Cathepsin B in human leukocytes.
We have applied a method using the fluorigenic substrate benzoloxycarbonyl-Arg-Arg-amido-4-methylcoumarin to measure cathepsin B, a thiol proteinase, in homogenates of human leukocytes. Data like pH optimum, stability, influence of thiol groups and effects of thiol proteinase inhibitors, lack of binding to Concanavalin A and lack of contribution to the fluorescence by other cathepsins indicate that cathepsin B is the enzyme measured. Although the activity of the enzyme was linear with respect to time at all protein concentrations measured, there was an acceptable 10% deviation of the enzyme activity from linearity as a function of protein concentration. The enzyme in the homogenate was stable at 0 degrees C but was rapidly inactivated at 50 degrees C and above pH 6.5-7. Very limited activation on the one hand and variable inhibition on the other was seen by reagents containing thiol groups and thiol proteinase inhibitors respectively. Latency (60% of the enzyme activity) indicates a probable subcellular lysosomal localization. There is no affinity towards the lectin Concanavalin A and the Km value was around 1 mmol/l. Normal enzyme activity values in leukocyte homogenates were determined.
Den Tandt
W R
WR
Faculty of Medicine, University of Antwerp, Antwerpen-Wilrijk, Belgium.
Scharpé
S
S
D000328:Adult; D002401:Cathepsin B / Q000037:antagonists & inhibitors / Q000097:blood*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007962:Leukocytes / Q000201:enzymology*; D008297:Male; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity
D015853:Cysteine Proteinase Inhibitors; D015842:Serine Proteinase Inhibitors; D002401:Cathepsin B