PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 3013781

A rapid, flexible method for biochemical assays using a microtiter plate reader and a microcomputer. Application for assays of protein, Na,K-ATPase and K-p-nitrophenylphosphatase.

T C Loomis·W L Stahl
View original source ↗Export metadata ↓

Abstract

A computerized system which greatly accelerates and eases the collection, storage, and analysis of data has been applied to several standard biochemical assays. The system uses a commercially available microtiter plate reader connected to an Apple IIe microcomputer via a standard serial port. Data are transmitted automatically from the reader to the microcomputer, where they can be viewed, printed, further analyzed immediately, or stored on a diskette for later retrieval and processing. Some or all of the data may be entered manually. The program calculates a linear least squares best fit to a standard curve after correcting all data for blanks, then determines the quantities of substrate or product contained in each well of a microtiter plate. Data from two plates may be combined, enabling calculation of enzyme specific activities. This system can be adapted to any assay whose final step can be performed by a microtiter plate reader. Its use is described for determination of protein concentration, Na,K-ATPase activity, and K-stimulated p-nitrophenylphosphatase activity.

Explore related subjects

4-NitrophenylphosphataseAnimalsComputersMicrocomputersPhosphoric Monoester HydrolasesProteinsSodium-Potassium-Exchanging ATPaseSoftwareJournal ArticleResearch Support, U.S. Gov't, Non-P.H.S.

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

T C Loomis, W L Stahl. 1986. A rapid, flexible method for biochemical assays using a microtiter plate reader and a microcomputer. Application for assays of protein, Na,K-ATPase and K-p-nitrophenylphosphatase.. https://doi.org/10.1016/0020-7101(86)90015-2

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2006

alpha-Amino acid derivatives as proton pump inhibitors and potent anti-ulcer agents.

In a program to identify new anti-ulcer compounds, a series of N-acyl derivatives of alpha-amino acids were screened for their in vitro H(+)/K(+) ATPase inhibitory activity, and in vivo efficacy in Pylorus ligation model. 3D-QSAR studies were carried out and a representative compound 13 was studied for the nature of its proton pump inhibition.

4-Nitrophenylphosphatase↗
PubMed2006

New evidence for ATP binding induced catalytic subunit interactions in pig kidney Na/K-ATPase.

Pig kidney Na/K-ATPase preparations showed a positive cooperative effect for pNPP in Na-pNPPase activity. Measurements of the Na-pNPPase activity, Na-ATPase activity and the accumulation of phosphoenzyme (EP) under conditions of pNPP saturation showed several different ATP affinities. The presence of pNPP reduced both the maximum amount of EP and Na-ATPase activity to half showing a value of 4 and a 3,700-fold reduced ATP affinity for EP formation, and a 7 and 1,300-fold reduced affinity for Na-ATPase activity. The presence of low concentrations of ATP in the phosphorylation induced a 2-fold enhancement in Na-pNPPase activity despite a reduction in available pNPP sites. However, higher concentrations of ATP inhibited the Na-pNPPase activity and a much higher concentration of ATP increased both the phosphorylation and Na-ATPase activity to the maximum levels. The maximum Na-pNPPase activity was 1.7 and 3.4-fold higher without and with ATP, respectively, than the maximum Na-ATPase activity. These data and the pNPP dependent reduction in both Na-ATPase activity and the amount of enzyme bound ATP provide new evidence to show that ATP, pNPP and ATP with pNPP, respectively, induce different subunit interactions resulting a difference in the maximum Na(+)-dependent catalytic activity in tetraprotomeric Na/K-ATPase.

4-Nitrophenylphosphatase↗
PubMed2006

An 18 kDa acid phosphatase from chicken heart possesses phosphotransferase activity.

A low molecular weight acid phosphatase was purified to homogeneity from chicken heart with a specific activity of 42 U/mg and a recovery of about 1%. Nearly 800 fold purification was achieved. The molecular weight was estimated to be 18 kDa by SDS-polyacrylamide gel electrophoresis. Para-nitrophenyl phosphate, phenyl phosphate and flavin mononucleotide were efficiently hydrolysed by the enzyme and found to be good substrates. Fluoride and tartrate had no inhibitory effect while phosphate, vanadate and molybdate strongly inhibited the enzyme. The acid phosphatase was stimulated in the presence of glycerol, ethylene glycol, methanol, ethanol and acetone, which reflected the phosphotransferase activity. When phosphate acceptors such as ethylene glycol concentrations were increased, the ratio of phosphate transfer to hydrolysis was also increased, demonstrating the presence of a transphosphorylation reaction where an acceptor can compete with water in the rate limiting step involving hydrolysis of a covalent phospho enzyme intermediate. Partition experiments carried out with two substrates, para-nitrophenyl phosphate and phenyl phosphate, revealed a constant product ratio of 1.7 for phosphotransfer to ethylene glycol versus hydrolysis, strongly supporting the existence of common covalent phospho enzyme intermediate. A constant ratio of K (cat)/K (m), 4.3 x 10(4), found at different ethylene glycol concentrations, also supported the idea that the rate limiting step was the hydrolysis of the phospho enzyme intermediate.

4-Nitrophenylphosphatase↗