A fluorometric-high-performance liquid chromatographic assay procedure for several enzymatic activities using formycin analogs of adenosine 5′-mono, 5′-tri, and cyclic 3′,5′-monophosphate as substrates

Rossomando, Edward F.; Jahngen, Jessica H.; Eccleston, John F.

doi:10.1016/0003-2697(81)90325-0

Cited by 22 publications

(7 citation statements)

References 15 publications

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“…Proteins were prepared as referenced in the accompanying paper (Jackson & Bagshaw, 1988). FTP and FDP were prepared by the enzymic method of Rossomando et al (1981). p[NH]ppF was synthesized by a method analogous to that of Rodbell et al (1971), with use of the exchange reaction of PPi and sodium imidodiphosphate as catalysed by a crude preparation of amino acid-activating enzymes (S-100).…”

Section: Materials and Methods Materialsmentioning

confidence: 99%

“…Fluorescent ATP analogues provide a means of increasing the amplitudes of the optical signals associated with the elementary steps of the ATPase reaction. In particular, formycin nucleotides have found wide application in biological systems (Ward et al, 1969b;Rossomando et al, 1981). On binding to rabbit skeletalmuscle and other myosin subfragments, FTP shows a large enhancement in fluorescence, followed by a decrease in signal concomitant with product release (Bagshaw et al, 1972;White et al, 1986).…”

Section: Introductionmentioning

confidence: 99%

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Kinetic trapping of intermediates of the scallop heavy meromyosin adenosine triphosphatase reaction revealed by formycin nucleotides

Jackson

Bagshaw

1988

Biochemical Journal

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The kinetics of interaction of formycin nucleotides with scallop myosin subfragments were investigated by exploiting the fluorescence signal of the ligand. Formycin triphosphate gives a 5-fold enhancement of the emission intensity on binding to heavy meromyosin, and the profile indicates that the kinetics of binding are Ca2+-insensitive. In contrast, the subsequent product-release steps show a marked degree of regulation by Ca2+. In the absence of Ca2+ formycin triphosphate turnover by the unregulated and the regulated heavy meromyosin fractions are clearly resolved, the latter showing a fluorescence decay rate of 0.002 s-1, corresponding to the Pi-release step. In the presence of Ca2+ this step is activated 50-fold. Formycin diphosphate release is also regulated by Ca2+, being activated from 0.008 s-1 to 5 s-1. In contrast with protein tryptophan fluorescence [Jackson & Bagshaw (1988) Biochem. J. 251, 515-526], formycin fluorescence is sensitive to conformational changes that occur subsequent to the binding step and demonstrate, directly, an effect of Ca2+ on both forward and reverse rate constants. Apart from a decrease in the apparent second-order association rate constants, formycin derivatives appear to mimic adenosine nucleotides closely in their interaction with scallop heavy meromyosin and provide a spectroscopic handle on steps that are optically silent with respect to protein fluorescence. A novel mechanism is discussed in which regulation of the formycin triphosphate activity by Ca2+ involves kinetic trapping of product complexes.

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Section: Materials and Methods Materialsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Kinetic trapping of intermediates of the scallop heavy meromyosin adenosine triphosphatase reaction revealed by formycin nucleotides

Jackson

Bagshaw

1988

Biochemical Journal

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“…43 It is phosphorylated in mammalian cells and can be readily converted to the 5′-phosphate by chemical phosphorylation. 44 Formycin 5′-phosphate gave a dissociation constant of 43 nM for the A. vinelandii AMP nucleosidase compared to a K m of 120 μM for the AMP substrate, a factor of 2800 tighter binding for the analogue (Figure 5). 44 The crystal structure of the E. coli AMP nucleosidase with formycin 5′-phosphate at the catalytic sites revealed Asp428 to be responsible for protonation of N7 in a bidentate interaction between the N6 amino group and N7 (Figure 3).…”

Section: Amp Nucleosidasementioning

confidence: 99%

“…Formycin (7-amino-3-(β- d -ribofuranosyl)pyrazolo-(4,3-d)-pyrimidine) is a natural product isolated from culture media of Nocardia interforma . It is phosphorylated in mammalian cells and can be readily converted to the 5′-phosphate by chemical phosphorylation . Formycin 5′-phosphate gave a dissociation constant of 43 nM for the A. vinelandii AMP nucleosidase compared to a K m of 120 μM for the AMP substrate, a factor of 2800 tighter binding for the analogue (Figure ).…”

Section: Amp Nucleosidasementioning

confidence: 99%

Enzymatic Transition States and Drug Design

Schramm

2018

Chem. Rev.

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Transition state theory teaches that chemically stable mimics of enzymatic transition states will bind tightly to their cognate enzymes. Kinetic isotope effects combined with computational quantum chemistry provides enzymatic transition state information with sufficient fidelity to design transition state analogues. Examples are selected from various stages of drug development to demonstrate the application of transition state theory, inhibitor design, physicochemical characterization of transition state analogues, and their progress in drug development.

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“…Care was taken to exclude smooth adductor muscle during the initial glycerination procedure. FTP was prepared as described by Rossomando et al (1981). Fura-2 was purchased from Molecular Probes, Oregon, U.S.A. Proteolytic enzymes and nucleotides were purchased from Sigma Chemical Co. Poole, U.K. Ail other reagents were obtained flore BDH chemicals, Poole or Fisons, Loughborough, U.K.…”

Section: Methodsmentioning

confidence: 99%

The calcium ion dependence of scallop myosin ATPase activity

Walmsley

Evans

Bagshaw

1990

J Muscle Res Cell Motil

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The ATPase activity of scallop (Pecten maximus) striated adductor myosin and heavy meromyosin (HMM) have been investigated as a function of [Ca2+] using formycin triphosphate (FTP) as a fluorescent ATP analogue. The FTPase activity of the regulated fraction of these preparations was activated steeply over the range of 0.1 to 1 microM [Ca2+], implying the existence of a form of cooperativity that is intrinsic to the myosin heads. In addition to the previously characterised heterogeneity with respect to an unregulated fraction, the regulated fraction of HMM was resolved into two populations whose activities showed a slightly different dependency on [Ca2+]. This was revealed unambiguously at intermediate levels of activation where, in some experiments, the product release rate constants differed for the two populations by more than fivefold. At maximum relaxation or maximum activation, these rate constants differed by two- to three-fold and were not clearly resolved by the multiexponential fitting procedure. The populations might arise as a consequence of isoenzymes, modification during preparation or slowly interconverting conformers; Ca2+ binding itself being a rapid equilibrium process in both populations. FTP turnover by myosin could not be analysed in such detail because of the technical problems of measuring the fluorescence of a suspension of filaments, but the rates of the elementary steps appeared similar to those of HMM. The fraction of unregulated molecules in myosin preparations was comparable to that of HMM indicating that if it is a consequence of preparative damage, the modification must occur prior to tryptic digestion.

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A fluorometric-high-performance liquid chromatographic assay procedure for several enzymatic activities using formycin analogs of adenosine 5′-mono, 5′-tri, and cyclic 3′,5′-monophosphate as substrates

Cited by 22 publications

References 15 publications

Kinetic trapping of intermediates of the scallop heavy meromyosin adenosine triphosphatase reaction revealed by formycin nucleotides

Kinetic trapping of intermediates of the scallop heavy meromyosin adenosine triphosphatase reaction revealed by formycin nucleotides

Enzymatic Transition States and Drug Design

The calcium ion dependence of scallop myosin ATPase activity

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