cleft region is more accessible to collisional quenchers at pH 7 than at higher pH and suggests that changes in the environment of the cleft might contribute to the faster polymerization rates observed at lower pH.Keywords: actin; collisional quenching; nucleotide site; polymerization; structure The recently published atomic structure of G-actin facilitates the search for specific conformational changes in the actin molecule at the atomic level. Because the X-ray crystallographic structure was solved for G-actin and not F-actin, a question of particular significance is how the conformation of G-actin changes upon its polymerization to F-actin. It is known that actin hydrolyzes ATP to ADP during polymerization

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Fluorescent modification of adenosine-containing coenzymes. Biological activities and spectroscopic properties

Cited by 517 publications

References 25 publications

Adenosine levels in the postimplantation mouse uterus: Quantitation by HPLC‐fluorometric detection and spatiotemporal regulation by 5′‐nucleotidase and adenosine deaminase

Adenosine levels in the postimplantation mouse uterus: Quantitation by HPLC‐fluorometric detection and spatiotemporal regulation by 5′‐nucleotidase and adenosine deaminase

Nicotinamide 1, N⁶‐ethenoadenine dinucleotide, a coenzyme for glutamate dehydrogenase

The accessibility of etheno‐nucleotides to collisional quenchers and the nucleotide cleft in G‐ and F‐actin

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Fluorescent modification of adenosine-containing coenzymes. Biological activities and spectroscopic properties

Cited by 517 publications

References 25 publications

Adenosine levels in the postimplantation mouse uterus: Quantitation by HPLC‐fluorometric detection and spatiotemporal regulation by 5′‐nucleotidase and adenosine deaminase

Adenosine levels in the postimplantation mouse uterus: Quantitation by HPLC‐fluorometric detection and spatiotemporal regulation by 5′‐nucleotidase and adenosine deaminase

Nicotinamide 1, N6‐ethenoadenine dinucleotide, a coenzyme for glutamate dehydrogenase

The accessibility of etheno‐nucleotides to collisional quenchers and the nucleotide cleft in G‐ and F‐actin

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Nicotinamide 1, N⁶‐ethenoadenine dinucleotide, a coenzyme for glutamate dehydrogenase