scholarly journals Indoleamine 2,3-dioxygenase. A new, rapid, sensitive radiometric assay and its application to the study of the enzyme in rat tissues

1980 ◽  
Vol 189 (3) ◽  
pp. 461-466 ◽  
Author(s):  
J S Cook ◽  
C I Pogson ◽  
S A Smith
Keyword(s):  
Enzyme Activity ◽  
Rat Tissues ◽  
Rat Intestine ◽  
Indoleamine 2,3 Dioxygenase ◽  
Crude Extracts ◽  
Large Numbers ◽  
Kidney Enzyme ◽  
Excess Substrate ◽  
Radiometric Assay

A simple and convenient assay for indoleamine 2,3-dioxygenase has been developed. This depends on the conversion of D-[ring-2-14C]tryptophan to [14C]formate, excess substrate is removed by adsorption onto charcoal. This assay, which is 20-fold more sensitive than previous procedures, is applicable both to crude extracts and to large numbers of samples. Activity in rat tissues is very much lower than in those of the rabbit; measureable activity is found only in the stomach, spleen, intestine and kidney. Enzyme activity in the rat intestine was increased by 50% in rats pretreated with L-tryptophan.

Photoelectric Method for Quantitative Evaluation of Mast-Cell-Associated Enzyme Activity in Human Gingival Tissues

1970 ◽  
Vol 49 (3) ◽  
pp. 480-486
Author(s):  
F.M. Sorenson ◽  
J.S. Bennett ◽  
D. Fujita ◽  
F.R. Poindexter ◽  
W.B. Hall
Keyword(s):  
Mast Cell ◽  
Enzyme Activity ◽  
Mast Cells ◽  
Quantitative Evaluation ◽  
Photoelectric Method ◽  
Scanning Method ◽  
Cell Counts ◽  
Biologic Activity ◽  
Large Numbers ◽  
Human Gingiva

Simple counts of mast cells per unit of human gingiva are often difficult to interpret because of the large numbers and varying sizes and shapes of the counted structures. The relatively simple photoelectric scanning method described herein eliminates tedious counting procedures while providing a measure of the relative quantity of stainable mast cell granules within the area scanned. Thus, the method may provide a better estimate of the total biologic activity than would simple mast cell counts.

Regulation of Phosphoenolpyruvate Carboxylase in Zea mays by Protein Phosphorylation and Metabolites and Their Roles in Photosynthesis

1996 ◽  
Vol 23 (1) ◽  
pp. 25 ◽  
Author(s):  
Y Gao ◽  
KC Woo
Keyword(s):  
Zea Mays ◽  
Enzyme Activity ◽  
Protein Phosphorylation ◽  
Photosynthetic Rate ◽  
Phosphoenolpyruvate Carboxylase ◽  
Strong Inhibitor ◽  
Physiological Concentration ◽  
Crude Extracts ◽  
Maize Leaves

The effects of metabolites, protein phosphorylation and malate inhibition on phosphoenolpyruvate carboxylase (PEPC) activity were investigated at pH 7.0 in partially purified enzyme from maize leaves. Glycine, glucose 6-phosphate or alanine stimulated the activity two- to three-fold. Glycine and glucose 6-phosphate increased the affinity for PEP by factors of eight and four respectively. These metabolites changed the response of the enzyme activity to pH. Activity increased between pH 6.8 and 8.0 by 10-fold in the absence and 26% in the presence of these metabolites. In vitro phosphorylation of PEPC increased the activity two-fold in the absence but not in the presence of these metabolites. Malate was a strong inhibitor of PEPC, the KI value being 0.25-0.5 mM. Protein phosphorylation and the above metabolites increased the Ki value by factors of three and 12 respectively, but they synergistically increased the Ki 50-fold, thus providing maximal protection against malate inhibition. In the crude extracts from light- and dark-adapted leaves in the presence of a physiological concentration of malate (20 mM), PEPC activity comparable to the photosynthetic rate was obtained only from the light-adapted leaves in the presence of metabolites indicating that both light-induced protein phosphorylation and metabolic activators were essential for PEPC activation during photosynthesis. We propose that both these factors act synergistically to modulate PEPC during photosynthesis in maize.

A Sensitive Radiometric Assay for Tryptophan Hydroxylase Applicable to Crude Extracts

1983 ◽  
Vol 40 (3) ◽  
pp. 894-897 ◽  
Author(s):  
S. Jane Beevers ◽  
Richard G. Knowles ◽  
Christopher I. Pogson
Keyword(s):  
Tryptophan Hydroxylase ◽  
Crude Extracts ◽  
Radiometric Assay

Applicability of the product isolation and the radiometric aromatase assays for the measurement of low levels of aromatase: lack of aromatase activity in the human endometrium

1990 ◽  
Vol 127 (3) ◽  
pp. 539-551 ◽  
Author(s):  
M. Prefontaine ◽  
C. Shih ◽  
C. C. Pan ◽  
B. R. Bhavnani
Keyword(s):  
High Performance ◽  
Human Endometrium ◽  
Heat Inactivation ◽  
Aromatase Activity ◽  
Excess Substrate ◽  
Release Assay ◽  
Radiometric Assay ◽  
Fetal Bovine ◽  
Low Levels

ABSTRACT The purpose of this investigation was to assess the applicability of two well established procedures: (i) the product isolation assay and (ii) the radiometric 3H2O assay for the determination of very low levels of aromatase activity. The methods were validated and used to assess the capacity of normal and neoplastic human endometrium to synthesize oestrogens from androgens. Using the product isolation assay, various specimens (n = 27) of normal and neoplastic endometrium were incubated with [1,2,6,7-3H]testosterone either by a standard incubation procedure or by a superfusion technique. Following the incubation, carrier oestrone and oestradiol or [14C]oestrone and [14C]oestradiol were added, and the oestrogens were isolated and purified by paper chromatography and high-performance liquid chromatography. The radiochemical purity of oestrone and oestradiol was checked by the isotope dilution technique. In all samples, the 3H associated with oestrone and oestradiol failed to recrystallize as oestrone and oestradiol. No radioactivity was detectable in the oestrone and oestradiol crystals after acetylation. Similarly, 16 endometrial samples were tested for aromatase activity by the 3H2O release assay using [1β-3H]androstenedione as substrate. The results indicate that 3H2O was indeed released during these incubations, but this activity could not be inhibited by the aromatase inhibitor 4-hydroxyandrostenedione, by excess substrate or by heat inactivation of the tissue. Furthermore, the release of 3H2O from [1β-3H]androstenedione under the incubation conditions used (Dulbecco's modified Eagle's medium or RPMI-1640 containing fetal bovine serum and NADPH) also occurred in the absence of any tissue. This activity was not inhibited by 4-hydroxyandrostenedione nor by excess substrate. The results demonstrate that the human endometrium does not contain detectable levels of aromatase activity and that the radiometric assay can give rise to false-positive results if used for detection of very low levels of aromatase activity. Journal of Endocrinology (1990) 127, 539–551

scholarly journals Cloning of rat brain succinyl-CoA:3-oxoacid CoA-transferase cDNA. Regulation of the mRNA in different rat tissues and during brain development

1987 ◽  
Vol 248 (3) ◽  
pp. 853-857 ◽  
Author(s):  
M K Ganapathi ◽  
M Kwon ◽  
P M Haney ◽  
C McTiernan ◽  
A A Javed ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Rat Brain ◽  
Ketone Bodies ◽  
Rat Kidney ◽  
Relative Rate ◽  
Cdna Clones ◽  
Rat Tissues ◽  
Transferase Activity ◽  
Coa Transferase ◽  
Old Rats

3-Oxoacid CoA-transferase, which catalyses the first committed step in the oxidation of ketone bodies, is uniquely regulated in developing rat brain. Changes in 3-oxoacid CoA-transferase activity in rat brain during the postnatal period are due to changes in the relative rate of synthesis of the enzyme. To study the regulation of this enzyme, we identified, with a specific polyclonal rabbit anti-(rat 3-oxoacid CoA-transferase), two positive cDNA clones (approx. 800 bp) in a lambda gtll expression library, constructed from poly(A)+ RNA from brains of 12-day-old rats. One of these clones (lambda CoA3) was subcloned into M13mp18 and subjected to further characterization. Labelled single-stranded probes prepared by primer extension of the M13mp18 recombinant hybridized to a 3.6 kb mRNA. Rat brain mRNA enriched by polysome immunoadsorption for a single protein of size 60 kDa which corresponds to the precursor form of 3-oxoacid CoA-transferase was also found to be similarly enriched for the hybridizable 3.6 kb mRNA complementary to lambda CoA3. Affinity-selected antibody to the lambda CoA3 fusion protein inhibited 3-oxoacid CoA-transferase activity present in rat brain mitochondrial extracts. The 3.6 kb mRNA for 3-oxoacid CoA-transferase was present in relative abundance in rat kidney and heart, to a lesser extent in suckling brain and mammary gland and negligible in the liver. The specific mRNA was also found to be 3-fold more abundant in the brain from 12-day-old rats as compared with 18-day-old foetuses and adult rats, corresponding to the enzyme activity and relative rate of synthesis profile during development. These data suggest that 3-oxoacid CoA-transferase enzyme activity is regulated at a pretranslational level.

scholarly journals sn-Glycero(3)phosphoinositol glycerophosphohydrolase. A new phosphodiesterase in rat tissues

1979 ◽  
Vol 182 (1) ◽  
pp. 39-45 ◽  
Author(s):  
R M C Dawson ◽  
N Hemington ◽  
D E Richards ◽  
R F Irvine
Keyword(s):  
Intestinal Mucosa ◽  
Microsomal Fraction ◽  
Temperature Stability ◽  
Rat Tissues ◽  
Alkaline Ph ◽  
Stability Studies ◽  
Ph Optimum ◽  
Excess Substrate ◽  
Membrane Bound ◽  
Cyclic Phosphate

1. A phosphodiesterase that cleaves glycerophosphoinositol into glycerophosphate and inositol has been detected in rat tissues. 2. The enzyme requires Mg2+ (Mn2+) and has a pH optimum of 7.7. 3. The richest sources of the enzyme are kidney and intestinal mucosa. In pancreas subcellular fractions it occurs largely in the microsomal fraction. 4. The enzyme is inhibited by excess substrate and by the reaction product glycerophosphate. 5. Temperature-stability studies and other observations distinguish the enzyme from other membrane-bound phosphodiesterases active at an alkaline pH e.g. glycerophosphoinositol inositophosphohydrolase, glycerophosphocholine diesterase, inositol cyclic phosphate phosphodiesterase and phosphodiesterase I.

Application of a novel human cervical mucin-based assay demonstrates the absence of increased mucinase activity in bacterial vaginosis

2002 ◽  
Vol 13 (11) ◽  
pp. 755-760 ◽  
Author(s):  
R Wiggins ◽  
M R Millar ◽  
P W Soothill ◽  
S J Hicks ◽  
A P Corfield
Keyword(s):  
Enzyme Activity ◽  
Bacterial Vaginosis ◽  
Gestational Age ◽  
Enzyme Activities ◽  
Significant Elevation ◽  
Term Birth ◽  
Large Numbers ◽  
Mucus Barrier ◽  
Individual Enzyme

Enzymes produced in bacterial vaginosis (BV) have been proposed as possible mediators of pre-term birth. Most studies have concentrated on mid-trimester measurements of enzyme activity, and utilize synthetic substrates to measure enzyme activity, which may not accurately represent mucinase activity in vivo. We have developed a novel ELISA mucinase assay using biotinylated human cervical mucin as a substrate. The assay is rapid, sensitive and can be used to screen large numbers of samples. The new assay has been used to assess vaginal mucinase activities in 92 women <14 weeks gestational age with and without BV. No differences in mucinase activity were detected between normal and BV groups while significant elevation of sialidase and other glycosidases was confirmed as reported before. This study shows that significant mucinase activity is a normal event in the mucus barrier, but does not reflect changes identified for individual enzyme activities associated with BV.

scholarly journals THE HISTOCHEMICAL DEMONSTRATION OF NUCLEASE ACTIVITY WITH FILMS OF SOLUBLE RIBONUCLEIC ACID AND POLYADENYLIC ACID

1972 ◽  
Vol 20 (5) ◽  
pp. 350-357 ◽  
Author(s):  
R. DAOUST ◽  
R. MORAIS
Keyword(s):  
Enzyme Activity ◽  
Ribonucleic Acid ◽  
Nuclease Activity ◽  
Histochemical Demonstration ◽  
The Other ◽  
Rat Tissues ◽  
Other Hand ◽  
Polyadenylic Acid ◽  
Normal Rat

Films of soluble ribonucleic acid (sRNA) and polyadenylic acid (poly-A) were used to investigate the distribution of nuclease activity in normal rat tissues. The reactions obtained with films of sRNA were similar to those previously observed with standard RNA and both substrates apparently reveal the same group of nucleases. On the other hand, the distribution of enzyme activity shown by films of poly-A differed markedly from that observed with RNA films, and it appears that films of poly-A demonstrate a different group of nucleases.

Identification of genetic variants in the human indoleamine 2,3-dioxygenase (IDO1) gene, which have altered enzyme activity

2009 ◽  
Vol 19 (6) ◽  
pp. 464-476 ◽  
Author(s):  
Million Arefayene ◽  
Santosh Philips ◽  
Donghua Cao ◽  
Sudharani Mamidipalli ◽  
Zeruesenay Desta ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Genetic Variants ◽  
Indoleamine 2,3 Dioxygenase
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