[6] Topics in the methodology of substitution reactions with agarose

Parikh, Indu; March, Steven C.; Cuatercasas, P

doi:10.1016/s0076-6879(74)34009-8

Cited by 202 publications

(65 citation statements)

References 15 publications

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“…Glazer, 1976) has made these compounds useful as components of heterobifunctional cross-linking reagents (Ji, 1983), reactive groups for attachment of protein ligands to immobilized supports (Parikh et al, 1974), blocking groups during peptide synthesis (Finn and Hofmann, 1976), and chemical-modifying reagents for enzymes (Stefanova et al, 1993). In the present study, AMCA-sulfo-NHS and sulfo-NHS-acetate caused rapid inactivation of Rubisco activase.…”

Section: Discussionmentioning

confidence: 60%

“…N-Hydroxysuccinimde esters react with unprotorlated amines, which for proteins includes the a-NH2 of the N-terminal amino acid and the eNH2 of interna1 Lys residues (Parikh et al, 1974). To identify the site(s) of modification from among the 25 possible Lys residues in tobacco Rubisco activase , tryptic peptides from the rrlodified Rubisco activase described in Figure 2 were separated by reverse-phase HPLC and analyzed for A355 (Fig.…”

Section: Ldentification Of the Modified Residuementioning

confidence: 99%

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Covalent Modification of a Highly Reactive and Essential Lysine Residue of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activase

Salvucci¹

1993

Plant Physiol.

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Chemical modification of ribulose-1,5-bisphosphate carboxylaseloxygenase (Rubisco) activase with water-soluble N-hydroxysuccinimide esters was used to identify a reactive lysyl residue that is essential for activity. lncubation of Rubisco activase with sulfosuccinimidyl-7-amino-4-methylcoumarin-3-acetate (AMCA-sulfo-NHS) or sulfosuccinimidyl-acetate (sulfo-NHS-acetate) caused progressive inactivation of ATPase activity and concomitant loss of the ability to activate Rubisco. AMCA-sulfo-NHS was the more potent inactivator of Rubisco activase, exhibiting a second-order rate constant for inactivation of 239 M-' s-l compared to 21 M-' s-' for sulfo-NHS-acetate. lnactivation of enzyme activity by AMCA-sulfo-NHS correlated with the incorporation of 1.9 mo1 of AMCA per mo1 of 42-kD Rubisco activase monomer. ADP, a competitive inhibitor of Rubisco activase, afforded considerable protection against inactivation of Rubisco activase and decreased the amount of AMCA incorporated into the Rubisco activase monomer. Sequence analysis of the major labeled peptide from AMCA-sulfo-NHS-modified enzyme showed that the primary site of modification was lysine-247 (K247) in the tetrapeptide methionine-glutamic acidlysine-phenylalanine. Upon complete inactivation of ATPase activity, modification of KZ4' accounted for 1 mo1 of AMCA incorporated per mo1 of Rubisco activase monomer. Photoaffinity labeling of AMCA-sulfo-NHS-and sulfo-NHS-acetate-modified Rubisco activase with ATP analogs derivatized on either the adenine base or on the y-phosphate showed that KZd7 is not essential for the binding of adenine nucleotides per se. Instead, the data indicated that the essentiality of KZ4' is probably due to an involvement of this highly reactive, species-invariant residue in an obligatory interaction that occurs between the protein and the nucleotide phosphate during catalysis.

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Section: Discussionmentioning

confidence: 60%

Section: Ldentification Of the Modified Residuementioning

confidence: 99%

Covalent Modification of a Highly Reactive and Essential Lysine Residue of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activase

Salvucci¹

1993

Plant Physiol.

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“…It was detected and estimated by titration with fluorescein, the fluorescence of which was quenched [21]. The purified antibody (200mg) was precipitated with ammonium sulphate (2 M), dialysed and coupled to Sepharose 4B (100 ml) which had been activated with CNBr [22]. The immunoadsorbent bound to 20 nmol fluorescein/ml column vol.…”

Section: Methodsmentioning

confidence: 99%

Identification of a labelled peptide after stoicheiometric reaction of fluorescein isothiocyanate with the Ca²⁺‐dependent adenosine triphosphatase of sarcoplasmic reticulum

et al. 1982

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Incorporation of 4.5 nmol fluorescein isothiocyanate/mg rabbit sarcoplasmic reticulum, or of 7.4 nmol/ mg purified ATPase, was sufficient to inhibit the activity completely. These results are not consistent with the suggestion (Pick, U. and Karlish, S.J.D. (1980) Biochim. Biophys. Acta 626, 255-261) that 2 mol ATPase were inhibited by each mole of reagent incorporated. A single labelled peptide was purified from the inhibited ATPase and it was shown that Lys 3/190, 10 residues from the N-terminus of tryptic fragment B, was the reactive lysine residue. This site is close to a potential nucleotide-binding fold in the ATPase sequence. A similar peptide showing only 2 conservative replacements was isolated from the sarcoplasmic reticulum of the lobster. (Ca 2 + + Mg 2 + )A TPase inhibition Reactive lysine residue Fluorescein isothiocyanateIntegral membrane protein Nucleotide-binding fold (Sarcoplasmic reticulum)

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“…The larger fragment, Bb, and C3b form the alternative pathway convertase, C3bBb (19,20,23 Affinity chromatography. 220 U Hirudin (Pentapharm AG, Basel, Switzerland) (1 U/,g) was covalently linked to 2 ml cyanogen bromide-activated Sepharose 6B (Pharmacia Fine Chemicals) by the method of Parikh et al (33). Samples containing thrombin, factor D), or buffer alone were incubated for 1 h at 22°C with hirudin-Sepharose, and were then centrifuged at 100 g for 5 min.…”

mentioning

confidence: 99%

Properdin factor D: effects on thrombin-induced platelet aggregation.

Davis¹,

Kenney²

1979

J. Clin. Invest.

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A B S T R A C T Factor 0, when preincubated with platelet suspensions, at concentrations as low as 1.2 ,sg/ml, inhibited thrombin-induced platelet aggregation. No inhibition of collagen or arachidonic acid-induced platelet aggregation was found. Inhibition occurred, but to a lesser extent, when thrombin and factor 0 were added to platelets at the same time.No inhibition occurred when factor D was added after thrombin. Thrombin was able to overcome inhibition by factor 0 by increasing its concentration. Diisopropylphosphorofluoridate-inactivated factor 0 also inhibited thrombin-induced platelet aggregation so that enzymatic activity of factor 0 was not required for inhibition. Factor o absorbed with hirudin coupled to Sepharose 6B showed no decrease in inhibitory capacity. 125I-Factor D bound to platelets in a manner suggesting an equilibrium reaction similar to thrombin. At low factor D input, binding was linear, whereas at higher input, binding began to approach saturation. Binding of 1251-labeled thrombin to platelets was inhibited by factor D. Analysis of these data show that factor D does not alter the total number of thrombin molecules which bind to the platelet surface at saturation. However, the dissociation constant for thrombin is altered from 2.78 to 6.90 nM in the presence of factor D (20 gg/ml). Factor D is thus a competitivie inhibitor of thrombin binding, although the affiinity of factor D for the platelet thrombin receptor is much less than that of thrombin. These phenomena occur at physiologic concentrations of factor 0. Therefore, factor D may function in vivo as an inhibitor of platelet aggregation.

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[6] Topics in the methodology of substitution reactions with agarose

Cited by 202 publications

References 15 publications

Covalent Modification of a Highly Reactive and Essential Lysine Residue of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activase

Covalent Modification of a Highly Reactive and Essential Lysine Residue of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activase

Identification of a labelled peptide after stoicheiometric reaction of fluorescein isothiocyanate with the Ca²⁺‐dependent adenosine triphosphatase of sarcoplasmic reticulum

Properdin factor D: effects on thrombin-induced platelet aggregation.

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[6] Topics in the methodology of substitution reactions with agarose

Cited by 202 publications

References 15 publications

Covalent Modification of a Highly Reactive and Essential Lysine Residue of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activase

Covalent Modification of a Highly Reactive and Essential Lysine Residue of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activase

Identification of a labelled peptide after stoicheiometric reaction of fluorescein isothiocyanate with the Ca2+‐dependent adenosine triphosphatase of sarcoplasmic reticulum

Properdin factor D: effects on thrombin-induced platelet aggregation.

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Identification of a labelled peptide after stoicheiometric reaction of fluorescein isothiocyanate with the Ca²⁺‐dependent adenosine triphosphatase of sarcoplasmic reticulum