Abstract
Cyclic inositol phosphohydrolase is a phosphodiesterase that cleaves the cyclic bond of cyclic inositol monophosphate. In 1990, Ross et al. (Ross, T. S., Tait, J. F., and Majerus, P. W. (1990) Science 248, 605-607) purified this enzyme from human placenta and reported that cyclic inositol phosphohydrolase is identical to annexin III. Independent confirmation of this finding has not been provided. The relative distribution of annexin III and cyclic inositol phosphohydrolase activity in rat kidney and spleen indicated that annexin III can be dissociated from cyclic inositol phosphohydrolase activity. Rat spleen contains large quantities of annexin III, but has very little cyclic inositol phosphohydrolase activity. In contrast, rat kidney, one of the richest sources of cyclic inositol phosphohydrolase activity, possesses very little (immunohistochemistry) or no (Western blot) annexin III. Similar to cytosol of human placenta, cytosol of guinea pig kidney contains both annexin III and cyclic inositol phosphohydrolase. On SDS-gel electrophoresis, guinea pig kidney annexin III has a slightly different mobility than the human placental annexin III. Human placental annexin III co-migrates with cyclic inositol phosphohydrolase on ion exchange chromatography, while guinea pig kidney annexin III is clearly dissociated from cyclic inositol phosphohydrolase on ion exchange chromatography. Both guinea pig kidney annexin III and human placental annexin III pellet with the addition of calcium and centrifugation, while cyclic inositol phosphohydrolase activity in both of these tissues remains in the supernatant. Our studies clearly show that cyclic inositol phosphohydrolase and annexin III are two different proteins.
Highlights
Lipocortin III has been referred to as placental anticoagulant protein III [16], calcimedin 35-␣ [17], and annexin III [18, 19]
Bathophenanthroline, inositol, cyclic inositol monophosphate, and NADϩ were obtained from Sigma; phenazine was from Aldrich; ferric chloride was from Mallinckrodt; rabbit polyclonal antibody to rat spleen annexin III was a gift from Dr John Dedman, University of Cincinnati, Cincinnati, OH; and rabbit polyclonal antibody raised against human neutrophil annexin III was a gift from Dr Joel Ernst, University of California at San Francisco
Distribution of Annexin III and cyclic inositol phosphohydrolase (cIPH)—Western blot with annexin III antibody to rat spleen shows a strong signal with rat spleen but no detectable band is observed in rat kidney and medulla (Fig. 1A)
Summary
Lipocortin III has been referred to as placental anticoagulant protein III [16], calcimedin 35-␣ [17], and annexin III [18, 19]. Annexin III is one of the 12 members of the annexin family of proteins that have been identified so far. Proteins belonging to this family have the following two characteristics: (a) Conserved 70-amino acid domain repeated either four or eight times in the overall structure and (b) ability to bind to phospholipids in a calcium-dependent manner. During the course of investigating cyclic inositol phosphate metabolism, we reinvestigated the putative identity of cIPH as annexin III [13]. Phospholipase C hydrolysis of phosphoinositide results in the generation of both cyclic and noncyclic inositol phosphates [1]. Dawson and co-workers [10] identified a phosphodiesterase that hydrolyzes the cyclic bond of cyclic inositol monophosphate (cIP) to yield inositol 1-phos-
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