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Naphthol AS-BI (7-bromo-3-hydroxy-2-naphtho-o-anisidine) phosphatase and naphthol AS-BI. beta. -D-glucuronidase in Chinese hamster ovary cells: biochemical and flow cytometric studies

Journal Article · · J. Histochem. Cytochem.; (United States)
DOI:https://doi.org/10.1177/27.1.35565· OSTI ID:5844700
;

Conditions for the biochemical and flow cytometric assay of 7-bromo-3-hydroxy-2-naphtho-o-anisidine phosphatase and ..beta..-D-glucuronidase activities in Chinese hamster ovary cells were studied. In the biochemical assays, the pH optimum for the phosphatase activity was pH 4.6 with a Km of 10/sup -5/ M; the pH optimum for ..beta..-D-glucuronidase activity was pH 5.0 with a Km of 2 x 10/sup -5/ M. For intact cells the derived constants were 3 to 10 times higher. The rate of hydrolysis of both substrates was also examined by flow cytometry. Cellular fluorescence increased linearly for only about 15 min. Diffusion of the fluorescent product probably caused nonlinearity of the fluorescence increase and was demonstrated by mixing cells incubated with substrate with those that had not been incubated. After 15 min, cells that had not been exposed previously to product or substrate contained the fluorescent product. Cells fractionated into size classes by centrifugal elutriation also were analyzed by flow cytometry for ..beta..-D-glucuronidase activity. The activity increased linearly with the increase in cell size corresponding to the progression from G/sub 1/ through S and into G/sub 2/-M phases of the cell cycle.

Research Organization:
Univ. of California, Livermore
DOE Contract Number:
W-7405-ENG-48
OSTI ID:
5844700
Journal Information:
J. Histochem. Cytochem.; (United States), Journal Name: J. Histochem. Cytochem.; (United States) Vol. 27:1; ISSN JHCYA
Country of Publication:
United States
Language:
English

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Related Subjects

550200 -- Biochemistry
550300* -- Cytology
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
BIOASSAY
BIOCHEMICAL REACTION KINETICS
BODY
CATALYSIS
CELL CULTURES
CELL CYCLE
CELL FLOW SYSTEMS
ENZYMES
ESTERASES
FEMALE GENITALS
FLUORESCENCE
GLUCURONIDASE
GLYCOSYL HYDROLASES
GONADS
HAMSTERS
HYDROLASES
KINETICS
LUMINESCENCE
MAMMALS
METABOLISM
ORGANS
OVARIES
PHOSPHATASES
REACTION KINETICS
RODENTS
SUBSTRATES
VERTEBRATES