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Title: 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 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:
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
Research Org:
Marshall Univ. School of Medicine, Huntington, WV
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