Purification of three forms of lipocortin from bovine lung

Experimental conditions are described for simultaneous purification of three forms of lipocortin (lipocortin I, lipocortin II and lipocortin-85) from bovine lung. The procedure yields milligram quantities of all three lipocortins. Using antisera against lipocortin I and lipocortin II, purified proteins show no cross contaminations. All forms of lipocortin exhibit equal potency as in vitro bovine pancreatic phospholipase A2 inhibitors. Protein kinase C catalyzes the in vivo incorporation of about 1.0, 0.7 and 0.4 mole of phosphate per mole of lipocortin I (p35), lipocortin II (p36) and lipocortin-85 (p36 oligomer) respectively. The phosphorylation is specific for protein kinase C and is dependent on the presence of both calcium and phospholipids. While lipocortin I is phosphorylated on threonine residues, lipocortin II and lipocortin-85 are phosphorylated on serine residues.     

Lipocortins are major substrates for protein kinase C in extracts of human neutrophils.

We have identified two major proteins in human neutrophils that are phosphorylated in vitro by protein kinase C (PKC) as lipocortins III and a fragment of a lipocortin-like 68-kDa protein. In electroporated cells, the 68-kDa protein was phosphorylated during stimulation of the cells with either FMLP or PMA. Lipocortins are of interest because of their Ca2(+)- and phospholipid-dependent actin binding properties and ability to inhibit phospholipase A2. Two crude fractions of enzymes and proteins exposed to [gamma-32]PATP in the presence of Ca2+, Mg2+, phosphatidylserine and 1,2-oleoyl-acetyl-rac-glycerol were analyzed by gel electrophoresis and autoradiography. A number of proteins in a detergent-free fraction, including proteins at 36 and 32 kDa, were phosphorylated in the presence of these cofactors. In contrast, only two major proteins (35 and 32 kDa) were phosphorylated in a detergent-extracted fraction. Phosphorylation of the 36, 35, and 32 kDa proteins required the presence of Ca2+, Mg2+, and phosphatidylserine in our soluble fraction and detergent extract, indicating PKC-dependent phosphorylation. The 32-kDa protein phosphorylated in both the soluble fraction and detergent extract was identified as lipocortin III by immunoprecipitation with a cross-reactive antibody that recognized lipocortin I and comparison of cyanogen bromide (CNBr) cleavage patterns of this protein with a lipocortin III standard. The 68-kDa protein was identified as a lipocortin VI-like protein by immunoprecipitation with anti-calelectrin. Additionally, the CNBr cleavage pattern of the 68-kDa protein was similar to that of the 36-kDa protein phosphorylated in our soluble fraction. Autoradiograms of the 68- and 36-kDa fragments immunoprecipitated from our soluble fraction with anticalelectrin and cleaved with CNBr showed that both of these proteins were phosphorylated in this sample. Because phosphorylation is known to change the functional characteristics of the lipocortins, the potential exists to link PKC and lipocortins in neutrophils to regulation of granulemembrane interactions or mediation of inflammation.     

Differential tissue expression of three 35-kDa annexin calcium-dependent phospholipid-binding proteins

We have purified three 35-kDa calcium- and phospholipid-binding proteins from rat liver. These three calcimedins bind to phosphatidylserine in a calcium-dependent manner and have been termed 35 alpha, 35 beta, and 35 gamma based on their relative charge as determined by isoelectric focusing. Purification of the three 35-kDa calcimedins is achieved by phenyl-Sepharose, ion exchange, and gel filtration chromatography. Antibody was produced against the annexin consensus peptide, Lys-Ala-Met-Lys-Gly-Leu-Gly-Thr-Asp-Glu, which was derived from the sequence of several Ca2+/phospholipid-binding proteins including calpactin, lipocortin, endonexin II, 67-kDa calelectrin, lymphocyte 68-kDa protein, and protein II. Recognition of each 35-kDa calcimedin by anticonsensus sequence antibody places them in this protein family. Antibodies against each 35-kDa calcimedin were raised and purified by antigen-affinity chromatography. Each antibody is monospecific for the respective 35-kDa calcimedin. Immunological cross-reactivity defines 35 alpha, 35 beta, and 35 gamma as lipocortins III, IV, and V, respectively. Surveys by immunoblot analysis using these monospecific antibodies demonstrate a markedly different tissue expression pattern for each 35-kDa calcimedin. Furthermore, the levels of 35 alpha, 35 beta, and 35 gamma are differentially regulated in maturing rat ovary and uterus. Each calcimedin has been localized by indirect immunofluorescence within specific cell types. These results support the concept that mediation of the intracellular calcium signal can occur via multiple pathways through several related yet independent mediator proteins.     

Correlation of gene and protein structure of rat and human lipocortin I

Lipocortins (annexins) are a family of calcium-dependent phospholipid-binding proteins with phospholipase A2 inhibitory activity. The characteristic primary structure of members of this family consists of a core structure of four or eight repeated domains, which have been implicated in calcium-dependent phospholipid binding. In two lipocortins (I and II) a short amino-terminal sequence distinct from the core structure has potential regulatory functions which are dependent on its phosphorylation state. We have isolated the rat and the human lipocortin I genes and found that they both consist of 13 exons with a striking conservation of their exon-intron structure and their promoter and amino acid sequences. Both lipocortin I genes are at least 19 kbp in length with exons ranging from 57 to 123 bp interrupted by introns as large as 5 kbp. Each of the four repeat units of lipocortin I are encoded by two consecutive exons while individual exons code for the highly conserved putative calcium-binding domains. The promoter sequences in the rat and in human genes are highly conserved and contain nucleotide sequences characterized as enhancer sequences in other genes. The structure of the lipocortin I gene lends support to the hypothesis that the lipocortin genes arose by a duplication of a single domain.     

Chromosomal localization of the human annexin III (ANX3) gene

The annexins or lipocortins are a new family of calcium-dependent phospholipid-binding proteins. Annexin III has been previously identified as inositol 1,2-cyclic phosphate 2-phosphohydrolase (EC 3.1.4.36), an enzyme of inositol phosphate metabolism, and also as placental anticoagulant protein III, lipocortin III, calcimedin 35-alpha, and an abundant neutrophil cytoplasmic protein. In this study, the gene (ANX3) encoding annexin III was localized to human chromosome 4 at band q21 (q13-q22) by (1) polymerase chain reaction analysis of a human-rodent hybrid cell panel, confirmed by genomic Southern blot analysis of the same panel with a cDNA probe and (2) in situ hybridization with a cDNA probe.     

Cloning and expression of cDNA for human endonexin II, a Ca2+ and phospholipid binding protein

Endonexin II is a member of the family of Ca2+-dependent phospholipid binding proteins known as annexins. We cloned human endonexin II cDNA and expressed it in Escherichia coli. The apparent size and Ca2+-dependent phospholipid binding properties of purified recombinant endonexin II were indistinguishable from those of the placental protein. A single mRNA of approximately 1.6 kilobase pairs was found to be expressed in human cell lines and placenta and was in close agreement with the length of the cDNA clone (1.59 kilobase pairs). The cDNA predicted a 320-amino acid protein with a sequence that was in agreement with the previously determined partial amino acid sequence of endonexin II isolated from placenta. Endonexin II contained 58, 46, and 43% sequence identity to protein II, calpactin I (p36, protein I), and lipocortin I (p35), respectively. The partial sequence of bovine endonexin I was aligned with the sequence of endonexin II to give 63% sequence identity. Like these other proteins, endonexin II had a 4-fold internal repeat of approximately 70 residues preceded by an amino-terminal domain lacking similarity to the repeated region. It also had significant sequence identity with 67-kDa calelectrin (p68), a protein with an 8-fold internal repeat. Comparing the amino-terminal domains of these four proteins of known sequence revealed that, in general, only endonexin II and protein II had significant sequence identity (29%). Endonexin II was not phosphorylated by Ca2+/phospholipid-dependent enzyme (protein kinase C) even though it contained a threonine at a position analogous to the protein kinase C phosphorylation sites of lipocortin I, calpactin I, and protein II.     

Differential expression of annexins I and II in normal and malignant human mammary epithelial cells

Abstract. Collagen-binding proteins ( CBPs ) of rat mammary tumors are identical to Ca²⁺-binding annexins [49]. We have now isolated a protein of 38 kDa from the human mammary tumor cell line ALAB by collagen type I affinity chromatography as well as by extraction of calcium-binding proteins. The 38-kDa band of both preparations was identified as annexin II (calpactin I) by its reaction with an annexin II-specific monoclonal antibody in Western blot analysis. Annexin I (lipocortin I) was not detectable in these cells. Two other human cell lines, the SV40-transformed cell line SV3 and cell line HBL-100, both established from normal mammary glands, were also positive for annexin II and negative for annexin I.
In vivo expression of annexins was investigated by immunohistological staining of normal and malignant human mammary tissue. The annexin II-specific mAb reacted with normal and tumor parenchyme whereas the annexin I-specific mAb reacted with acini and ductal myoepithelium of the normal mammary gland but showed no reaction with tumor tissue. Immunolocalization studies also showed annexin II expression in both normal and tumor stroma while only tumor stromal cells were found to be reactive with the antibody against annexin I. The differential expression of annexins in normal and malignant human mammary tissue suggests special functions of these proteins in the mammary gland.     

Calcium-independent phospholipases from guinea pig digestive tract as probes to study the mechanism of lipocortin

Two calcium-independent phospholipases isolated from guinea pig pancreas (lipase Ia, 37 kDa) and from guinea pig intestine (phospholipase B, 97 kDa) have been used to probe the mechanism of phospholipase inhibition by lipocortin. In the presence of calcium, both enzymes were inhibited by lipocortin I in a manner very similar to the inhibition of pig pancreas phospholipase A2. By using phospholipases that lack a requirement for calcium, we have for the first time been able to dissociate enzymatic activity from the role of calcium in the inhibitory process. It was found that lipocortin was without effect against phospholipase A1 and phospholipase B in the absence of calcium, under which conditions the inhibitory protein is unable to interact with anionic phospholipid surfaces. The same behavior toward phospholipase A1 was observed with two other related proteins, endonexin II or lipocortin V, and p68/67-kDa calelectrin or lipocortin VI. Together with the observation that lipocortins are active only in the presence of limited amounts of substrate, these data give further support to the "surface depletion model" of lipocortin inhibition, rather than to a mechanism involving a direct interaction between enzyme and inhibitor.     

Five distinct calcium and phospholipid binding proteins share homology with lipocortin I

We have purified two 35-kDa proteins from rat peritoneal lavages that inhibit phospholipase A2 activity. Both are calcium/phospholipid-dependent membrane binding proteins and share similar structural and biochemical properties with lipocortins I and II. By sequence analysis we confirmed that they are lipocortin-related, and we refer to the two inhibitors as lipocortins III and V. Using partial sequence information obtained from the purified rat proteins, full length cDNA clones for both proteins and for their human counterparts were isolated. As with lipocortins I and II, the amino acid sequences of lipocortins III and V which were deduced from the cDNA clones are highly conserved, sharing 50% identity with other family members. Related proteins were also purified from bovine intestinal mucosa and characterized by peptide mapping, sequence, and immunological analyses. In addition to lipocortins III and V the bovine preparation contained a third 35-kDa inhibitor and a 68-kDa inhibitor, extending the number of known lipocortins to six distinct proteins. While the various lipocortins are structurally similar, distinct differences in their cellular distribution indicate specialized roles for the individual proteins.     

Possible involvement of a lipocortin in the initiation of DNA synthesis by human endothelial cells.

This work focused on three themes. First, evidence was obtained for the presence of proteins of 34, 35, 32, and 69 kDa immunologically related to lipocortins I, II, V, and VI, respectively, in human umbilical vein endothelial (HUVE) cells. The 69-kDa protein (p69), but not proteins related to lipocortins I, II, and V, exhibited an increased phosphorylation after exposure of cells to basic fibroblast growth factor (bFGF) and phorbol ester PMA. Second, treatment of HUVE cell particulate fractions with EGTA and hydrophobic affinity chromatography in combination with conventional techniques provided extracts rich in p69 and purified p69. p69 from control cells and extracts from control, bFGF-treated, and PMA-treated cells were found to possess anti-phospholipase A2 (PLA2) activity of lipocortin. In contrast, a striking reverse effect occurred when extracts were obtained from cells exposed to bFGF plus PMA. Third, the combination of bFGF and PMA induced a stimulated PLA2-catalyzed release of arachidonic acid in HUVE cells. This arachidonate production was shown to be involved in the decision of cells to enter into DNA synthesis. Taken together, the present results suggest that phosphorylation of p69 is causally involved in the control of commitment to growth in HUVE cells by acting as a coupling mechanism between surface stimuli and arachidonate pathways.     

Cross-linking of lipocortin I and enhancement of its Ca2+ sensitivity by tissue transglutaminase

The stimulation of human epidermoid carcinoma A431 cells with the calcium ionophore A23187 resulted in the formation of high-molecular-weight lipocortins I, having apparent molecular weights of 75 kDa and 160 kDa as detected with specific anti-lipocortin I antibody. These immunoreactive proteins were identified to be covalently cross-linked multimers of lipocortin I, since essentially the same cross-linked multimers were observed when purified lipocortin I was incubated with tissue transglutaminase (TGase) in vitro. Classical amine substrates for TGase, such as dansylcadaverine and putrescine, were also incorporated stoichiometrically into lipocortin I. Cross-linking or amine incorporation was not observed with lipocortin II. Des 1-26 lipocortin I did not serve as a substrate for TGase, indicating that the N-terminal region of lipocortin I plays an important role in the formation of lipocortin I multimers. The cross-linking of lipocortin I by TGase resulted in a remarkable enhancement of calcium sensitivity for phospholipid binding; i.e., the free calcium concentration required for the cross-linked lipocortin I to attain 50% maximal binding to phosphatidylserine vesicles was as little as 3 microM, while that required for intact monomeric lipocortin I was 20 microM.     

Lipocortin I and lipocortin II inhibit phosphoinositide- and polyphosphoinositide-specific phospholipase C. The effect results from interaction with the substrates

Lipocortins I and II, known to inhibit phospholipase A2, have been purified from bovine lung and tested with respect to their ability to affect the enzymatic activities of phosphoinositide- and polyphosphoinositide-specific phospholipase C from human platelets, rat liver cytosol or rat brain membranes. At 0.67 microM, both lipocortins led to complete inhibition of phospholipase C activity with either phosphatidylinositol or phosphatidylinositol 4,5-bisphosphate as substrate. The inhibition could be overcome by increasing the substrate concentration. Ultracentrifugation studies with lipocortin II showed a direct interaction between phosphatidylinositol and the lipocortin, indicating that the lipocortins inhibit phospholipase C not directly but by interacting with the substrate. In experiments with plasma membranes from [3H]inositol-labeled HL-60 cells, lipocortin II did not affect PI-specific phospholipase C activity in the absence or presence of calcium plus or minus GTP-gamma-S.     

Isolation of a calcium-sensitive, 35,000-dalton microfilament- and liposome-binding protein from ascites tumor cell microvilli: identification as monomeric calpactin

Microvilli isolated from the MAT-C1 ascites subline of the 13762 rat mammary adenocarcinoma contain a major calcium-sensitive microfilament-binding protein, AMV-p35 (ascites microvillar p35). Association of AMV-p35 with microfilament cores during Triton X-100 extraction of the microvilli is half-maximal at 0.1-0.2 mM calcium. The protein, which comprises 6% of the total microvillar protein, can be isolated from microfilament cores prepared in the presence of calcium by extraction with EGTA and purification by ion-exchange chromatography. Alternatively, the protein can be isolated from Triton extracts of microvilli prepared in the absence of calcium by precipitation with calcium, solubilization of the precipitate with EGTA, and chromatography on an ion-exchange column. AMV-p35 binds to phosphatidylserine liposomes and F-actin with half-maximal calcium concentrations of about 10 microM and 0.2 mM, respectively. Treatment of AMV-p35 with chymotrypsin yields a 33,000-dalton fragment, behavior similar to the tyrosine kinase substrates calpactins I and II and lipocortins I and II. Immunoblot analyses using antibodies directed against calpactin I, lipocortin I, and lipocortin II showed strong reactivity of AMV-p35 with anti-calpactin I and anti-lipocortin II, but little reactivity toward anti-lipocortin I. The close relationship between AMV-p35 and calpactin I was verified by amino acid sequence analyses of peptides isolated from cyanogen bromide digests of AMV-p35. By gel filtration and velocity sedimentation analyses purified AMV-p35 is a 35,000-dalton monomer. Moreover, AMV-p35 extracted directly from microvilli in Triton/EGTA also behaves as a 35,000-dalton menomer. These findings indicate that AMV-p35 is closely related to the pp60src kinase substrate calpactin I (p36). However, AMV-p35 occurs in the microvilli as a monomer rather than as the heterotetrameric calpactin found in several other cell types.     

Purification, characterization, and partial sequence analysis of 32-kDa calcimedin from chicken gizzard

Calcimedin is a group of proteins which has a binding ability to several hydrophobic matrices or cellular membrane fractions in the presence of Ca2+. Although the molecular properties were partially clarified, the physiological functions of calcimedins have not been clearly defined. In this study, we describe the isolation and characterization of 32-kDa calcimedin from chicken gizzard. Both structural and functional studies establish that 32-kDa calcimedin is a member of the calpactin/lipocortin family. The 32-kDa calcimedin displays phospholipase A2 inhibitory activity, Ca2(+)-dependent F-actin binding activity, and phospholipid binding activity similar to those of calpactins/lipocortins. Antiendonexin II antibody recognized 32-kDa calcimedin. However, antibodies against calpactin I (lipocortin II), calpactin II (lipocortin I), 35-kDa calcimedin, and 67-kDa calcimedin did not cross-react with 32-kDa calcimedin. One-dimensional peptide maps of the 32-kDa calcimedin and the 35-kDa calcimedin are different, confirming that they are distinct proteins. By comparing the sequence of 32-kDa calcimedin with the predicted sequence of endonexin II, we concluded that the primary structure of the 32-kDa protein is highly conserved. In particular, the sequences AMKGMGTDDEXEIXL, GMGTDEEEIL, VLTEILASR, and ILTSR conform to the endonexin consensus sequence, which is characteristic of the calpactin/lipocortin family.     

Changes in annexin (lipocortin) content in human amnion and chorion at parturition.

Arachidonic acid is mobilized from fetal membrane phospholipids at parturition leading to increased production of oxytocic prostaglandins which may initiate or maintain myometrial contractions. Phospholipid mobilization requires activation of phospholipase A2 or C, both of which require calcium for activity. The annexins (lipocortins) are a superfamily of proteins which bind to calcium and phospholipids and thereby may alter phospholipase activity through two mechanisms: modulation of intracellular free Ca2+ concentrations or regulation of the accessibility of phospholipids to hydrolyzing enzymes. Using Western immunoblotting with monospecific polyclonal antibodies, annexins I-VI were identified in human amnion and chorion/decidua at term in tissues obtained from patients in labor or not in labor. Each annexin was present in two distinct pools: a pool which only associated with the membrane in the presence of calcium (calcium-dependent pool) and a calcium-independent pool that remained membrane bound in the presence of calcium chelators. Annexin I was present as two species, resolving at 36 kDa and 68 kDa. The total concentration of annexin I in both amnion and chorion/decidua was significantly decreased with labor, while the total concentration of annexin V in chorion significantly increased with labor. The size of individual pools of annexins also changed with labor: the calcium-dependent pool of annexins I and II in both amnion and chorion significantly decreased; the calcium-dependent pool of annexin V increased in chorion; and calcium-independent pools of annexin I in amnion and annexins I, II, and V in chorion significantly decreased with labor. The decrease in total annexin I concentration with labor in amnion reflects a substantial decrease (80-90%) in the pool tightly bound to the membrane in a calcium-independent manner. This striking change distinguishes annexin I as a potential candidate inhibitor which is specifically downregulated at parturition, potentially leading to increased access of phospholipases to substrate phospholipids and increased prostaglandin production at labor.     

Differential Subcellular Distribution of p36 (the Heavy Chain of Calpactin I) and Other Annexins in the Adrenal Medulla

The annexins are a group of highly related Ca2(+)-dependent membrane-binding proteins that are present in a wide variety of cells and tissues. We have examined the subcellular distribution of five members of the annexin family in the adrenal medulla. Bovine adrenal medullary tissue was homogenized in buffers containing EGTA and fractionated on sucrose gradients. p36 (the large subunit of calpactin I) was found to be predominantly membrane associated, with approximately 20% present in fractions enriched in chromaffin granules. In contrast, lipocortin I was localized primarily to the cytosol, with only a small proportion found in plasma membrane-containing fractions. Like lipocortin I, endonexin I was found to be present almost entirely in the soluble fractions. The 67-kDa calelectrin was localized primarily to the plasma membrane fractions, with a small amount present in the chromaffin granule and cytoplasmic fractions. Synexin was present in both membranous and cytoplasmic fractions. p36 appeared to be a peripherally associated granule membrane protein in that it was dissociated from the membrane by addition of base and it partitioned with the aqueous phase when granule membranes were treated with Triton X-114. Antiserum against p10 (the small subunit of calpactin I) reacted with a protein of 19 kDa that is specifically localized in chromaffin granule membrane fractions. The differences in subcellular distributions of the annexins suggest that these proteins have distinct cellular functions. The finding that p36 is associated with chromaffin granule and plasma membrane fractions provides further support for a possible role of calpactin in exocytosis.     

A 32 kDa lipocortin from human mononuclear cells appears to be identical with the placental inhibitor of blood coagulation.

A 32 kDa protein isolated from human mononuclear cells is a member of the lipocortin family, a new group of Ca2+-dependent lipid-binding proteins thought to be involved in the regulation of phospholipase A2, in exocytosis and in membrane-cytoskeleton interactions. Purification of this protein was based on its ability to associate with membrane phospholipids in a Ca2+-dependent manner and its capacity to inhibit purified phospholipase A2 from pig pancreas. Using immunological detection, we show that it is present in various cells involved in the inflammatory and coagulation processes. We present extensive amino acid data that strongly suggest that this protein is identical with a recently described inhibitor of blood coagulation, with endonexin II and with lipocortin V. Sequence alignment with other known proteins show a significant degree of homology with lipocortins I and II, the substrates of the epidermal-growth-factor receptor tyrosine kinase and the oncogene pp60src tyrosine kinase respectively, and with protein II. The possible physiological role of this 32 kDa lipocortin is discussed.     

Inhibition of phospholipase A2 by protein I

The 36 kDa substrate of several tyrosine protein kinases has been shown to exist in monomeric and oligomeric (362102) forms. Partial sequence data has suggested that the oligomer, referred to as protein I, is homologous to a group of phospholipase A2 inhibitory proteins, collectively called lipocortins. In the present communication we demonstrate that protein I inhibits bovine pancreas phospholipase A2 with similar potency to that of lipocortin. Approximately 44 pmol protein I was required to produce 50% inhibition of 7.2 pmol of phospholipase A2. Inhibition of phospholipase A2 activity by calmodulin, S-100, calregulin, parvalbumin, troponin-C, or CAB-48 was not observed. These results indicate that protein I is a potent and specific inhibitor of phospholipase A2 activity, and thus shares functional homology with the lipocortin proteins. We therefore propose that this protein be named lipocortin-85.     

Primary structure of the human, membrane-associated Ca2+-binding protein p68 a novel member of a protein family.

cDNA clones encoding human 'p68', a membrane-associated Ca2+-binding protein, were isolated from a lambda gt11 expression library of the human T-leukaemia cell line J6, by using a rabbit antiserum against denatured purified lymphocyte p68, and from a liver cDNA library by using 32P-labelled p68 cDNA fragments. The amino acid sequence of p68, deduced from the sequences of overlapping cDNA clones, is described. The results show that p68 is closely related to a family of proteins which includes intracellular substrates of the EGF receptor and pp60src tyrosine kinases. The p68 amino acid sequence is internally repetitive, being constructed from eight repeats of varying lengths, each of which contains a highly conserved sequence. Multiple copies of the latter sequence are also present in the related proteins p36, lipocortin I and protein II. We discuss how the common structural features of these proteins may reflect common functions and, furthermore, how the eight repeat structure of p68 may have evolved. The sequences of independent cDNAs suggest that alternatively-spliced mRNAs could encode different p68 protein species. This suggestion is consistent with the observation that purified p68 migrates as a closely-spaced doublet when analysed by SDS-PAGE.     

Identification and characterization of phospholipase A2 inhibitory proteins in human mononuclear cells

Calcium-dependent phospholipid binding and phospholipase A2 inhibitory proteins were isolated from human mononuclear cells. Lipocortins I and II were present whereas lipocortin IV (endonexin I) was not. The other proteins were purified to homogeneity and shown to have molecular masses of 35, 36, 32 and 73 kDa. The 36-kDa and 73-kDa proteins are related, the smaller appears to be part of the larger. The 73-kDa protein is related to the 67-kDa calelectrin and to lipocortin VI; the 32-kDa protein is different from endonexin I but related to chromobindin 7 and to lipocortin V. The 35-kDa protein has been identified by tryptic peptide sequencing as lipocortin III. All these proteins inhibit phospholipase A2 activity in vitro and the three smaller ones inhibit the [3H]arachidonic acid release from prelabelled monocytes induced by the calcium ionophore A23187 in a dose-dependent manner.