Proteins separated by SDS gel electrophoresis and transferred to a nitrocellulose sheet can be visualised by 'probing' with peroxidase-linked reagents which are detected by luminescence. A modified luminescent substrate is described containing 4-methylumbelliferone which enhances light emission four-fold. Using the modified substrate, luminescent detection was found to be more sensitive ... More
High intracellular pH accompanies mitotic activity in murine lymphocytes.
AuthorsGerson DF, Kiefer H
JournalJ Cell Physiol
PubMed ID6980886
'Intracellular pH is crucial to the control of many cellular processes such as glycolysis, protein synthesis, and cell division. To study the relation between intracellular pH and mitotic activity in actively dividing Con A- or LPS-stimulated splenic lymphocytes, a method was developed to determine intracellular pH using a fluorescence-activated cell ... More
Diffusion coefficients of CO2 molecule and bicarbonate ion in hemoglobin solution measured by fluorescence technique.
AuthorsUchida K, Mochizuki M, Niizeki K
JournalJpn J Physiol
PubMed ID6417383
'The diffusion process of CO2 within a thin layer of hemoglobin (Hb) solution was followed by pH-sensitive fluorescence of 4-methylumbelliferone. In the presence of sufficient carbonic anhydrase in the layer, the pH change was accelerated and determined only by the diffusion. The diffusion rate was reduced as the Hb concentration ... More
Determination of 4-methylumbelliferone after separation from its conjugates by high-performance liquid chromatography. Application to lysosomal enzyme activity assays.
AuthorsSandman R
JournalJ Chromatogr
PubMed ID6841548
'A high-performance liquid chromatographic method is described for the separation and estimation of 4-methylumbelliferone in the presence of its conjugates. The technique utilizes a simple, isocratic eluent and fluorometric detection. Percentages of 4-methylumbelliferone in the conjugates and fluorescences of the conjugates are reported. 4-Methylumbelliferone, liberated under currently used conditions for ... More
A sensitive liquid chromatographic assay of ethoxycoumarin deethylase with fluorescence detection.
AuthorsZitting A
JournalAnal Biochem
PubMed ID6975585
Fluorescence of substituted 7-hydroxycoumarins.
AuthorsSherman WR, Robins E
JournalAnal Chem
PubMed ID5641323
Fluorescence spectroscopic profiling of compound libraries.
AuthorsSimeonov A, Jadhav A, Thomas CJ, Wang Y, Huang R, Southall NT, Shinn P, Smith J, Austin CP, Auld DS, Inglese J,
JournalJ Med Chem
PubMed ID18363325
Chromo/fluorophoric properties often accompany the heterocyclic scaffolds and impurities that comprise libraries used for high-throughput screening (HTS). These properties affect assay outputs obtained with optical detection, thus complicating analysis and leading to false positives and negatives. Here, we report the fluorescence profile of more than 70,000 samples across spectral regions ... More
Enzyme cytochemical techniques for metabolic mapping in living cells, with special reference to proteolysis.
AuthorsBoonacker E, Van Noorden CJ
JournalJ Histochem Cytochem
PubMed ID11724895
Specific enzymes play key roles in many pathophysiological processes and therefore are targets for therapeutic strategies. The activity of most enzymes is largely determined by many factors at the post-translational level. Therefore, it is essential to study the activity of target enzymes in living cells and tissues in a quantitative ... More
Quantitative assay of senescence-associated beta-galactosidase activity in mammalian cell extracts.
AuthorsGary RK, Kindell SM
JournalAnal Biochem
PubMed ID16004951
Senescence-associated beta-galactosidase activity is a widely used biomarker for assessing replicative senescence in mammalian cells. This enzymatic activity has generally been measured by staining cells with the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (X-gal) at pH 6.0, a reaction condition that suppresses lysosomal beta-galactosidase activity sufficiently to ensure that most nonsenescent cells will ... More
Rate of CO2 diffusion in the human red blood cell measured with pH-sensitive fluorescence.
AuthorsNiizeki K, Mochizuki M, Uchida K
JournalJpn J Physiol
PubMed ID6417384
The diffusion rate of CO2, into and out of the red cell, was measured by using a stopped flow method with pH-sensitive fluorescence of 4-methylumbelliferone. A red cell suspension of 15% hematocrit with the PCO2 of 8 Torr (or 65 Torr) was mixed with the same amount of saline solution ... More
Flow cytometric measurement of cytoplasmic pH: a critical evaluation of available fluorochromes.
AuthorsMusgrove E, Rugg C, Hedley D
JournalCytometry
PubMed ID3488180
Three pH-sensitive fluorochromes-4-methyl-umbelliferone(4MU),2, 3-dicyano-hydroquinone (DCH), and 2',7'-bis(carboxyethyl)-5,6-carboxy fluorescein (BCECF)--were evaluated for their resolution, range, and stability of cellular fluorescence. Flow cytometric techniques for determining cytoplasmic pH (pHi) have been fully described for 4MU and DCH; BCECF has previously been used for fluorimetric estimation of pHi, and was adapted to flow ... More
A continuous fluorescence-based assay for the human high-molecular-weight cytosolic phospholipase A2.
AuthorsHuang Z, Laliberté F, Tremblay NM, Weech PK, Street IP
JournalAnal Biochem
PubMed ID7856835
A sensitive method for continuously monitoring the activity of the human cytosolic phospholipase A2 (cPLA2) is described. Recombinant cPLA2 efficiently hydrolyzes fatty acid esters of 7-hydroxycoumarin, producing the free fatty acid and the highly fluorescent 7-hydroxycoumarin. All of the observed 7-hydroxycoumarinyl ester hydrolase activity (7-HCEase) in a preparation of the ... More
Characteristics of fluoroprobes for measuring intracellular pH.
AuthorsGraber ML, DiLillo DC, Friedman BL, Pastoriza-Munoz E
JournalAnal Biochem
PubMed ID3740410
We evaluated four different fluoroprobes to determine their capabilities and limitations in measuring intracellular pH by the fluorescent indicator technique. In vitro, carboxyfluorescein, dimethylcarboxyfluorescein, biscarboxyethyl carboxyfluorescein, and 4-methylumbelliferone (4MU) all showed comparably intense fluorescence and excellent pH sensitivity near their respective pKa values. Major differences were found between 4MU and ... More
Ligand exclusion on acetylcholinesterase.
AuthorsBerman HA, Leonard K
JournalBiochemistry
PubMed ID2271673
This paper examines covalent reactivity of AchE with respect to cationic and uncharged methylphosphonates and substrates in the absence and presence of cationic ligands selective for the active center and the peripheral anionic site. The organophosphorus inhibitors are enantiomeric alkyl methylphosphonothioates (1-5) containing cycloheptyl and isopropyl phosphono ester groups and ... More
Parathyroid hormone decreases HCO3 reabsorption in the rat proximal tubule by stimulating phosphatidylinositol metabolism and inhibiting base exit.
AuthorsPastoriza-Munoz E, Harrington RM, Graber ML
JournalJ Clin Invest
PubMed ID1314850
The mechanism of inhibition of HCO3 transport by parathyroid hormone (PTH) in the proximal tubule is not clearly defined. Previous studies in vitro have suggested that this effect is mediated via cAMP generation, which acts to inhibit Na/H exchange, resulting in cell acidification. To examine this question in vivo, intracellular ... More