In situ biochemical studies of intestinal alkaline phosphatase in normal and phosphate-depleted rats by microdensitometry.

Bader, Cyrille; Nasr, Leila Ben; Monet, Jean-Dominique; Bachelet, Maria; Assailly, J.; Ulmann, A

doi:10.1016/s0021-9258(20)71256-1

Journal of Biological Chemistry

1984

DOI: 10.1016/s0021-9258(20)71256-1

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In situ biochemical studies of intestinal alkaline phosphatase in normal and phosphate-depleted rats by microdensitometry.

Cyrille Bader¹,

Leila Ben Nasr²,

Jean-Dominique Monet³

et al.

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Cited by 11 publications

References 14 publications

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Δ⁵3β hydroxysteroid dehydrogenase activity of the rat corpus luteum exhibits positive substrate‐binding co‐operativity: A continuous monitoring microdensitometric study with unfixed ovarian tissue sections

Gordon

Robertson

1987

Cell Biochemistry & Function

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delta (5)3 beta hydroxysteroid dehydrogenase activity transforms biologically inactive delta (5)3 beta hydroxy steroids into the active delta (4)3-keto products (e.g. pregnenolone to progesterone). Using a cytochemical procedure which allows for the continuous microdensitometric monitoring of an enzyme reaction as it proceeds and a well described cytochemical assay for delta (5)3 beta HSD we have analysed the initial velocity rates (Vo) for dehydroepiandrosterone (DHEA) binding to this enzyme in regressing (i.e. 20 alpha hydroxy steroid dehydrogenase positive) corpus luteum (CL) cells in unfixed tissue sections (5 micron) of the dioestrous and proestrous rat ovary. The results are mean +/- S.E.M. The relationship between DHEA concentration (0 to 50 microM) and delta (5)3 beta HSD activity in the dioestrous corpora lutea was sigmoidal and had an atypical 1/Vo versus 1/S plot, the x intercept being positive. Using a 1/Vo versus 1/S2 plot the Vmax was determined to be 1.0 +/- 0.08 mumol min-1 mg-1 CL (n = 6). The Hill constant was 2.7 +/- 0.02 (n = 6) suggesting a high degree of positive co-operativity for DHEA binding. The S concentration for half maximal activity was 17 +/- 1 mumoles (n = 6). In the corpora lutea cells of the proestrous ovary, the Vmax for DHEA transformation was unchanged (0.95 +/- 0.04 mumol min-1 mg-1, n = 3) whilst the S0.5 was significantly increased to 27 +/- 0.1 (p less than 0.01, n = 3). The Hill constant remained positive being 2.9 +/- 0.2 (n = 3).(ABSTRACT TRUNCATED AT 250 WORDS)

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Δ⁵3β hydroxysteroid dehydrogenase activity of the rat corpus luteum exhibits positive substrate‐binding co‐operativity: A continuous monitoring microdensitometric study with unfixed ovarian tissue sections

Gordon

Robertson

1987

Cell Biochemistry & Function

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The application of continuous monitoring microdensitometry to an analysis of NAD+ binding and 3β-hydroxy-Δ5-steroid dehydrogenase activity in the regressing corpus luteum of the pro-oestrous rat ovary

Gordon

Robertson

1986

Histochem J

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A technique which allows for the continuous microdensitometric monitoring (at 3.3 s intervals) of an enzyme reaction has been applied to a study of 3 beta-hydroxy-delta 5-steroid dehydrogenase activity in regressing corpora lutea cells of unfixed tissue sections (5 micron thick) of the pro-oestrous rat ovary. Initial reaction rates for NAD+ reduction by the activity of the enzyme were maintained for less than 3 min at all the concentrations (0-500 mumol/l) of co-factor employed. The relationship between NAD+ concentration and enzyme activity under initial velocity rate conditions was hyperbolic and maximum enzyme activity was seen when the NAD+ concentration was greater than 250 mumol/l. The apparent Michaelis-Menten constant (Km) was 29 mumol/l and Vmax 0.63 mumol NAD+ reduced/min/cm3 corpora lutea.

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Quantification of the histochemical reaction for alkaline phosphatase activity using the indoxyl—tetranitro BT method

Noorden

Jonges

1987

Histochem J

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The indoxyl-tetranitro BT method for the demonstration of alkaline phosphatase activity has been optimized and its validity for quantitative histochemistry tested. The study has been performed with model films of polyacrylamide gel incorporating homogenate of rat liver and with cryostat sections from the same livers. Addition of polyvinyl alcohol to the incubation medium greatly improved the localization of the final reaction product in cryostat sections. In polyacrylamide films, the formazan production specifically due to alkaline phosphatase was highest when using a medium containing 100 mM Tris-HCl buffer, pH 9.0, 0.2-1.0 mM substrate, 0.32 mM 1-methoxyphenazine methosulphate, 10 mM MgCl2, 5 mM sodium azide and 1 mM tetranitro BT. For the incubation of cryostat sections in the presence of polyvinyl alcohol, the same medium could be used but the optimum concentrations of substrate and tetranitro BT appeared to be 1-2 mM and 5 mM respectively. The test minus control reaction was specific for alkaline phosphatase activity and could be inhibited completely with tetramisole. The test minus control reaction was linear with time up to 30 min with model films and up to 15 min with cryostat sections. The formazan production was also linear with the amount of homogenate incorporated in model films and with section thickness up to 18 micron and therefore, the reaction obeyed the Beer-Lambert law. Variation of the substrate concentration yielded a KM of 0.05 mM for aqueous media and a KM of 0.55 mM for polyvinyl alcohol-containing media.(ABSTRACT TRUNCATED AT 250 WORDS)

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In situ biochemical studies of intestinal alkaline phosphatase in normal and phosphate-depleted rats by microdensitometry.

Cited by 11 publications

References 14 publications

Δ⁵3β hydroxysteroid dehydrogenase activity of the rat corpus luteum exhibits positive substrate‐binding co‐operativity: A continuous monitoring microdensitometric study with unfixed ovarian tissue sections

Δ⁵3β hydroxysteroid dehydrogenase activity of the rat corpus luteum exhibits positive substrate‐binding co‐operativity: A continuous monitoring microdensitometric study with unfixed ovarian tissue sections

The application of continuous monitoring microdensitometry to an analysis of NAD+ binding and 3β-hydroxy-Δ5-steroid dehydrogenase activity in the regressing corpus luteum of the pro-oestrous rat ovary

Quantification of the histochemical reaction for alkaline phosphatase activity using the indoxyl—tetranitro BT method

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In situ biochemical studies of intestinal alkaline phosphatase in normal and phosphate-depleted rats by microdensitometry.

Cited by 11 publications

References 14 publications

Δ53β hydroxysteroid dehydrogenase activity of the rat corpus luteum exhibits positive substrate‐binding co‐operativity: A continuous monitoring microdensitometric study with unfixed ovarian tissue sections

Δ53β hydroxysteroid dehydrogenase activity of the rat corpus luteum exhibits positive substrate‐binding co‐operativity: A continuous monitoring microdensitometric study with unfixed ovarian tissue sections

The application of continuous monitoring microdensitometry to an analysis of NAD+ binding and 3β-hydroxy-Δ5-steroid dehydrogenase activity in the regressing corpus luteum of the pro-oestrous rat ovary

Quantification of the histochemical reaction for alkaline phosphatase activity using the indoxyl—tetranitro BT method

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Δ⁵3β hydroxysteroid dehydrogenase activity of the rat corpus luteum exhibits positive substrate‐binding co‐operativity: A continuous monitoring microdensitometric study with unfixed ovarian tissue sections

Δ⁵3β hydroxysteroid dehydrogenase activity of the rat corpus luteum exhibits positive substrate‐binding co‐operativity: A continuous monitoring microdensitometric study with unfixed ovarian tissue sections