Purification and characterization of a 32-kDa protein that localizes to the sea urchin extraembryonic matrix, the hyaline layer.

We have purified a 32 kilodalton (kDa) protein that localized with isolated, intact hyaline layers prepared from 1-h-old embryos. The protein appeared not to bind calcium and was not quantitatively released from 1-h-old embryos in the absence of Ca2+ and Mg2+. Using polyclonal antiserum prepared against the 32-kDa protein, the antigen was detected throughout embryonic development. By the hatched blastula stage of development, the 32-kDa protein was replaced by a species of slightly smaller molecular mass. Quantitative determination indicated that the 32-kDa protein accounted for approximately 6% of the total protein present in the sea urchin egg. This result is suggestive of a structural role for the 32-kDa protein that is required throughout embryonic development, although perhaps in a modified form from the hatched blastula stage on.     

Identification and characterization of gelatin-cleavage activities in the apically located extracellular matrix of the sea urchin embryo.

We have identified and partially characterized several gelatinase activities associated with the sea urchin extraembryonic matrix, the hyaline layer. A previously identified 41-kDa collagenase/gelatinase activity was generally not found to be associated with isolated hyaline layers but was dissociated from the surface of 1-h-old embryos in the absence of Ca2+ and Mg2+. While hyaline layers, freshly prepared from 1-h-old embryos, were devoid of any associated gelatinase activities, upon storage at 4 degrees C for 4 days, a number of gelatin-cleavage activities appeared. Comparative analysis of these activities with the 41-kDa collagenase/gelatinase revealed that all species were inhibited by ethylenediamine tetraacetic acid but were refractory to inhibition with the serine protease inhibitors, phenylmethyl sulfonyl fluoride and benzamidine. In contrast, the largely Zn2+ specific chelator 1,10-phenanthroline had markedly different effects on the gelatinase activities. While several of the storage-induced, hyaline-layer-associated gelatinase activities were inhibited, the 41-kDa collagenase/gelatinase was refractory to inhibition as was a second gelatinase species with an apparent molecular mass of 45 kDa. We also examined the effects of a series of divalent metal ions on the gelatin-cleavage activities. In both qualitative and quantitative assays, Ca2+ was the most effective activator while Mn2+, Cu2+, Cd2+, and Zn2+ were all inhibitory. In contrast, Mg2+ had a minimal inhibitory effect on storage-induced gelatinase activities but significantly inhibited the 41-kDa collagenase/gelatinase. These results identify several distinct gelatin-cleavage activities associated with the sea urchin extraembryonic hyaline layer and point to diversity in the biochemical nature of these species.     

Calcium–protein interactions in the extracellular environment: Calcium binding,activation, and immunolocalization of a collagenase/gelatinase activity expressed in the sea urchin embryo

We have purified and characterized a collagenase/gelatinase activity expressed during sea urchin embryonic development. The native molecular mass was determined to be 160 kDa, while gelatin substrate gel zymography revealed an active species of 41 kDa, suggesting that the native enzyme is a tetramer of active subunits. Incubation in the presence of EGTA resulted in nearly complete loss of activity and this effect could be reversed by calcium. Calcium-induced reactivation appeared to be cooperative and occurred with an apparent kd value of 3.7 mM. Two modes of calcium binding to the 41-kDa subunit were detected; up to 80 moles of calcium bound with a kd value of 0.5 mM, while an additional 120 moles bound with a kd value of 5 mM. Amino acid analysis revealed a carboxy plus carboxyamide content of 24.3 mol/100 mol, indicating the availability of substantial numbers of weak Ca²⁺-binding sites. Calcium binding did not result in either secondary or quaternary structural changes in the collagenase/gelatinase, suggesting that Ca²⁺ may facilitate activation through directly mediating the binding of substrate to the enzyme. The collagenase/gelatinase activity was detected in blastocoelic fluid and in the hyalin fraction dissociated from 1-h-old embryos. Immunolocalization studies revealed two storage compartments in the egg; cortical granules and small granules/vesicles dispersed throughout the cytoplasm. After fertilization, the antigen was detected in both the apical and basal extracellular matrices, the hyaline layer, and basal lamina, respectively. J. Cell. Biochem. 71:546–558, 1998. © 1998 Wiley-Liss, Inc.     

Hyalin, a sea urchin extraembryonic matrix protein: relationship between calcium binding and hyalin gelation.

The protein hyalin, a major component of the sea urchin extraembryonic hyaline layer, was previously shown to undergo a Ca(2+)-induced self-association into large aggregates (gelation). This reaction represented a major step in assembly of the layer. In the experiments reported here, digestion with trypsin resulted in a rapid dissociation of hyalin into a mixture of peptides which retained the capacity to bind Ca2+. However, unlike intact hyalin, none of these peptides associated into large aggregates (gelation) in the presence of Ca2+, Mg2+, and NaCl. Loss of the ability to undergo gelation was not accompanied by any significant change in the content of acidic plus amide amino acid residues. Decreasing the pH to 5.6 resulted in a loss of 25% of hyalin's Ca(2+)-binding capacity but had no effect on the ability of the protein to undergo gelation. Peptide fragments were only partially effective at inhibiting hyalin gelation. Clearly, not all the Ca(2+)-binding sites were required for hyalin gelation and Ca2+ binding alone was insufficient to drive this reaction. In addition, hyalin appeared to possess two classes of protein-protein interaction domains, one of which was essential for gelation.     

The apical lamina of the sea urchin embryo: Major glycoproteins associated with the hyaline layer

The hyaline layer (HL) surrounding the sea urchin blastula appears to dissolve in 1 M glycine. However, after this treatment, there persists over the surfaces of the blastomeres a layer of material, referred to here as the apical lamina (AL), that sloughs off as an adhesive convoluted bag upon gradual dissociation of the embryo. Isolated hyaline layers, referred to as HL-AL complexes, were analyzed by urea-SDS-polyacrylamide gel electrophoresis. A major protein of the HL-AL complex, hyalin, bands or precipitates in the stacking gel. Two other major proteins, both strongly PAS positive, migrate with apparent molecular weights of 175K and 145K daltons. As with intact embryos, the glycine wash removes the hyalin protein from the isolated HL-AL complex, leaving the undissolved AL which consists primarily of the 175K- and 145K-dalton proteins. The embryo's own perivitelline-localized cortical granule peroxidase heavily radioiodinates the proteins of the HL-AL complex, further verifying their apical, extracellular location. Unlike hyalin, the AL proteins do not precipitate with calcium ions. Compared to the entire HL-AL complex, the AL contains a greater percentage of carbohydrate. No sialic acid is associated with the HL-AL complex, but the AL contains some sulfate. In contrast to a published report based on ultrastructural staining, no biochemical evidence was found in this study for the presence of collagen or significant glycosaminoglycan within the HL-AL complex. No developmental differences were observed in AL proteins from 1-hr-old embryos compared to those from blastulae. However, there is evidence suggesting heterogeneity and developmental differences in hyalin. The possible organization of hyalin and the AL proteins into separate layers surrounding the embryo is discussed. The influence of the AL proteins in morphogenesis and cell adhesion is considered, and hypothetical roles attributed to the HL and hyalin are critically questioned.     

Sea urchin morphogenesis and cell-hyalin adhesion are perturbed by a monoclonal antibody specific for hyalin

We have generated and characterized a monoclonal antibody (McA Tg-HYL) that recognizes sea urchin hyalin as evidenced by immunofluorescence staining of the hyaline layer (HL) and immunoblot staining of the hyalin protein band. On immunoblots of HL extracts only the hyalin protein reacted with McA Tg-HYL. Immunoprecipitates of radioactive proteins from embryos incubated with [35S]methionine yielded radioactive hyalin and 190, 140 and 105 x 10(3) Mr proteins associated with hyalin. McA Tg-HYL was generated against Tripneustes gratilla embryos but reacts with hyalin from the distantly related sea urchin species, Colobocentrotus atratus, Strongylocentrotus purpuratus, Arbacia punctulata, Lytechinus variegatus and Lytechinus pictus. Developing embryos of the above-mentioned six species were treated with McA Tg-HYL and did not gastrulate or form arms. Observations of treated embryos revealed areas of separation of the hyaline layer from the underlying embryonic cells, suggesting that McA Tg-HYL was interfering with binding of the cells to the HL. Using the centrifugation-based adhesion assay of McClay et al. (Proc. natn. Acad. Sci. U.S.A. 78, 4975-4979, 1981), Fab' fragments of McA Tg-HYL were found to inhibit cell-hyalin binding. McA Tg-HYL did not inhibit hyalin gelation in vitro or the reaggregation of dissociated blastula cells. We postulate that McA Tg-HYL recognizes an evolutionarily conserved hyalin domain involved in cell-hyalin binding and required for normal epithelial folding.     

Relationship between p62 and p56, two proteins of the mammalian cortical granule envelope, and hyalin, the major component of the echinoderm hyaline layer, in hamsters

Mammalian cortical granules contain two polypeptides (p62 and p56) that are incorporated into the cortical granule envelope after fertilization and function in cleavage of the zygote and the preimplantation blastomeres. Since the echinoderm hyaline layer and mammalian cortical granule envelope are analogous, and since the hyaline layer protein, hyalin, functions in early echinoderm embryogenesis, this study was done to determine whether p62 and p56 and/or other components of the mammalian cortical granule envelope are related to hyalin. A polyclonal antibody (IL2) against purified S. purpuratus hyalin was shown by confocal scanning laser microscopy to bind to hamster cortical granules and to the cortical granule envelope of fertilized hamster oocytes and preimplantation embryos up to the blastocyst stage. In immunoblots, IL2 bound only to 62- and 56-kDa cortical granule proteins that were incorporated into the cortical granule envelope after fertilization. IL2 binding antigens appeared to be resynthesized by preimplantation embryos starting at the 2-cell stage of development. In vivo treatment of 2-cell-stage hamster embryos with IL2 inhibited blastomere cleavage, but treatment of morulae did not inhibit blastocyst implantation. These results support the idea that the mammalian cortical granule envelope proteins, p62/p56, share a common antigenic epitope(s) with echinoderm hyalin, and that p62/p56, like hyalin, play a role in early embryogenesis.     

Hyalin release during normal sea urchin development and its replacement after removal at fertilization

All stages of sea urchin embryos through pluteus can be dissociated to their component cells through the use of a 1 M solution of glycine containing EDTA, and a similar glycine solution can be used to prevent the formation of the fertilization membrane and remove the hyaline layer material released at fertilization. The protein hyalin, which makes up the bulk of the hyaline layer, can be recovered from these glycine solutions by calcium addition and quantitative agreement was found between the hyalin released at fertilization and the hyalin present at all later developmental stages. However, embryos stripped of their hyalin at fertilization often develop normally, which is unexpected in view of the apparent involvement of the hyaline layer in developmental mechanics. Such embryos are found to have regenerated an appreciable fraction of the hyalin removed at fertilization and this regeneration occurs at the time of blastulation. Thus the regeneration appears to be stimulated by hyaline layer removal at fertilization, but it does not take place until several hours later, at the time this layer has been postulated to play a role in development.     

A hyaline layer protein that becomes localized to the oral ectoderm and foregut of sea urchin embryos

An antigen is described which is a marker for the oral ectoderm and foregut of the sea urchin embryo. In Lytechinus variegatus, the antigen is first detectable by immunofluorescence on the surface of fertilized eggs, and remains globally distributed through the early stages of gastrulation. Thereafter the antigen is localized to the oral ectoderm and foregut, coincident with the morphogenesis of these regions. The antigen is a large, detergent-insoluble, filamentous glycoprotein associated with the tips of the microvilli in the hyaline layer. This glycoprotein is present in two forms, a approximately 350-kDa form that is maternally synthesized and a much larger form which is synthesized at late gastrula stage as a 350-kDa precursor before becoming modified and assembled into the hyaline layer. The timing of synthesis of the zygotic form of the molecule correlates precisely with the localized expression of the antigen. The antigen copurifies with intact hyaline layers and cosediments with hyalin in the presence of calcium, suggesting that it is a structural component of the hyaline layer.     

Exogenous hyalin and sea urchin gastrulation, Part II: hyalin, an interspecies cell adhesion molecule.

The 330 kDa fibrillar glycoprotein hyalin is a well known component of the sea urchin embryo extracellular hyaline layer. Only recently, the main component of hyalin, the hyalin repeat domain, has been identified in organisms as widely divergent as bacteria and humans using the GenBank database and therefore its possible function has garnered a great deal of interest. In the sea urchin, hyalin serves as an adhesive substrate in the developing embryo and we have recently shown that exogenously added purified hyalin from Strongylocentrotus purpuratus embryos blocks a model cellular interaction in these embryos, archenteron elongation/attachment to the blastocoel roof. It is important to demonstrate the generality of this result by observing if hyalin from one species of sea urchin blocks archenteron elongation/attachment in another species. Here we show in three repeated experiments, with 30 replicate samples for each condition, that the same concentration of S. purpuratus hyalin (57 microg/ml) that blocked the interaction in living S. purpuratus embryos blocked the same interaction in living Lytechinus pictus embryos. These results correspond with the known crossreactivity of antibody against S. purpuratus hyalin with L. pictus hyalin. We propose that hyalin-hyalin receptor binding may mediate this adhesive interaction. The use of a microplate assay that allows precise quantification of developmental effects should help facilitate identification of the function of hyalin in organisms as divergent as bacteria and humans.     

Microplate assay for quantifying developmental morphologies: effects of exogenous hyalin on sea urchin gastrulation

It is often difficult to determine the effects of various substances on the development of the sea urchin embryo due to the lack of appropriate quantitative microassays. Here, a microplate assay has been developed for quantitatively evaluating the effects of substances, such as hyalin, on living sea urchin embryos. Hyalin (330 kDa) is a major constituent of the sea urchin hyaline layer, an extracellular matrix that develops 20 min postinsemination. Function of the hyaline layer and its major constituent, is the adhesion of cells during morphogenesis. Using wide-mouthed pipette tips, 25 microl of 24-h Strongylocentrotus purpuratus embryos were transferred to each well of a 96-well polystyrene flat-bottom microplate yielding about 12 embryos per well. Specific concentrations of purified hyalin diluted in low calcium seawater were added to the wells containing the embryos, which were then incubated for 24 h at 15 degree C. The hyalin-treated and control samples were observed live and after fixation with 10% formaldehyde using a Zeiss Axiolab photomicroscope. The small number of embryos in each well allowed quantification of the developmental effects of the added media. Specific archenteron morphologies-attached, unattached, no invagination and exogastrula-were scored and a dose-dependent response curve was generated. Hyalin at high concentrations blocked invagination. At low concentrations, it inhibited archenteron elongation/attachment to the blastocoel roof. While many studies have implicated hyalin in a variety of interactions during morphogenesis, we are not aware of any past studies that have quantitatively examined the effects of exogenous hyalin on specific gastrulation events in whole embryos.     

Isolation and characterization of calcium binding glycoproteins of cardiac sarcolemmal vesicles

Two major Ca2(+)-binding glycoproteins Mr 120,000 and 100,000 were isolated from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid -solubilized bovine heart sarcolemma membrane. Peroxidase-conjugated concanavalin A and wheat germ agglutinin lectins bind strongly to the isolated 120- and 100-kDa glycoproteins. Treatment with endoglycosidase F resulted in conversion of the 120-kDa glycoprotein to a form migrating at about 97 kDa. Treatment of the 100-kDa band with endoglycosidase F produced form of about 80 kDa. Endoglycosidase H digestion removes only 5% of the mass of both glycoproteins. the carbohydrate structure of both glycoproteins, is therefore, predicted to be at least 75% complex structure and 25% high mannose or hybrid structure. The 120- and 100-kDa glycoproteins are the major Ca2(+)-binding proteins in the sarcolemma membranes. Intact and endoglycosidase-treated glycoproteins bind 45Ca2+ as analyzed by a 45Ca2+ overlay technique. Using polyclonal antibodies, the 120- and 100-kDa glycoproteins were identified in muscle plasma membranes (ventricles, atria, and uterus smooth muscle). They were, however, not present in non-muscle tissues such as pancreas, liver, and kidney. The 120- and 100-kDa glycoproteins appear to be homologous molecules as judged by their similar V8 protease peptide maps, cross-reactivity with polyclonal antibody, and other physicochemical properties.     

Roles for Ca2+, Mg2+ and NaCl in modulating the self-association reaction of hyalin, a major protein component of the sea-urchin extraembryonic hyaline layer.

The self-association reaction of hyalin, a major protein component of the sea-urchin extraembryonic hyaline layer, was examined. Concentrations of Ca2+ below 1 mM had little effect on the hyalin gelation reaction, but higher concentrations of the cation induced protein aggregation. Quantitative aggregate formation required a Ca2+ concentration in excess of 10 mM. This reaction was modulated by both NaCl and Mg2+. The effectiveness of Ca2+ in inducing hyalin gelation was markedly enhanced in the presence of 500 mM-NaCl, the concentration found in sea water. Similarly, 20 mM-Mg2+ also enhanced Ca2+-induced hyalin gelation. Neither NaCl nor Mg2+ alone induced hyalin gelation. Concentrations of Ca2+ as low as 1 mM effectively protected hyalin from tryptic digestion both in the presence and in the absence of 500 mM-NaCl. The latter result suggested that, although higher concentrations of Ca2+ were required to induce the hyalin gelation reaction, lower concentrations of the cation could mediate a protein-protein interaction in an NaCl-independent fashion. These results identify the parameters that modulate hyalin self-association, a reaction that is essential for hyaline-layer assembly around the developing sea-urchin embryo.     

Two major antigens of heart sarcolemma are Ca2(+)-binding glycoproteins that copurify with the dihydropyridine receptor.

Ca2+ binding has been studied in isolated heart sarcolemmal membranes using the 45Ca overlay technique. 45Ca bound to two sarcolemmal polypeptides of 125 kDa and 97 kDa in preparations from dog, rabbit, cow and pig. During fractionation on DEAE ion-exchange and wheat-germ lectin affinity columns, the two Ca2(+)-binding polypeptides copurified with the dihydropyridine receptor associated with the voltage gated Ca2+ channel. These polypeptides were the major proteins in the isolated fraction as judged by silver staining in SDS-PAGE. Antisera raised against purified dog heart, sarcolemma indicated that the 125 and 97 kDa polypeptides were highly antigenic components of this membrane. The antisera cross-reacted with similar polypeptides in cardiac sarcolemmal preparations from rabbit, cow and pig, but not sarcoplasmic reticulum membranes. Purified antibodies against the 125 kDa polypeptide did not cross-react with the 97 kDa polypeptide, while antibodies against the 97 kDa polypeptide did not cross-react with the 125 kDa polypeptide. Both the 125 kDa and 97 kDa polypeptides bound wheat-germ lectin, suggesting both were glycoproteins. It is unlikely that these Ca2+ binding glycoproteins represent subunits of the dihydropyridine receptor-Ca2+ channel in this membrane.     

Identification and partial characterization of two inducible gelatin-cleavage activities localized to the sea urchin extraembryonic matrix,the hyaline layer

We have identified two inducible, gelatin-cleaving activities in the sea urchin extraembryonic matrix, the hyaline layer. Isolated hyaline layers, incubated in the presence of benzamidine, were devoid of gelatin-cleavage activities with apparent molecular mass less then 80k. However, when layers were incubated for 9-11 h in the absence of benzamidine, gelatin-cleavage activities, with apparent molecular mass 40- and 50k, were detected. Induction required the presence of NaCl and CaCl(2) at concentrations similar to those found in seawater and readdition of the reversible serine protease inhibitor benzamidine prevented induction. Both gelatin-cleaving activities were activated by calcium at a concentration similar to the calcium concentration found in seawater. Magnesium, also a major cationic species present in seawater, could not replace calcium as the activating ion. In addition, magnesium could not compete with calcium for binding to the gelatinases. Both cleavage activities showed substrate specificity and each failed to cleave bovine serum albumin, bovine hemoglobin or casein. Cleavage activity towards gelatin was inhibited by benzamidine and aminoethyl benzenesulfonyl fluoride, indicating that both activities belonged to the serine class of proteases. The induced 40-kDa activity displayed similar properties to those of a comigrating, gelatin-cleaving activity present in 69-h-old embryos.     

Cleavage of A-CAM by endogenous proteinases in cultured lens cells and in developing chick embryos

We describe two truncated forms of A-CAM (N-cadherin) and present evidence suggesting that both forms are proteolytically derived from the intact A-CAM molecule. The first is a membrane-bound fragment of A-CAM displaying an apparent molecular weight of 78 kDa. This polypeptide, containing the C-terminal portion of the protein, may be generated in cultured chicken lens cells, either by a short treatment with trypsin-EGTA, or by endogenous proteinase(s) during incubation in low Ca2+ medium. Immunofluorescent labeling of normal and EGTA-treated cells indicated that the 78-kDa fragment is uniformly distributed over the cell surface. Moreover, staining of developing chick embryos with pairs of antibodies which distinguish the 78-kDa fragment from intact A-CAM indicated that, at early stages of sclerotome dissociation in developing somites, a truncated derivative of the molecule is generated. The second truncated form of A-CAM is a 97-kDa polypeptide which is constitutively released by cultured lens cells into the culture medium in the presence of normal medium. We present evidence that the 97-kDa molecule is proteolytically derived from A-CAM by the action of an endogenous proteinase. We discuss possible mechanisms leading to the formation of these two truncated derivatives and their possible involvement in the physiological modulation of A-CAM-mediated interactions.     

A Ca2+-dependent tryptic cleavage site and a protein kinase A phosphorylation site are present in the Ca2+ regulatory domain of scallop muscle Na+-Ca2+ exchanger

Digestion of scallop muscle membrane fractions with trypsin led to release of soluble polypeptides derived from the large cytoplasmic domain of a Na(+)-Ca(2+) exchanger. In the presence of 1 mm Ca(2+), the major product was a peptide of approximately 37 kDa, with an N terminus corresponding to residue 401 of the NCX1 exchanger. In the presence of 10 mm EGTA, approximately 16- and approximately 19-kDa peptides were the major products. Polyclonal rabbit IgG raised against the 37-kDa peptide also bound to the 16- and 19-kDa soluble tryptic peptides and to a 105-110-kDa polypeptide in the undigested membrane preparation. The 16-kDa fragment corresponded to the N-terminal part of the 37-kDa peptide. The conformation of the precursor polypeptide chain in the region of the C terminus of the 16-kDa tryptic peptide was thus altered by the binding of Ca(2+). Phosphorylation of the parent membranes with the catalytic subunit of protein kinase A and [gamma-(32)P]ATP led to incorporation of (32)P into the 16- and 37-kDa soluble fragments. A site may exist within the Ca(2+) regulatory domain of a scallop muscle Na(+)-Ca(2+) exchanger that mediates direct modulation of secondary Ca(2+) regulation by cAMP.     

Selective metal ion binding at the calcium-binding sites of the sea urchin extraembryonic coat protein hyalin

The interaction of metal ions with the sea urchin extraembryonic coat protein hyalin was investigated. Hyalin, immobilized on nitrocellulose membrane, bound Ca2+ and this interaction was disrupted by ruthenium red and selective metal ions. The divalent cations Cd2+ and Mn2+, when present at a concentration of 30 microM, displaced hyalin-bound Ca2+. In competition assays, 1 mM Cd2+ or 3 mM Mn2+ were effective competitors with Ca2+ for binding to hyalin. Cobalt, at a concentration of 30 microM, was unable to displace protein-bound Ca2+, but was effective in competition assays at a concentration of at least 10 mM. Magnesium and the monovalent cation Cs+ were unable to disrupt Ca2(+)-hyalin interaction. Interestingly, Cd2+, Mn2+, and Co2+ mimicked the biological effects of Ca2+ on the hyalin self-association reaction. These results clearly demonstrate that the Ca2(+)-binding sites on hyalin can selectively accommodate other divalent cations in a biologically active configuration.     

Properties, biosynthesis and processing of a sulfur-rich protein in Brazil nut (Bertholletia excelsa H.B.K.)

An abundant seed protein, which is exceptionally rich in the sulfur-containing amino acids, methionine (18%) and cysteine (8%), is synthesized in Brazil nut embryos about 9 months after flowering. This sulfur-rich protein consists of two low-molecular-mass polypeptide components, a 9-kDa polypeptide and a 3-kDa polypeptide. The two-subunit polypeptides associate through disulfide linkage(s) to form a 12-kDa protein molecule. We have demonstrated through in vitro translation studies, using RNA from 9-month-old embryos, that the sulfur-rich protein is synthesized as a larger precursor polypeptide of 18 kDa. In addition, data from in vivo labelling studies of 9-month-old Brazil nuts suggest that there are two intermediate precursors of the sulfur-rich protein, one of 15 kDa and another of 12 kDa. One of these precursors, the 12-kDa polypeptide, accumulates for a 2-month period in the developing embryos. From these data we infer that at least three stepwise cleavages are involved in the maturation of the sulfur-rich protein from its 18-kDa precursor.     

Cytoplasmic gels from macrophages. Evidence for the involvement of four proteins in the calcium-dependent solation of actin networks

A method has been devised to study the influence of Ca2+ on the in vitro formation of actin gel networks. Under appropriate conditions low-Ca2+ cytosolic extracts (less than 1 nM) from macrophages rapidly formed a macromolecular complex composed of actin, filamin, alpha-actinin and two new proteins of 70 kDa and 55 kDa. [Pacaud, M. (1986) Eur. J. Biochem. 156, 521-530]. Increasing concentrations of free Ca2+ to 1-2 microM resulted in complete inhibition of the association of 70-kDa protein, a protein which associates actin filaments into parallel arrays. Concentrations of Ca2+ greater than 3 microM caused incorporation of two additional proteins, gelsolin and a 18-kDa polypeptide, with no change in either the actin or alpha-actinin content of the cytoskeletal structures. Use of a polyacrylamide gel overlay technique with 125I-calmodulin revealed that a high-Mr calmodulin-binding protein analogous to spectrin was also associated with these structures when micromolar Ca2+ was present. Similar assays with 45CaCl2 indicated that the 70-kDa protein binds Ca2+ with high affinity. It is thus suggested that Ca2+ might regulate the dynamic assembly of microfilaments through several target proteins, gelsolin, the 70-kDa protein and calmodulin.