Calcium-dependent properties of peptidylarginine deiminase from rabbit skeletal muscle.

Abstract

Peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, required Ca2+ as an essential co factor and the half-maximal activity was attained at 40~60/-iM Ca2+. Other divalent cations were practically inactive except for Sr2+, which was about 50% as active as Ca2+ when tested at 10mM. However, Sr2+ at less than the concentration of 100/wr had little or no activity. The direct Ca2+-binding for the enzymeshowed a sigmoidal curve with a transition midpoint of about 1 10 /iM, indicating that the binding is cooperative. Analysis of Hill plots of the data revealed that the enzyme binds 3mol of Ca2+/mol of protein with an apparent dissociation constant of 1 10 /iM. A conformational change upon Ca2+-binding was also described for the enzymeusing UV-difference spectra. The alteration could be attributed to an increased exposure of the aromatic residues to a more aqueous environment, as has been described for Ca2 +binding proteins such as calmodulin. Phosphatidylserine enhanced the reaction velocity and concomitantly reduced the Ca2 +-requirement for the enzyme. These effects were stimulated by the addition of diacylglycerol. Diacylglycerol alone had little or no effect. On the other hand, calmodulin had no effect on the enzymatic activity over a wide range of Ca2+ concentrations. These suggest that the activity and Ca2 +-sensitivity of peptidylarginine deiminase is increased at the cell membrane.

Expression and activity of citrullinating peptidylarginine deiminase enzymes in monocytes and macrophages

“…The cytosolic and nucleoplasmic Ca 2+ concentration under normal physiological conditions is much too low (about 100fold) for PAD activity. 45 Raising the cytosolic and nucleoplasmic calcium concentration by treatment with either ionomycin or thapsigargin induced activation of PAD and subsequent citrullination of intracellular proteins. As shown in fig 6, monocytes and macrophages had different banding patterns of citrullinated proteins.…”

“…It is known, however, that calcium ions, and possibly other factors, are required for activity of the PAD enzymes. The threshold Ca 2+ concentration for PAD activity is about 10 25 mol/l (Nijenhuis S, Nijmegen, The Netherlands, personal communication) 45 ; the cytosolic calcium concentration in normal cells is much lower (10 27 mol/l). The cytosolic calcium concentration can be artificially raised by addition of various agents like ionomycin or thapsigargin.…”

Protein Arginine Deiminases and Associated Citrullination: Physiological Functions and Diseases Associated with Dysregulation

“…Under physiological conditions, PADs are usually inactive because calcium concentration is maintained at very low levels in the cell (10 −8 to 10 −6 M) [19, 50]. Normal functioning PADs become activated during certain events, such as apoptosis and terminal epidermal differentiation, where calcium levels are above the physiological concentration [19, 51].…”

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“…Normally, PAD enzymes are present intracellularly as inactive enzymes [11]. Calcium ions are required for activation of the PAD enzymes, but the intracellular calcium concentration in normal cells (10 -7 mol/l) is much lower than the threshold calcium concentration for PAD activity (approximately 10 -5 mol/l; our unpublished observations and data from Takahara and coworkers [12]). During cell death, however, the integrity of the plasma membrane is lost [13,14], causing influx of calcium from the extracellular space and subsequent activation of intracellular PAD [11,15-18].…”

“…During cell death, however, the integrity of the plasma membrane is lost [13,14], causing influx of calcium from the extracellular space and subsequent activation of intracellular PAD [11,15-18]. Alternatively, PAD enzymes may leak out of the dying cells, become activated (the extracellular calcium concentration is approximately 10 -3 mol/l, which is sufficient for PAD activity [12]), and cause citrullination of extracellular proteins.…”