Identification of Multiple Metabolic Enzymes from Mice Cochleae Tissue Using a Novel Functional Proteomics Technology
Animal tissues; Beef; Biology; cancer; cancer metabolism; cell metabolism; Cochlea; draft; Drug therapy; E coli; Electrophoresis; Elution; Enzymatic activity; Enzyme assays; Enzyme metabolism; Enzymes; Feasibility studies; Functional analysis; Gel electrophoresis; Genes; Genomics; Hearing impairment; Kinases; Mass spectrometry; Mass spectroscopy; medicine; messenger-rna; metabolism; mice; NAD(P)H oxidase; NADH; Neurobiology; Neurosciences; Nicotinamide adenine dinucleotide; Noise; Otolaryngology; Oxidation-reduction reactions; Phosphatase; Protein expression; proteins; Proteomes; Proteomics; Science & Technology - Other Topics; Sciences: Comprehensive Works; Scientific imaging; Studies; Substance abuse treatment; Technology; United States--US
A new type of technology in proteomics was developed in order to separate a complex protein mixture and analyze protein functions systematically. The technology combines the ability of two-dimensional gel electrophoresis (2-DE) to separate proteins with a protein elution plate (PEP) to recover active proteins for functional analysis and mass spectrometry (MS)-based identification. In order to demonstrate the feasibility of this functional proteomics approach, NADH and NADPH-dependent oxidases, major redox enzyme families, were identified from mice cochlear tissue after a specific drug treatment. By comparing the enzymatic activity between mice that were treated with a drug and a control group significant changes were observed. Using MS, five NADH-dependent oxidases were identified that showed highly altered enzymatic activities due to the drug treatment. In essence, the PEP technology allows for a systematic analysis of a large enzyme family from a complex proteome, providing insights in understanding the mechanism of drug treatment.
Wang D L; Li H; Liang R Q; Bao J X
PLOS ONE
2015
2015-03
Journal Article
<a href="http://doi.org/10.1371/journal.pone.0121826" target="_blank" rel="noreferrer noopener">10.1371/journal.pone.0121826</a>
Cholesterol 7 alpha-hydroxylase activities from human and rat liver are modulated in vitro posttranslationally by phosphorylation/dephosphorylation
bile-acid synthesis; cloning; dietary-cholesterol; enzyme; Escherichia coli; expression; Gastroenterology & Hepatology; hmg-coa reductase; messenger-rna levels; Phosphatase; purification
Purified cholesterol 7 alpha-hydroxylases (C7 alpha H) from human and rat liver microsomes, and from transformed Escherichia coli expression systems, were incubated with 0.3 mmol/L [gamma-P-32] adenosine triphosphate (ATP) in the presence and absence of bacterial alkaline phosphatase (AP) or rabbit muscle adenosine 3',5'-cyclic monophosphate (cAMP) dependent protein kinase. The amounts of P-32 incorporation after separation of human and rat C7 alpha H proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were related to C7 alpha H catalytic activities (determined by a radioisotope incorporation method) and enzyme protein mass (determined by Western blotting and laser densitometry). Both human and rat C7 alpha H activities significantly decreased after dephosphorylation by AP (-57%--72%) and increased up to twofold with phosphorylation by rabbit muscle cAMP-dependent protein kinase. The increases in C7 alpha H activities were proportional to the amounts of cAMP-dependent protein kinase used, and were coupled to P-32 incorporation into the purified enzymes, Both the activation of C7 alpha H and the amounts of P-32 incorporation were time-dependent and reached a maximum after 1 hour of incubation with 5 U of cAMP-dependent protein kinase. In a second set of experiments, purified human and rat Liver C7 alpha H were dephosphorylated by 30-minute incubation with AP, followed by inactivation of the phosphatase by the inhibitor NaF, and rephosphorylation of C7 alpha H by 30-minute incubation with rabbit muscle cAMP-dependent protein kinase or bovine heart cAMP-independent protein kinase. Rephosphorylation of the dephosphorylated C7 alpha H proteins by cAMP-dependent protein kinase increased C7 alpha H catalytic activities up to fourfold, and the stimulation in catalytic activities paralleled the increases in P-32 incorporation into the purified enzymes. Bovine heart protein kinase was as potent as rabbit muscle cAMP-dependent protein kinase in stimulating catalytic activity and P-32 incorporation into the human C7 alpha H protein. Because the protein mass of these purified enzymes did not change, the short-term regulation or catalytic efficiency of C7 alpha H (activity per protein mass unit) is modulated, in vitro, posttranslationally by a phosphorylation/ dephosphorylation mechanism in both the human and the rat enzymes.
Nguyen L B; Shefer S; Salen G; Chiang J Y L; Patel M
Hepatology
1996
1996-12
Journal Article
<a href="http://doi.org/10.1053/jhep.1996.v24.pm0008938182" target="_blank" rel="noreferrer noopener">10.1053/jhep.1996.v24.pm0008938182</a>