Effect of Accessible Immobilized NAD+ Concentration on the Bioaffinity Chromatographic Behavior of NAD+-Dependent Dehydrogenases Using the Kinetic Locking-on Strategy

Mulcahy, Patricia; O’Flaherty, Martina; McMahon, Mary; Oakey, Laura

doi:10.1006/prep.1999.1050

Cited by 11 publications

(17 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As the concentration of leading ligand (cofactor) is increased, the K m for the specific substrate decreases and the K i value for the specific substrate analogue decreases. This is a general property of multisubstrate enzymes following ordered kinetic mechanisms and has been demonstrated for other dehydrogenases studied including GDH (56), L-LDH (59), and TBADH (62). The investigations presented in Fig.…”

Section: Interrelationship Between [Immobilized Cofactor] and Effectisupporting

confidence: 61%

“…The investigations presented in Fig. 2 also underline the importance of determining not only the total immobilized NAD(P) ϩ concentration when developing purification systems based on the kinetic locking-on strategy, but also the accessible immobilized NAD(P) ϩ concentration (59). For example, the yeast enzyme does not distinguish between the two S 6 -linked immobilized NAD ϩ derivatives with total substitution levels of 0.4 and 0.7 mol NAD ϩ /g wet wt because these two matrices have the same concentration of accessible immobilized NAD ϩ (Figs.…”

Section: Interrelationship Between [Immobilized Cofactor] and Effectimentioning

confidence: 73%

“…Another important factor that influences the K i value of the enzyme for the specific substrate analogue is the substitution level of the immobilized cofactor derivative (56,57,59,63). For example, when YADH is chromatographed on an S 6 -linked immobilized NAD ϩ derivative in the presence of a decreasing locking-on ligand (hydroxylamine) concentration gradient, the enzyme elutes at progressively higher concentrations of soluble locking-on ligand as the substitution level of the affinity adsorbent is decreased (Fig.…”

Section: Interrelationship Between [Immobilized Cofactor] and Effectimentioning

confidence: 99%

See 2 more Smart Citations

Kinetic Locking-On Strategy and Auxiliary Tactics for Bioaffinity Purification of NAD(P)+-Dependent Dehydrogenases

Mulcahy

O’Flaherty

2001

Analytical Biochemistry

Self Cite

View full text Add to dashboard Cite

Section: Interrelationship Between [Immobilized Cofactor] and Effectisupporting

confidence: 61%

Section: Interrelationship Between [Immobilized Cofactor] and Effectimentioning

confidence: 73%

Section: Interrelationship Between [Immobilized Cofactor] and Effectimentioning

confidence: 99%

See 1 more Smart Citation

Kinetic Locking-On Strategy and Auxiliary Tactics for Bioaffinity Purification of NAD(P)+-Dependent Dehydrogenases

Mulcahy

O’Flaherty

2001

Analytical Biochemistry

Self Cite

View full text Add to dashboard Cite

“…The limitations of affinity systems based on immobilized cofactor derivatives for the purification of NAD(P) ϩ -dependent dehydrogenases are well established (1). However, many of these limitations can be overcome using the kinetic locking-on and auxiliary tactics (1)(2)(3).…”

mentioning

confidence: 99%

“…However, many of these limitations can be overcome using the kinetic locking-on and auxiliary tactics (1)(2)(3). The locking-on strategy involves the use of soluble-specific substrates or substrate analogues to increase selective adsorption of individual dehydrogenases on their complementary immobilized cofactor derivatives (NAD ϩ or NADP ϩ ).…”

mentioning

confidence: 99%

The Kinetic Locking-on Strategy for Bioaffinity Purification: Further Studies with Bovine Liver Glutamate Dehydrogenase

O’Flaherty

McMahon

Forde

et al. 1999

Protein Expression and Purification

Self Cite

View full text Add to dashboard Cite

Bioaffinity purification of NADP⁺‐dependent dehydrogenases: Studies with alcohol dehydrogenase from Thermoanaerobacter brockii

McMahon

Mulcahy

2002

Biotech & Bioengineering

Self Cite

View full text Add to dashboard Cite

This study describes the development and application of a bioaffinity chromatographic system for the one-step purification of an NADP(+)-dependent secondary alcohol dehydrogenase from the obligate anaerobe, Thermoanaerobacter brockii (TBADH, EC 1.1.1.2). The general approach is based upon improving the selectivity of immobilized cofactor derivatives (general ligand approach to bioaffinity chromatography) through using soluble enzyme-specific substrate analogues in irrigants to promote biospecific adsorption (the kinetic locking-on tactic). Specifically, the following is described: Evaluation of 8'-azo-linked, C(8)-linked, N(1)-linked, and N(6)-linked immobilized NADP(+) derivatives for use with the kinetic locking-on strategy for bioaffinity purification of TBADH; evaluation of 2', 5'-ADP as a stripping ligand for TBADH bioaffinity purifications using an 8'-azo-linked immobilized NADP(+) derivative in the locking-on mode; and application of the developed bioaffinity chromatographic system to the purification of TBADH from a crude cellular extract. Surprizingly, of the four immobilized NADP(+) derivatives investigated, only the 8'-azo-linked immobilized NADP(+) derivative proved effective for TBADH affinity purification when used in conjunction with pyrazole (a competitive inhibitor of TBADH activity) as the locking-on ligand. This is in contrast to other NADP(+)-dependent dehydrogenases where the immobilized N(6)-linked cofactor proved to be suitable. While the one-step purification of TBADH to electrophoretic homogeneity is described in the present study (92% yield), results from the model chromatographic studies point to improvements that could be made to the immobilized cofactor derivative to improve its suitability for TBADH bioaffinity purification and to facilitate future large scale protein purification operations.

show abstract

Effect of Accessible Immobilized NAD+ Concentration on the Bioaffinity Chromatographic Behavior of NAD+-Dependent Dehydrogenases Using the Kinetic Locking-on Strategy

Cited by 11 publications

References 27 publications

Kinetic Locking-On Strategy and Auxiliary Tactics for Bioaffinity Purification of NAD(P)+-Dependent Dehydrogenases

Kinetic Locking-On Strategy and Auxiliary Tactics for Bioaffinity Purification of NAD(P)+-Dependent Dehydrogenases

The Kinetic Locking-on Strategy for Bioaffinity Purification: Further Studies with Bovine Liver Glutamate Dehydrogenase

Bioaffinity purification of NADP⁺‐dependent dehydrogenases: Studies with alcohol dehydrogenase from Thermoanaerobacter brockii

Contact Info

Product

Resources

About

Effect of Accessible Immobilized NAD+ Concentration on the Bioaffinity Chromatographic Behavior of NAD+-Dependent Dehydrogenases Using the Kinetic Locking-on Strategy

Cited by 11 publications

References 27 publications

Kinetic Locking-On Strategy and Auxiliary Tactics for Bioaffinity Purification of NAD(P)+-Dependent Dehydrogenases

Kinetic Locking-On Strategy and Auxiliary Tactics for Bioaffinity Purification of NAD(P)+-Dependent Dehydrogenases

The Kinetic Locking-on Strategy for Bioaffinity Purification: Further Studies with Bovine Liver Glutamate Dehydrogenase

Bioaffinity purification of NADP+‐dependent dehydrogenases: Studies with alcohol dehydrogenase from Thermoanaerobacter brockii

Contact Info

Product

Resources

About

Bioaffinity purification of NADP⁺‐dependent dehydrogenases: Studies with alcohol dehydrogenase from Thermoanaerobacter brockii