Hydrophobic affinity chromatography of human thrombin.

Hydrophobic affinity chromatography on p-chlorobenzylamido-agarose (p-CBA-agarose) was used to characterize various modified forms of human thrombin. Native alpha-thrombin bound tightly to the column and was eluted with either acetonitrile or 1,4-dioxane, while the catalytically inactive prethrombin 2 did not bind to the matrix. Site-specific chemical modification with pyridoxal 5'-phosphate resulted in the loss of at least 80% of fibrinogen clotting activity but did not influence the binding of thrombin to p-CBA agarose.

Identification of tissue kallikrein messenger RNA in human neutrophils.

Expression of tissue kallikrein in human neutrophils has been suggested by previous studies using enzymatic and immunochemical techniques. Secretion of this potent biological factor by neutrophils would be of marked significance in the inflammatory process. The present study utilized the polymerase chain reaction following reverse transcriptase generation of total neutrophils cDNA to demonstrate the presence of tissue kallikrein mRNA in the human neutrophils.

Expression of tissue kallikrein in normal and SV40-transfected human endometrial stromal cells.

The polymerase chain reaction with specific tissue kallikrein primers was utilized to demonstrate the presence of tissue kallikrein mRNA in human endometrial stromal cells. Enzymatic analysis measured with a specific tripeptide nitroanilide substrate demonstrated the presence of tissue kallikrein in the conditioned medium obtained from both normal stromal cells and stromal cells transfected with an origin-defective temperature-sensitive SV40 large T antigen. The transfected stromal cell supernatant exhibited approximately twice as much tissue kallikrein activity as normal stromal cells at 60-100% of cell confluence.

Factors in biological fluids affect gel filtration behavior of epidermal growth factor.

There have been conflicting reports on the presence of multiple forms of epidermal growth factor (EGF) in human saliva. The present study was initiated to study the gel filtration behavior of EGF in saliva and other biological fluids. In addition, studies were performed to determine if there were factors in saliva or other biological fluids that would influence the gel filtration behavior of EGF.

Affinity chromatography of platelets on immobilized thrombin: retention of catalytic activity by platelet-bound thrombin.

Radioactivity from I125-labeled human platelets was measured to estimate the extent of binding of platelet surface proteins to immobilized thrombin. 1-3% of the radioactivity was bound with 10-20% of this amount apparently irreversibly bound to the thrombin matrix. Site-specific chemical modification of thrombin with pyridoxal-5'-phosphate, N-bromosuccinimide or tetranitromethane resulted in a variable reduction of the amount of radiolabel bound.