Partial purification of nicotinamide adenine dinucleotide (NAD) pyrophosphatase from Salmonella typhimurium
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/.
- Research Organization:
- Marshall Univ. School of Medicine, Huntington, WV
- OSTI ID:
- 6079018
- Report Number(s):
- CONF-870644-; TRN: 87-037156
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 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
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Related Subjects
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