Abstract
Cell-free extracts of a strain of Proteus vulgaris degrade NADH to reduced nicotinamide riboside, adenosine and two molecules of phosphate. The system is weakly active in fresh cell extracts, but activity is increased about 10-fold on rapid heating to 70-100 degrees C. On returning to room temperature, the activity returns rapidly to its initial low value but can be re-activated by again heating to 70-100 degrees C. Reversible activation can also be effected by extremes of pH or by teatment with 8M-urea. Activation appears to be due to reversible changes in conformation of the protein of the enzyme rather than to combination of the enzyme with a heat-labile inhibitor. The active form can be stabilized by addition of PPi. The system, which also possesses 5'-nucleotidase activity not separable from the NADH pyrophosphatase, requires Co2+ (0.4mM) for maximum activity. Although activated at relatively high temperatures, it is not enzymically active until cooled to 50-60 degrees C. It may be purified by affinity chromatography (with NAD+ as ligand) to an activity over 400 times that of the crude cell extract, and yields only one major band on polyacrylamide-gel electrophoresis.
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Selected References
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- Andrews P. The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem J. 1965 Sep;96(3):595–606. doi: 10.1042/bj0960595. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cuatrecasas P. Protein purification by affinity chromatography. Derivatizations of agarose and polyacrylamide beads. J Biol Chem. 1970 Jun;245(12):3059–3065. [PubMed] [Google Scholar]
- LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
- Larsson P. O., Mosbach K. Preparation of a NAD(H)-polymer matrix showing coenzyme function of the bound pyridine nucleotide. Biotechnol Bioeng. 1971 May;13(3):393–398. doi: 10.1002/bit.260130306. [DOI] [PubMed] [Google Scholar]
- Mather I. H., Knight M. A heat-stable nicotinamide adenine dinucleotidase from Pseudomonas fluorescens. J Gen Microbiol. 1969 Jun;56(3):i–ii. [PubMed] [Google Scholar]
- Neu H. C. The 5'-nucleotidase of Escherichia coli. I. Purification and properties. J Biol Chem. 1967 Sep 10;242(17):3896–3904. [PubMed] [Google Scholar]
- Neu H. C. The 5'-nucleotidase of Escherichia coli. II. Surface localization and purification of the Escherichia coli 5'-nucleotidase inhibitor. J Biol Chem. 1967 Sep 10;242(17):3905–3911. [PubMed] [Google Scholar]
- Neu H. C. The 5'-nucleotidases (uridine diphosphate sugar hydrolases) of the Enterobacteriaceae. Biochemistry. 1968 Oct;7(10):3766–3773. doi: 10.1021/bi00850a059. [DOI] [PubMed] [Google Scholar]
- SWARTZ M. N., KAPLAN N. O., FRECH M. E. Significance of heat-activated enzymes. Science. 1956 Jan 13;123(3185):50–53. doi: 10.1126/science.123.3185.50. [DOI] [PubMed] [Google Scholar]
- SWARTZ M. N., KAPLAN N. O., LAMBORG M. F. A heat-activated diphosphopyridine nucleotide pyrophosphatase from Proteus vulgaris. J Biol Chem. 1958 Jun;232(2):1051–1063. [PubMed] [Google Scholar]
- SWARTZ M. N., MERSELIS J. A "heat-activated" diphosphopyridine nucleotide pyrophosphatase activity of Staphylococcus aureus. Proc Soc Exp Biol Med. 1962 Feb;109:384–388. doi: 10.3181/00379727-109-27211. [DOI] [PubMed] [Google Scholar]