Comparison of NADH and NADPH oxidase activities in granules isolated from human polymorphonuclear leukocytes with a fluorometric assay
Journal Article
·
· J. Clin. Invest.; (United States)
A fluorometric method for the determination of pyridine nucleotides has been adapted for use in studying the reduced pyridine nucleotide oxidases in human polymorphonuclear leukocytes. In the presence of strong base the oxidized forms of the pyridine nucleotides form a highly fluorescent product. The small amounts of NAD(P) formed by the oxidase reactions can be determined with great sensitivity. This method has been compared to the radioisotopic assay for NADPH oxidation. Both methods gave essentially the same results in terms of nanomoles NADP produced by control, resting, and phagocytizing samples. Both NADPH and NADH oxidase activities were insensitive to cyanide. NADPH oxidation had a pH optimum of 5.5, while that for NADH appeared to be 6.0. Granules isolated from phagocytizing cells routinely showed more activity toward both substrates (two to threefold) than granules from resting cells. Both activities were located primarily in a granule fraction prepared by differential centrifugation. Oxidation of NADPH was routinely four to five times that of NADH at all except very high substrate levels. Measurable NADH oxidation was rarely seen below 0.80 mM NADH, while NADPH oxidation was easily measurable at 0.20 mM.
- Research Organization:
- Bowman Gray School of Medicine, Winston-Salem, NC
- OSTI ID:
- 6777620
- Journal Information:
- J. Clin. Invest.; (United States), Journal Name: J. Clin. Invest.; (United States) Vol. 59; ISSN JCINA
- Country of Publication:
- United States
- Language:
- English
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Thu Sep 01 00:00:00 EDT 1983
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Fri Dec 31 23:00:00 EST 1976
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Sun Dec 31 23:00:00 EST 1978
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Related Subjects
550200* -- Biochemistry
550300 -- Cytology
59 BASIC BIOLOGICAL SCIENCES
BIOLOGICAL MATERIALS
BIOMEDICAL RADIOGRAPHY
BLOOD
BLOOD CELLS
BODY FLUIDS
CENTRIFUGATION
CHEMICAL REACTIONS
COENZYMES
COMPARATIVE EVALUATIONS
CYANIDES
DIAGNOSTIC TECHNIQUES
ENZYME ACTIVITY
ENZYMES
FLUOROSCOPY
LEUKOCYTES
MEDICINE
METABOLISM
NADP
NUCLEAR MEDICINE
NUCLEOTIDES
ORGANIC COMPOUNDS
OXIDASES
OXIDATION
OXIDOREDUCTASES
PH VALUE
PHAGOCYTOSIS
RADIOLOGY
SENSITIVITY
SEPARATION PROCESSES
550300 -- Cytology
59 BASIC BIOLOGICAL SCIENCES
BIOLOGICAL MATERIALS
BIOMEDICAL RADIOGRAPHY
BLOOD
BLOOD CELLS
BODY FLUIDS
CENTRIFUGATION
CHEMICAL REACTIONS
COENZYMES
COMPARATIVE EVALUATIONS
CYANIDES
DIAGNOSTIC TECHNIQUES
ENZYME ACTIVITY
ENZYMES
FLUOROSCOPY
LEUKOCYTES
MEDICINE
METABOLISM
NADP
NUCLEAR MEDICINE
NUCLEOTIDES
ORGANIC COMPOUNDS
OXIDASES
OXIDATION
OXIDOREDUCTASES
PH VALUE
PHAGOCYTOSIS
RADIOLOGY
SENSITIVITY
SEPARATION PROCESSES