More than two-thirds of all known G-protein coupled receptors are known to modulate the function of adenylate cyclase resulting in altered levels of cAMP. In turn, cAMP fluctuations transform agonist binding events into physiological changes in cell behavior. The advent of nonradioactive, homogeneous methods of measuring intracellular cAMP has enabled the rapid growth of drug discovery and research applications for these GPCR targets. In this chapter, we describe a nonradioactive, chemiluminescent cAMP detection method using enzyme fragment complementation technology to detect a wide range of GPCR modulators which is also suitable for high-throughput screening.

The IQ Technology has been developed to serve as a homogeneous, universal detection platform for HTS of kinases and phosphatases. The technology is a direct, noncompetitive assay format that does not require antibodies or radioactive reagents to measure phosphorylation state. Fluorophore-labeled peptides are used as enzyme substrates, and kinase or phosphatase activity is quantitated by direct measurement of the phosphorylation state of the substrate. Phosphorylation is measured by the change in fluorescence intensity that occurs when a proprietary iron-containing compound binds specifically to phosphoryl groups on peptides. This change in observed fluorescence is proportional to the extent of phosphorylation of the fluorophore-labeled peptide. The technology provides a universal method that can be used with any peptide sequence and is insensitive to high concentrations of ATP. Inhibition at the ATP-binding site versus the phosphorylation site can be differentiated and compound selectivity identified using the same detection method as in the primary screen. The technology has been tested against a large number of detergents, organics, and other reagents found in reaction mixtures, and the detection method eliminates common issues associated with fluorescent and chromogenic compounds. The technology has been formatted for 96-, 384-, and 1,536-well microplate formats, and a representative Z' value of 0.7 was obtained. IC(50) values generated using this platform correlate with previously reported values, and screening of a small compound library was performed to evaluate the assay further.

The objective of this study was to demonstrate that weakened pelvic floor support of the lower genitourinary tract in women with stress urinary incontinence (SUI) is due to increased collagenolysis. When fibrillar collagen is degraded, pyridinium (PYD) crosslinks are released and excreted in the urine. Degradation of collagen also results in peptide fragments of various lengths which are excreted in the urine. Degradation of mature fibrillar collagen and collagen which has not been crosslinked can be assessed independently by measurement of both PYD and collagen‐derived peptides in the urine.

IQ® Technology, a homogeneous, universal-detection platform, originally designed for high-throughput screening (HTS) of kinases and phosphatases, has now been applied to protease screening. Representative enzymes from the major classes of proteases have been assayed in the IQ® format. Enzyme activity and compound inhibition data are presented for such enzymes as Trypsin, Matrix Metalloproteinase 3 (MMP-3) and Calpain 1. The technology has been tested in 96- to 384- to 1536-well microplate formats and is universally suited for automated screening. IQ® Technology is a direct, noncompetitive assay that does not require antibodies or radioisotopes. Fluorophore-labeled peptides are used as enzyme substrates. Kinase or phosphatase activity is quantified by direct measurement of the phosphorylation state of the substrate. For protease activity, cleavage is quantified with a peptide substrate containing a phospho-residue distal to the fluorphore. Cleavage of the substrate liberates the fluorphore-labeled terminus from the terminus containing the phospho-residue. Protease activity is measured by the change in fluorescence intensity that occurs when a proprietary compound binds specifically to phosphoryl groups on peptides and quenches the fluorescence. Iq® Technology can be used with any peptide sequence and is insensitive to high concentrations of ATP and substrate. The IQ® Technology has been validated against a large number of detergents, organics, and other reagents found in reaction mixtures and has been optimized for HTS applications exhibiting representative Z' values of 0.7. (JALA 2004;9:171-6)

L'invention concerne des elastases associees a l'incontinence urinaire d'effort. Elle concerne aussi des procedes determinant si une femme est atteinte d'incontinence urinaire d'effort, et des methodes de traitement ou de prevention de ce type d'incontinence.

Matrix degrading enzymes are implicated in several disease processes such as abdominal aortic aneurysms and emphysema, however, monitoring proteolytic activity in a single assay is not well-established. Numerous assays have been developed to measure matrix degrading enzymes, which use artificial substrates or substrates derived from natural substrate protein. We have recently developed an assay for elastolytic activity based on the detection of primary amines, using trinitrobenzene sulfonic acid (TNBSA), following the digestion of succinylated elastin. The assay is also versatile enough to allow the detection of other proteases through the use of succinylated substrate specific for given protease.In order to improve the sensitivity and versatility of the assay we have refined the buffer conditions (PBS pH 7.2/1 mM CaCl2) to provide a 60 % increase in sensitivity to elastolytic activity and also formulated a substrate mixture containing succinylated elastin, collagen and gelatin. The combination of a substrate mixture and an optimum buffer will allow a spectrum of enzymes to be detected in a single reaction, providing a more complete picture of total proteolytic activity in a biological sample. This assay may also provide a tool to use proteolytic activity as a marker to monitor pathologic conditions involving matrix turn-over.

A spectrophotometric assay using succinylated gelatin as substrate is described for measuring the catalytic activity of gelatinases. The assay is based on measurement of primary amines exposed as a result of hydrolysis of the substrate by gelatinases. Comparison of hydrolysis by matrix metalloproteinase (MMP) 1, 2, 3, 7, 9 indicated that succinylated gelatin was primarily digested by MMP-2 and -9. The assay is rapid (<60 min), specific, suitable for measuring gelatinolytic activity of enzymes and high volume screening of MMP-2 and -9 inhibitors. Sensitivity of the assay is comparable to that of gelatin zymography, under similar experimental conditions. Thus, the assay combines ease and rapidity of assays based on synthetic peptide substrates with specificity of the gelatin zymography technique.

Background. Loss of elastin in the aortic wall is an early event in abdominal aortic aneurysm (AAA). An imbalance in the protease-antiprotease system is proposed to be one of the factors that promote connective tissue destruction. We hypothesize that plasma from AAA patients will have a reduced inhibitory capacity in comparison to normal controls.Materials and methods. Using an assay we developed, plasma (10 mu l), collected from AAA patients (n = 14) and normal controls (n = 13), was added to the elastase inhibition assay containing succinylated elastin substrate. The reaction was initiated with 13.9 units porcine pancreatic elastase (PPE). Elastase activity in the presence and absence of plasma was compared. Plasma elastase was also determined using the Merck PMN-elasease kit.Results. The relative activity of exogenous elastase (%) in the presence of AAA plasma (n = 14, mean age 73.4 years +/- 1.7 SEM) was 42.59% +/- 4.3 SEM, while that in the presence of control plasma (n = 13, mean age 73.9 years +/- 2.1 SEM) was 10.23% +/- 2.1 SEM (P < 0.0001). Analysis of plasma elastase (mu g/L) indicated that there was no significant difference between normal (n = 9, 207.33 mu g/L +/- 58.67 SEM) and AAA (n = 9, 145.34 mu g/L +/- 29.54 SEM) (P = 0.359).Conclusion. There is a significant reduction in the plasma inhibitory capacity of elastase in AAA patients in comparison to normal controls, though plasma elastase level was not significantly different. The data presented here give experimental evidence to the protease-antiprotease imbalance in AAA patient plasma and may lead to the development of a measurable parameter to monitor AAA. (C) 1999 Academic Press.

Uncontrolled tissue destruction by protease release plays a role in some pathological conditions, and an imbalance in the protease–antiprotease system is hypothesized to be an important contributing factor. Elastase, one of the matrix-degrading enzymes found in plasma, is closely associated with the elastic tissue destruction found in conditions such as abdominal aortic aneurysm (1), emphysema (2), pancreatitis (3)(4), and inflammatory conditions (5). A possible factor in the progression of these conditions may be an imbalance in the protease–antiprotease system. Excess production of protease or loss of inhibition is a factor in the alteration of the protease–antiprotease imbalance. To investigate the elastase inhibitory potential of plasma we have developed a method of evaluating the ability of plasma to inhibit purified elastase. This assay is a modification of a technique recently developed by us, which detects the primary amines that are exposed because of the enzymatic degradation of elastin substrate (6). The inhibitory effect of plasma is determined by introducing 10 μL of plasma into a reaction mixture containing succinylated elastin, and the reaction is initiated by the addition of a known amount of elastase. The inhibitory effect of plasma is evaluated by comparing the activity of elastase in the presence and absence of plasma. The inhibition assay contained 100 μg of succinylated elastin, 10 μL of plasma, and 50 μL of porcine pancreatic elastase (PPE; EC 3.4.21.36; 1 g/L made up in 50 mmol/L sodium borate buffer, pH 8.5; 297 U/mg; Calbiochem), and was made up to a final volume of 150 μL with 50 mmol/L sodium borate buffer, pH 8.5, in a 96-well microplate. [One U of enzyme will hydrolyze 1 μmol of N-Ac-(Ala)3-methyl ester per minute at 25 °C, pH 8.5.] The assay was carried out at 37 °C for 30 min. Then 50 …

We have developed a rapid, versatile, and sensitive elastase assay that is based on the measurement of primary amines that are exposed due to enzymatic degradation of proteins, using succinylated elastin as the substrate for elastase. After incubation with elastase the degree of digestion is determined with trinitrobenzene sulfonic acid. The assay is rapid and sensitive, detecting elastase down to 1 ng/ml, and is linear up to enzyme concentrations of 10 microg/ml. The assay is carried out in microtiter plate wells and therefore offers the potential for assaying numerous samples of small volume. The use of succinylated elastin shows specificity for elastase over the control protease, trypsin. This assay is also versatile because it can be applied to samples such as cell culture supernatants, blood plasma, tissue biopsies, and tissue homogenates.

A phage contig of 400 kb that extends from the brain-specific promoter at the 5′-end of the human dystrophin gene, through the muscle-specific promoter over 100 kb further downstream, and across most of intron 1 has been assembled. To achieve this, a yeast artificial chromosome (YAC) subcloning approach was used. Total DNA from a yeast strain containing a 400-kb YAC from the dystrophin gene was cloned using a λ phage vector containing RNA polymerase promoters flanking the cloning sites. Phage containing human DNA inserts were then ordered into an overlapping set by hybridization of end-specific RNA probes from individual clones back to plaque lifts of gridded phage subclones. The clones generated will be useful as reagents for detailed structural and functional analyses of this region of the dystrophin gene.

Classical Western blotting is considered a qualitative method; however, it can be used for quantitative analysis of proteins pro- vided that specific controls are included. Using SuperSignal ® West Dura Chemiluminescent Substrate we qualitatively and quan- titatively detected changes in the regulation of IkBα and p53 in A431 cells treated with EGF (and doxorubicin or cisplatin) by comparing to a standard curve generated using pure protein. ELISAs for both proteins enabled confirmation of the Western data. Additionally, using an imager provided greater linear range than film and, therefore, greater flexibility. SuperSignal West Sub- strates are therefore useful for qualitative and quantitative Western blotting when the proper controls are used.