Richard G. Woodbury

The structure of rat mast cell protease II at 1.9-A resolution.

The structure of rat mast cell protease II (RMCP II), a serine protease with chymotrypsin-like primary specificity, has been determined to a nominal resolution of 1.9 A by single isomorphous replacement, molecular replacement, and restrained crystallographic refinement to a final R-factor of 0.191. There are two independent molecules of RMCP II in the asymmetric unit of the crystal. The rms deviation from ideal bond lengths is 0.016 A and from ideal bond angles is 2.7 degrees. The overall structure of RMCP II is extremely similar to that of chymotrypsin, but the largest differences between the two structures are clustered around the active-site region in a manner which suggests that the unusual substrate specificity of RMCP II is due to these changes. Unlike chymotrypsin, RMCP II has a deep cleft around the active site. An insertion of three residues between residues 35 and 41 of chymotrypsin, combined with concerted changes in sequence and a deletion near residue 61, allows residues 35-41 of RMCP II to adopt a conformation not seen in any other serine protease. Additionally, the loss of the disulfide bridge between residues 191 and 220 of chymotrypsin leads to the formation of an additional substrate binding pocket that we propose to interact with the P3 side chain of bound substrate. RMCP II is a member of a homologous subclass of serine proteases that are expressed by mast cells, neutrophils, lymphocytes, and cytotoxic T-cells. Thus, the structure of RMCP II forms a basis for an explanation of the unusual properties of other members of this class.

Structure, specificity and localization of the serine proteases of connective tissue

It is the purpose of this review to describe several well characterized proteases present in tissues and cells which appear to be related to the pancreatic serine proteases. Much of the early work concerning the nature of tissue proteases was done during the late 19th century by German physiologists who were studying tissue autolysis [ 11. Hedin and Rowland [2] examined homogenates of various mammalian organs for proteolytic activity and observed that, with the exception of muscle, autolysis was greatest at acid pH. The term ‘cathepsin’ was introduced by Willsttitter and Bamann [3] in 1929 to describe proteolytic activity of tissues in the weakly acid pH range. In recent years, however, this term has sometimes been applied to include tissue proteases in general, such as cathepsin G which has an optimum activity at pH 8 and cathepsin E which is most active at pH 2.5. The rapidly growing literature dealing with tissue and cellular proteases has been recently reviewed by Barrett [4]. respiratory and gastrointestinal diseases [4]. Malignant tissues also show significant changes in proteolytic activity compared to normal, particularly that due to collagenases [ 111. As important as abnormal proteolytic activity in tissues may be in disease states, intraand extracellular proteases are necessary for maintaining normal tissue homeostasis. The steady-state concentration of proteins in cells and tissues is controlled by the rates of their synthesis and degradation [ 12,131 and although lysosomal proteases play a major role in intracellular protein degradation [ 141, there is considerable evidence that other proteases are of equal importance [15].

Construction of biosensors using a gold-binding polypeptide and a miniature integrated surface plasmon resonance sensor

Surface plasmon resonance (SPR) biosensors were constructed on miniature integrated sensors. Recognition elements were attached to the sensor surface using a gold-binding repeating polypeptide. Biosensors with fluorescyl groups attached to their surfaces were functional for at least 1 month of daily use with little decrease in response to the binding of an anti-fluorescyl monoclonal antibody. The coupling of protein A to the gold-binding polypeptide on the sensor surface enabled the biosensor to detect the binding of antibodies to the protein A and provided a sensor with convertible specificity. The system described herein provides a simple and rapid approach for the fabrication of highly specific, durable, portable and low cost SPR-based biosensors.

[45] Mast cell proteases

Publisher Summary This chapter presents a procedure for purification and assay of mast cell proteases. For Purification procedure mast cells are collected from Sprague-Dawley rats by washing the peritoneal cavities with 10 ml of ice-cold phosphate-buffered saline solution at pH 7.2. All subsequent steps are carried out at 4°C, including affinity-adsorption chromatography and adsorption to barium sulfate. During the purification of the mast cell protease, it is important to maintain the solutions at relatively high ionic strength to prevent the enzyme from adsorbing to surfaces. The assay measures chymotrypsin-like esterase activity. The procedure involves mixing of substrate and diluted enzyme appended with the buffer. The absorption change at 256 nm is recorded for about 5 rain. An enzyme unit is defined as an amount of enzyme activity that results in the hydrolysis of 1 μmol of substrate per minute at pH 7.8, 25°C.

Fundamental system for biosensor characterization: application to surface plasmon resonance (SPR)

The aim of the described research is to develop a general system for characterizing and developing signal transduction systems for microbiosensors. The approach that we are using is applicable to signal transduction systems based on surface plasmon resonance, chemiluminescence, fluorescence, mass as well as other phenomena. The specific goal of our approach is to develop a general system that will allow for the systematic characterization of the effects of the affinity of the sensor specificity element for the target analyte, the effect of analyte mass on signal size and the general performance of the sensor system with respect to sensitivity and selectivity. At the same, time this system should allow for the characterization of the distribution of biospecificity elements on the sensor surface. We chose the anti-fluorescein monoclonal antibody approach for this development system, since the antigen fluorescein can be attached to many different molecules and organisms through free amine groups via reaction with fluorescein isothiocyanate. Also, well characterized monoclonal antibodies with a broad range of Kd values are available. We also describe rapid procedures for generating proteins for use in biosensor applications.