ABSTRACT
Imbalanced protease activity has long been recognized in the progression of disease states such as cancer and inflammation. Serpins, the largest family of endogenous protease inhibitors, target a wide variety of serine and cysteine proteases and play a role in a number of physiological and pathological states. The expression profiles of 20 serpins and 105 serine and cysteine proteases were determined across a panel of normal and diseased human tissues. In general, expression of serpins was highly restricted in both normal and diseased tissues, suggesting defined physiological roles for these protease inhibitors. A high correlation in expression for a particular serpin-protease pair in healthy tissues was often predictive of a biological interaction. The most striking finding was the dramatic change observed in the regulation of expression between proteases and their cognate inhibitors in diseased tissues. The loss of regulated serpin-protease matched expression may underlie the imbalanced protease activity observed in pathological states.
Subject(s)
Cysteine Endopeptidases/genetics , Gene Expression Profiling/methods , Gene Expression Regulation, Enzymologic , Polymerase Chain Reaction/methods , Serine Endopeptidases/genetics , Serpins/genetics , Amino Acid Sequence , Cell Line , Cell Line, Transformed , Cysteine Endopeptidases/metabolism , Disease Progression , Female , Gene Expression Regulation , Humans , Male , Molecular Sequence Data , Neoplasms/genetics , Neoplasms/metabolism , Serine Endopeptidases/metabolism , Serpins/metabolism , Species SpecificityABSTRACT
Adenine phosphoribosyltransferase plays a role in purine salvage by catalyzing the direct conversion of adenine to adenosine monophosphate. The involvement of the purine salvage pathway in tumor proliferation and angiogenesis makes adenine phosphoribosyltransferase a potential target for oncology drug discovery. We have expressed and characterized recombinant, N-terminally His-tagged human adenine phosphoribosyltransferase. Two assay formats were assessed for use in a high throughput screen: a spectrophotometric-based enzyme-coupled assay system and a radiometric ionic capture scintillation proximity bead assay format. Ultimately, the scintillation proximity assay format was chosen because of automated screening compatibility limitations of the coupled assay. We describe here the biochemical characterization of adenine phosphoribosyltransferase and the development of a robust, homogeneous, 384-well assay suitable for high throughput screening.
Subject(s)
Adenine Phosphoribosyltransferase/metabolism , Scintillation Counting/methods , Adenine/metabolism , Adenine Phosphoribosyltransferase/antagonists & inhibitors , Adenylate Kinase/metabolism , Humans , L-Lactate Dehydrogenase/metabolism , NAD/metabolism , Pyruvate Kinase/metabolism , Recombinant Proteins/metabolism , TritiumABSTRACT
Inositol-specific PLCs comprise a family of enzymes that utilize phosphoinositide substrates, e.g., PIP(2), to generate intracellular second messengers for the regulation of cellular responses. In the past, monitoring this reaction has been difficult due to the need for radiolabeled substrates, separation of the reaction products by organic-phase extraction, and finally radiometric measurements of the segregated products. In this report, we have studied the enzymatic characteristics of two novel PLCs that were derived from functional genomic analyses using a phospholipid-modified solid scintillating support. This method allows for the hydrophobic capture of the [(3)H]phosphoinositide substrate on a well defined scintillation surface and the homogenous measurement of the enzymatic hydrolysis of the substrate by proximity effects. Our results show that the assay format is robust and well suited for this class of lipid-metabolizing enzymes.
