Purification and characterization of calpain from human polymorphonuclear leukocytes.

Recent studies have demonstrated that a calcium-sensitive protease converts Ca2+/phospholipid-dependent protein kinase C to a Ca2+/phospholipid-independent form during the activation of human neutrophils. In this paper, the results of the purification and characterization of a calcium-dependent cytosolic protease from neutrophils is reported. Calcium-dependent protease has been purified 1062-fold from human neutrophils and behaves as a single species on native polyacrylamide gels. The protease is active in the neutral pH range with no observable activity amide gels. The protease is active in the neutral pH range with no observable activity at pH values greater than 8.0, has an absolute requirement for calcium for expression of activity with half-maximal activity observed at 12 microM free calcium, and has an apparent molecular weight of 110,000 based on gel filtration. The protease requires the presence of dithiothreitol for activity and is inhibited by sulfhydryl inhibitors, leupeptin, and antipain but not by serine protease inhibitors, pepstatin, or orthophenanthroline. The protease is also susceptible to inactivation by autoproteolysis. Based on the similarities of this calcium-dependent protease with calpains from a variety of other mammalian tissues, the protease isolated from human neutrophils appears to be a calpain I.

Calmodulin-dependency of human neutrophil phosphodiesterase.

A recent study has reported that the phosphodiesterases of human neutrophils are calmodulin-insensitive (Smolen and Geosits, Inflammation 8:193-199, 1984). In the present study, two forms of human neutrophil phosphodiesterase were separated by chromatography on DEAE-52. Peak I phosphodiesterase is activated 2.3-fold by calcium and calmodulin but is not stimulated by either calcium or calmodulin alone. Calmodulin-dependent activation of the phosphodiesterase is blocked by both 20 microM trifluoperazine and 20 microM W-7. Peak II is not stimulated by calmodulin. These findings suggest that calmodulin may play an important role in regulating alterations in cyclic nucleotide metabolism that accompany neutrophil activation.

Identification of calcium-dependent proteolytic activity in human polymorphonuclear leukocytes.

Calcium-dependent proteolytic activity has been identified in extracts of human polymorphonuclear leukocytes. The activity is most pronounced in the neutral pH range with a pH optimum of 7.3. Maximal activation of the protease occurs at a free calcium concentration of 190 microM; it is half maximal at 91 microM. This protease activity is strongly inhibited by aprotinin and phenylmethylsulfonyl fluoride (PMSF) and more weakly inhibited by antipain, leupeptin, and o-phenanthroline. The protease is not activated by calmodulin nor is it inhibited by the calmodulin antagonist trifluoperazine. Gel filtration suggests a molecular weight of 74,100 daltons.

Di- and trihydroxy analogs of chlorpromazine influence the central activity of adenylate cyclase and cyclic AMP phosphodiesterase.

We previously demonstrated that chlorpromazine (CPZ), 7,8 diOH-CPZ and 3,7,8 triOH-CPZ were potent inhibitors of central adenylate cyclase systems. The present studies were thus designed to observe whether further substitutions of dihydroxy groups on the CPZ molecule would influence adenylate cyclase in broken cell preparations of the rat frontal cortex. The inhibition of adenylate cyclase by 7,8 diOH-CPZ was further found to be noncompetitive with respect to ATP concentration. Raising the Ca++ concentration obliterated adenylate cyclase activation by either dopamine or 5'-guanylylimidodiphosphate (Gpp(NH)p) alone or in combination, while inhibition by 7,8 diOH-CPZ was overcome. The enzyme inhibition by 7,8 diOH-CPZ was not influenced, however, by increasing Mg++ concentration. Incubation of rat cortical homogenates with CPZ, 3,7,8 triOH-CPZ or 6,9 diOH-CPZ inhibited stimulation of adenylate cyclase activity while either 3,7- or 3,8 diOH-CPZ resulted in an enhancement of enzyme activity. In further study, using the high speed cortical supernatant, the high Km form of cyclic AMP phosphodiesterase and its activation by the Ca++-dependent, heat stable regulator protein were inhibited by CPZ, 6,9 dioxo-, 7,8 dioxo-, 3,7diOH-, 3,8 diOH-, 6,9 diOH-, 7,9 diOH- and 7,8 diOH-CPZs, but not by 3,7,8 triOH-CZP. The high affinity-low Km form of the enzyme was inhibited to a considerably lesser extent by these analogs. The studies reveal rather diverse and complicated actions of CPZ and its putative metabolites on central enzyme systems.