Michael R. Carpenter

Amino acid sequence of Streptomyces griseus protease B, a major component of pronase

Summary Streptomyces griseus Protease B is a close homologue of Protease A, another serine protease isolated from Pronase. Homology based on identity of residues in the two enzymes is 61%. Extensive identical sequences are found in the vicinities of histidine-57, aspartic acid-102, serine-195, the two disulfide bridges, the NH2-terminal and COOH-terminal ends as well as in the region of the presumed substrate binding sites. However, certain regions of the Protease B sequence are markedly different and include a heptapeptide insertion between residues 88 and 89 and a tetrapeptide deletion between residues 129 and 136 when compared with Protease A. These differences are probably responsible for the remarkable stability of Protease B in concentrated solutions of urea and guanidine hydrochloride.

Miniaturized protein microsequencer with PTH amino acid identification by capillary electrophoresis I. An argon pressurized delivery system for adsorptive and covalent sequencing.

The design of a simple, highly miniaturized instrument for manual microsequence analysis of proteins and peptides is described. The reaction chamber is made of fused silica capillary tubing with all reagents and solvents necessary for coupling and cleavage delivered via two valves and a syringe-based dispenser. Only two pressure regulators are required. A section of the flow-through reaction chamber is heated by thermoelectric modules to control the sequencing reaction temperature. Conversion of the extracted amino acid product to the more stable phenylthiohydantoin (PTH) form is performed off-line so that it may be dissolved in 1 mu1 buffer for identification. Approximately 0.1% of this PTH product is analyzed by micellar electrokinetic capillary chromatography (MECC) with thermo-optical absorbance detection (TOAD), providing femtomole detection of the phenylthiohydantoin amino acids. Preliminary results at the 50 pmol level for both adsorptive and covalent sequencing methods are presented.

Co-purification of proteases with basic fibroblast growth factor (FGF)

Acidic and basic fibroblast growth factors (FGFs) are proteins of 16-18 kDa. Other forms of 25-30 kDa related to this growth factor family have recently been described. All these components bind tightly to heparin-Sepharose, a property that allows the purification of several FGF-related proteins. During the purification of acidic and basic FGFs from bovine pituitary glands, we detected the presence of 28-30 kDa components that are immunoreactive against anti-basic FGF antisera. However, microsequencing analysis revealed that the 28-30 kDa components are lysosomal proteases that co-elute with basic FGF from heparin-Sepharose columns. The involvement of these proteases in the etiology of microheterogenous forms of FGFs and/or release of FGFs from the extracellular matrix is discussed.

High Sensitivity Analysis of PTH Amino Acids

Capillary electrophoresis and laser-based photothermal detection are used to analyze minute amounts of PTH amino acids. This technology is demonstrated for analysis of manual Edman degradation reactions. This technology is also used to analyze the products generated by a highly miniaturized automated protein sequencer.

A Xylanase from Schizophyllum commune

Having previously described (1) the production and isolation of cellulases and xylanases from a white rot fungus, Schizophyllum commune, we now report some details of the structure of one of the xylanases.