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Title: Partial purification of nicotinamide adenine dinucleotide (NAD) pyrophosphatase from Salmonella typhimurium

Abstract

NAD is an extremely important compound in cellular physiology. In the pyridine nucleotide cycle of S. typhimurium NAD pyrophosphatase, located in the inner membrane, carries out the cleavage of NAD prior to the transport of nicotinamide mononucleotide (NMN) into the cell. The partial purification of this enzyme is reported here. A cell suspension of S. typhimurium was passed twice through a French pressure cell, centrifugated at 5000 xg, and at 200,000 xg, for 1 hr. The pellet containing the crude membrane fraction was extracted with a novel detergent extraction using the differential solubility of NAD pyrophosphatase at various concentrations of the non-ionic detergent n-octyl glucoside (nOG). Extraction of the membrane fraction with 0.5% nOG in the presence of 10mM MgCl/sub 2/ removed 60% of the protein with no loss in activity. A second extraction with 2% nOG and 10mM MgCl/sub 2/ removed 20% of the protein and 71% of the activity from the membrane fraction. Ammonium sulfate fractionation at 45 to 50% sat. gave a partially purified enzyme preparation having a specific activity of about 2500 units/mg with a 94% recovery compared to the crude extract. One unit of activity is the cleavage of 1 nmole /sup 14/C NAD tomore » /sup 14/C NMN per minute. The enzyme appears to have a MW of 200,000 on Sephacryl S-200, is temperature labile, and stabilized by 1mM Mg/sup + +/ and storage at -70/sup 0/.« less

Authors:
; ;
Publication Date:
Research Org.:
Marshall Univ. School of Medicine, Huntington, WV
OSTI Identifier:
6079018
Report Number(s):
CONF-870644-
Journal ID: CODEN: FEPRA; TRN: 87-037156
Resource Type:
Conference
Resource Relation:
Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States); Journal Volume: 46:6; Conference: 78. annual meeting of the American Society of Biological Chemists conference, Philadelphia, PA, USA, 7 Jun 1987
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; PHOSPHATASES; ENZYME ACTIVITY; FRACTIONATION; CARBON 14 COMPOUNDS; CELL MEMBRANES; MEMBRANE TRANSPORT; NAD; NUCLEOTIDES; SALMONELLA TYPHIMURIUM; TRACER TECHNIQUES; BACTERIA; CELL CONSTITUENTS; COENZYMES; ENZYMES; ESTERASES; HYDROLASES; ISOTOPE APPLICATIONS; LABELLED COMPOUNDS; MEMBRANES; MICROORGANISMS; ORGANIC COMPOUNDS; SALMONELLA; SEPARATION PROCESSES; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Putt, M.M., Foster, J.W., and Kasvinsky, P.J. Partial purification of nicotinamide adenine dinucleotide (NAD) pyrophosphatase from Salmonella typhimurium. United States: N. p., 1987. Web.
Putt, M.M., Foster, J.W., & Kasvinsky, P.J. Partial purification of nicotinamide adenine dinucleotide (NAD) pyrophosphatase from Salmonella typhimurium. United States.
Putt, M.M., Foster, J.W., and Kasvinsky, P.J. 1987. "Partial purification of nicotinamide adenine dinucleotide (NAD) pyrophosphatase from Salmonella typhimurium". United States. doi:.
@article{osti_6079018,
title = {Partial purification of nicotinamide adenine dinucleotide (NAD) pyrophosphatase from Salmonella typhimurium},
author = {Putt, M.M. and Foster, J.W. and Kasvinsky, P.J.},
abstractNote = {NAD is an extremely important compound in cellular physiology. In the pyridine nucleotide cycle of S. typhimurium NAD pyrophosphatase, located in the inner membrane, carries out the cleavage of NAD prior to the transport of nicotinamide mononucleotide (NMN) into the cell. The partial purification of this enzyme is reported here. A cell suspension of S. typhimurium was passed twice through a French pressure cell, centrifugated at 5000 xg, and at 200,000 xg, for 1 hr. The pellet containing the crude membrane fraction was extracted with a novel detergent extraction using the differential solubility of NAD pyrophosphatase at various concentrations of the non-ionic detergent n-octyl glucoside (nOG). Extraction of the membrane fraction with 0.5% nOG in the presence of 10mM MgCl/sub 2/ removed 60% of the protein with no loss in activity. A second extraction with 2% nOG and 10mM MgCl/sub 2/ removed 20% of the protein and 71% of the activity from the membrane fraction. Ammonium sulfate fractionation at 45 to 50% sat. gave a partially purified enzyme preparation having a specific activity of about 2500 units/mg with a 94% recovery compared to the crude extract. One unit of activity is the cleavage of 1 nmole /sup 14/C NAD to /sup 14/C NMN per minute. The enzyme appears to have a MW of 200,000 on Sephacryl S-200, is temperature labile, and stabilized by 1mM Mg/sup + +/ and storage at -70/sup 0/.},
doi = {},
journal = {Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)},
number = ,
volume = 46:6,
place = {United States},
year = 1987,
month = 5
}

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  • The kinetic ..cap alpha..-deuterium secondary isotope effect on the second-order rate constant has been measured for the nonenzymatic direct hydride transfer reduction of 4-cyano-2,6-dinitrobenzenesulfonate by NADH (deuterium substitution of the hydrogen bonded to the 4 carbon of NADH which is not transferred to the acceptor). Values of 1.156 +- 0.018 and 1.1454 +- 0.0093 were obtained using direct and intramolecular competition methods, respectively. The corresponding (enzyme catalyzed) equilibrium isotope effects were found to be 1.013 +- 0.020 and 1.0347 +- 0.0087 as determined by direct and intermolecular competition methods, respectively. Thus, the value of the kinetic effect is significantly greatermore » than that on the equilibrium. It is suggested that this may arise either from participation of the ..cap alpha.. hydrogen in a hyperconjugative stabilization of an early transition state or from its participation in the reaction coordinate motion of a nonlinear activated complex. The values of the equilibrium effect allow calculation of a fractionation factor (relative to acetylene) for hydrogen bonded to the 4 carbon of NAD/sup +/ of 1.448 +- 0.028 or 1.418 +- 0.020. This is larger than expected based on comparison with hydrogen bound to sp/sup 2/ carbon in propene (1.336) or benzene (1.368) but is consistent with the decreased aromatic character of pyridinium vibrational spectra. The lack of a significant inverse value for the equilibrium ..cap alpha..-dueterium effect suggests complications in the interpretation of reported kinetic secondary effects of 0.85 and 1.2 for the forward (sp/sup 3/ ..-->.. sp/sup 2/) and reverse (sp/sup 2/ ..-->.. sp/sup 3/) rate constants for the nonenzymatic transhydrogenation of N-benzyl-1,4-dihydronicotinamide and its nicotinamide salt.« less
  • The dicarboxylate radical was generated in an N/sub 2/O-saturated fumarate solution by high energy ionizing radiation. When NADH was present in the solution, product analysis indicated a stoichiometry of 2 molecules of the radical reacted with 1 NADH molecule to form 2 malate and 1 enzymatically active NAD/sup +/ molecules. In a similar experiment using tritium label on position A of NADH, due to an isotope effect, only 10 percent of the label was transferred to malate; most of the remaining tritium was found in the NAD/sup +/ formed. When lactate dehydrogenase was added, however, no label was detectable inmore » NAD/sup +/, and over 80 percent of the tritium lost from NADH was found in malate. The stereospecific transfer of the hydrogen atom from lactate dehydrogenase-bound NADH to the dicarboxylate radical suggested that the free radical reaction must have taken place at the active site. The hydrogen atom transfer was inhibited by oxamate. Results from flow experiments in which an irradiated fumarate solution was mixed with a solution of lactate dehydrogenase and NADH are in support of a mechanism in which the hydrogen atom transfer occurs in the first oxidation step. (auth)« less
  • The pH dependence of the kinetic parameters and the primary deuterium isotope effects with nicotinamide adenine dinucleotide (NAD) and also thionicotinamide adenine dinucleotide (thio-NAD) as the nucleotide substrates were determined in order to obtain information about the chemical mechanism and location of rate-determining steps for the Ascaris suum NAD-malic enzyme reaction. The maximum velocity with thio-NAD as the nucleotide is pH-independent from pH 4.2 to 9.6, while with NAD, V decreases below a pK of 4.8. V/K for both nucleotides decreases below a pK of 5.6 and above a pK of 8.9. Both the tartronate pKi and V/Kmalate decrease belowmore » a pK of 4.8 and above a pK of 8.9. Oxalate is competitive vs. malate above pH 7 and noncompetitive below pH 7 with NAD as the nucleotide. The oxalate Kis increases from a constant value above a pK of 4.9 to another constant value above a pK of 6.7. The oxalate Kii also increases above a pK of 4.9, and this inhibition is enhanced by NADH. In the presence of thio-NAD the inhibition by oxalate is competitive vs. malate below pH 7. For thio-NAD, both DV and D(V/K) are pH-independent and equal to 1.7. With NAD as the nucleotide, DV decreases to 1.0 below a pK of 4.9, while D(V/KNAD) and D(V/Kmalate) are pH-independent. Above pH 7 the isotope effects on V and the V/K values for NAD and malate are equal to 1.45, the pH-independent value of DV above pH 7. Results indicate that substrates bind to only the correctly protonated form of the enzyme. Two enzyme groups are necessary for binding of substrates and catalysis. Both NAD and malate are released from the Michaelis complex at equal rates which are equal to the rate of NADH release from E-NADH above pH 7. Below pH 7 NADH release becomes more rate-determining as the pH decreases until at pH 4.0 it completely limits the overall rate of the reaction.« less
  • The /sup 13/C primary kinetic isotope effect on the decarboxylation of malate by nicotinamide adenine dinucleotide malic enzyme from Crassula argentea is 1.0199 +/- 0.0006 with proteo L-malate-2-H and 1.0162 +/- 0.0003 with malate-2-d. The primary deuterium isotope effect is 1.45 +/- 0.10 on V/K and 1.93 +/- 0.13 on V/sub max/. This indicates a stepwise conversion of malate to pyruvate and CO/sub 2/ with hydride transfer preceding decarboxylation, thereby suggesting a discrete oxaloacetate intermediate. This is in agreement with the stepwise nature of the chemical mechanism of other malic enzymes despite the Crassula enzyme's inability to reduce or decarboxylatemore » oxaloacetate. Differences in morphology and allosteric regulation between enzymes suggest specialization of the Crassula malic enzyme for the physiology of crassulacean and acid metabolism while maintaining the catalytic events founds in malic enzymes from animal sources.« less