Some properties of reactions catalyzed by pig brain NAD glycohydrolase

Apitz, Rafael; Mickelson, Kenneth E.; Shriver, K K; Cordes, E. H.

doi:10.1016/0003-9861(71)90222-0

Cited by 23 publications

(8 citation statements)

References 20 publications

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“…The observed curves for these two latter representations could conceivably reflect the perturbation of an ionizing group (pK = 6) of the enzyme during the formation of the Michaelis complex. The curves obtained in this study are similar to the one published by Apitz et al for pig brain NAD glycohydrolase [28]. Below pH 4.5 the buffering capacity of the nicotinamide released during the hydrolysis of NAD does not allow precise rate measurements by the pH-stat method.…”

Section: Effict Of Ionic Strengthsupporting

confidence: 76%

“…The CMcellulose chromatography step was found to be critical in our purification scheme. Several authors have experienced difficulties at this point [28,33]. The stability of the NADase on the CM-cellulose column is low and the duration of the chromatographic procedure becomes an important factor ; flow rates that are too slow should be avoided and altogether this step should not exceed 6-8 h. The best purification was achieved with a Servacel CM-53 cellulose, while Whatman CM-52 gave consistently lower specific activities.…”

Section: Solubilization and Purification Of N A D Glycohydrolasementioning

confidence: 99%

“…The protein fractionation for NADases up to DEAEcellulose chromatography was as has been previously described. Some authors have mentioned the possibility of applying a CM-cellulose chromatography as a further step in the purification procedure, but the method described previously, using low ionic strength, proved to result in substantial losses of enzyme activity [28,33]. We developed a technique based on a pH gradient which proved to be highly successful.…”

Section: Soluhilization and Purijication Of N A D Glycohydrolasrmentioning

confidence: 99%

“…For our kinetic studies we used the titrimetric method (pH-stat), which was shown to be of sufficient sensitivity to follow the kinetics of this enzyme directly [28,54]. Parallel runs performed according to the titrimetric method and the cyanide method show excellent agreement in determination of initial rates.…”

Section: Kineticsmentioning

confidence: 99%

“…Despite its potential significance in cell biology and pharmacology and the growing importance of the related ADP-ribosylation reactions almost nothing is known about the molecular mechanism of the reactions catalyzed by the NADases. The kinetics of the enzymic hydrolysis of NAD rarely have been explored [28,29] and the catalytic groups involved in the process have not been identified. The regulation of the NAD glycohydrolase activity in vivo and in vitvo remains unexplained [30].…”

mentioning

confidence: 99%

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Calf-Spleen Nicotinamide - Adenine Dinucleotide Glycohydrolase Solubilization Purification and Properties of the Enzyme

Schuber¹,

Travo²

1976

Eur J Biochem

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NAD glycohydrolase of calf spleen was solubilized with pancreatic lipase and purified approximatively 800-fold to a specific activity of 7 units/mg of protein by successive DEAE-cellulose and carboxymethyl-cellulose chromatography. The purified enzyme has a molecular weight of 24 000 and is characterized by a double band on disc gel electrophoresis. Some kinetic properties of the NADglycohydrolase-catalyzed hydrolysis of NAD have been examined using a titrimetric assay for enzyme activity. The reaction is subject to inhibition by excess of substrate, which disappears at high ionic strength and low pH. At a pH below 5 the kinetic displays an apparent activation by substrate. The effects of pH (4.5-9.0) on the kinetic parameters do not reveal an essential ionizable group in the catalytic process.NAD glycohydrolases hydrolyze NAD at the ribose -nicotinamide bond :

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Section: Effict Of Ionic Strengthsupporting

confidence: 76%

Section: Solubilization and Purification Of N A D Glycohydrolasementioning

confidence: 99%

Section: Soluhilization and Purijication Of N A D Glycohydrolasrmentioning

confidence: 99%

Section: Kineticsmentioning

confidence: 99%

mentioning

confidence: 99%

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Calf-Spleen Nicotinamide - Adenine Dinucleotide Glycohydrolase Solubilization Purification and Properties of the Enzyme

Schuber¹,

Travo²

1976

Eur J Biochem

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Transition States for Hydrolysis of Acetals, Ketals, Glycosides, and Glycosylamines

Cordes

Bull

1978

Transition States of Biochemical Processes

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Self-inactivation of an erythrocyte NAD glycohydrolase

1980

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NAD glycohydrolase activity was studied using bovine erythrocytes, erythrocyte ghosts and partially purified enzyme preparations. During catalysis the enzyme becomes irreversibly inactivated in a process related to substrate turnover. Self-inactivation was observed with intact cells, ghosts and solubilized enzyme and could be demonstrated with NAD, NADP and nicotinamide 1,N6 ethenoadenine dinucleotide as substrates. Thionicotinamide adenine dinucleotide and NADH, which are not substrates for the enzyme, do not inactivate but are reversible substrate-competitive inhibitors. Added thiols had no effect on enzyme self-inactivation. Of the reaction products, added nicotinamide partially protected the enzyme while added ADPR had no effect. Thermodynamic parameters calculated from Arrhenius plots for rate constants of self-inactivation indicate a large negative delta S for transition state formation suggesting a process other than extensive denaturation. Erythrocyte ghost NADases from several other mammalian sources have been demonstrated to undergo a self-inactivation similar to that observed with the bovine enzyme.

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Some properties of reactions catalyzed by pig brain NAD glycohydrolase

Cited by 23 publications

References 20 publications

Calf-Spleen Nicotinamide - Adenine Dinucleotide Glycohydrolase Solubilization Purification and Properties of the Enzyme

Calf-Spleen Nicotinamide - Adenine Dinucleotide Glycohydrolase Solubilization Purification and Properties of the Enzyme

Transition States for Hydrolysis of Acetals, Ketals, Glycosides, and Glycosylamines

Self-inactivation of an erythrocyte NAD glycohydrolase

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