Identification of calcium-dependent proteolytic activity in human polymorphonuclear leukocytes

Area of Science:

• Biochemistry
• Cell Biology
• Enzymology

Background:

• Human polymorphonuclear leukocytes (PMNs) are key immune cells involved in inflammatory processes.
• The presence and characterization of intracellular proteases within PMNs are crucial for understanding cellular functions and pathology.

Purpose of the Study:

• To identify and characterize calcium-dependent proteolytic activity in human polymorphonuclear leukocytes.
• To determine the optimal conditions and properties of this newly identified protease.

Main Methods:

• Extraction of proteolytic activity from human polymorphonuclear leukocytes.
• Assays to determine pH optimum, calcium concentration dependence, and inhibition by protease inhibitors.
• Gel filtration for molecular weight estimation.

Main Results:

• Calcium-dependent proteolytic activity was detected, with optimal activity at neutral pH (optimum 7.3).
• Maximal protease activation occurred at 190 µM free calcium, with half-maximal activation at 91 µM.
• The protease was strongly inhibited by aprotinin and phenylmethylsulfonyl fluoride (PMSF), and weakly by antipain, leupeptin, and o-phenanthroline.
• The activity was not modulated by calmodulin or trifluoperazine, suggesting it is not a calmodulin-dependent enzyme.
• Gel filtration indicated a molecular weight of approximately 74,100 daltons.

Conclusions:

• Human polymorphonuclear leukocytes possess a distinct calcium-dependent protease.
• This protease is active under neutral physiological conditions and its activity is modulated by specific calcium concentrations.
• The enzyme's inhibition profile and lack of calmodulin dependence distinguish it from other known proteases.