Aggregation of sponge cells. Isolation and characterization of an inhibitor of aggregation receptor from the cell surface.

From the cell membranes of the sponge Geodia cydonium a component was isolated and purified which inhibits the aggregation factor isolated from the same source; the component was termed anti-aggregation receptor. This molecule was characterized as a glycoprotein (54% neutral carbohydrate) and its molecular weight is in the range of 180,000 One biological site of the anti-aggregation receptor was determined to be D-galactose. Indirect evidence presented seems to indicate that this molecule is present in an active form in aggregation-deficient cells and absent in aggregation-susceptible cells.     

Purification and characterization of beta-galactoside (alpha 2 leads to 6)sialyltransferase from rat liver and hepatomas

Asialofetuin sialyltransferase from Triton X-100 extracts of rat liver was resolved by phosphocellulose chromatography into two fractions, designated I and II in order of elution. When previously treated with Arthrobacter ureafaciens neuraminidase, fraction I eluted at about the same position as II while no alteration occurred in II. Primary rat hepatomas contained only a single asialofetuin sialyltransferase, identical to fraction I in chromatographic behavior. Transferases I and II were purified to near homogeneity. Transferase II, as well as neuraminidase-treated I, could be sialylated auto-catalytically, indicating that the lack of sialic acid in II is not due to the lack of a sialic-acid-accepting site. Both enzymes formed an (alpha 2 leads to 6)sialylgalactoside linkage with asialo-glycoproteins of the glycosylamine-type and with lactose, and were indistinguishable immunologically. Nevertheless, the transferases exhibited different molecular weights of 37000 (I) and 43000 (II). When heated at 50 degrees C, transferase I lost half its original activity within 20 min while II was scarcely inactivated. Kinetically, transferase I showed three-times higher affinity than II for CMP-N-acetylneuraminic acid and for desialylated plasma membrane. Asialofetuin sialyltransferase was also purified from primary rat hepatoma. The purified enzyme was identical to transferase I in every respect examined. We conclude that hepatomas contain transferase I but lack transferase II.     

Species-specific aggregation factor in sponges. VI. Aggregation receptor from the cell surface.

An aggregation receptor from the siliceous sponge Geodia cydonium has been isolated and purified in an almost pure form. It sediments at about 2-6s, has a buoyant density of 1-51 g/ml in CsCl and elutes from Sephadex G-50 at a Ve/V0 value of 1-311. Chemical analysis revealed that the receptor consists of 81% neutral carbohydrate and 7-5% protein. The activity of the receptor is rapidly destroyed by Na-periodate. The receptor is released from the cell surface after removal of Ca2+ from the medium or after incubation of the cells with trypsin. The depleted cells can be charged again with isolated receptor molecules. The binding of the receptor molecules on the cell surface is prevented in the presence of trypsin. For optimal binding, physiological salt concentrations with respect to NaCl (540 mM NaCl) and Ca2+ ions are necessary. The receptor whose isolation is described in this report, is involved in secondary aggregation processes, which are initiated by a soluble aggregation factor. The primary aggregation of the cells is not influenced by the receptor. Time-course studies with receptor-depleted cells revealed that new aggregation receptor molecules are formed during the aggregation process. By competition experiments it could be shown that high concentrations of soluble aggregation receptor molecules inhibit secondary aggregation. The soluble receptor molecules can complete with surface-bound receptor molecules only if these are not linked with the aggregation factor.     

Species-specific aggregation factor in sponges. VII. Its effect on cyclic amp and cyclic gmp metabolism in cells of Geodia cydonium.

In dissociated single cells from the sponge Geodia cydonium, DNA synthesis is initiated after incubation with a homologous, soluble aggregation factor. During the DNA-initiation phase the cyclic AMP- and cyclic GMP levels vary drastically; the cyclic AMP content drops from 2.2 pmol/10(6) cells to 0.3 pmol/10(6) cells while the cyclic GMP content increases from 0.6 pmol to 3.7 pmol/10(6) cells. The activity of neither the adenylate cyclase nor of the guanylate cyclase isolated from cells which have been incubated for different periods of time with the aggregation factor, is changed. The soluble as well as the particulate enzyme activities were checked in vitro. The cyclic nucleotide receptors have been isolated from the sponge cells and characterized with respect to their molecular weight, dissociation constant for cyclic AMP or cyclic GMP and intracellular concentration. None of these parameters are altered during aggregation factor-mediated DNA initiation. From these data it is concluded that the regulation of cyclic nucleotide levels is a consequence of a changed activity of nucleotide cyclases or of phosphodiesterases, but this is presumably not caused by a changed rate of synthesis of nucleotide cyclases or of cyclic nucleotide receptors.     

The serum sialyltransferase activity in alpha 1-antitrypsin deficiency.

alpha 1-Antitrypsin phenotypes Pi M and Z, purified by the thiol-disulfide exchange procedure, were desialylated by treatment with neuraminidase covalently coupled to Sepharose and used as acceptors of sialic acid in an assay system for serum sialic acid transferase (CMP-N-acetylneuraminate:D-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1) activity. Both asialoantitrypsins were equally effective as acceptors in contrast to native Pi Z antitrypsin which did not accept any sialic acid. Serum sialyltransferase activity was determined in 38 adult alpha 1-antitrypsin deficient individuals (Pi Z, MZ, FZ, SZ) with normal liver function and was found to be of the same magnitude as the activity in normal individuals (Pi M). Equal activities were also found in 5 Pi Z patients with cirrhosis of the liver. The results strongly argue against the concept that sialyltransferase deficiency provides the molecular basis for alpha 1-antitrypsin deficiency.     

Identification and further characterization of the specific cell binding fragment from sponge aggregation factor

Monoclonal antibodies (McAbs) were raised against the aggregation factor (AF) from the marine sponge Geodia cydonium. Two clones were identified that secrete McAbs against the cell binding protein of the AF complex. Fab fragments of McAbs: 5D2-D11 completely abolished the activity of the AF to form secondary aggregates from single cells. The McAbs were determined to react with the AF in vitro; this interaction was prevented by addition of the aggregation receptor, isolated and purified from the same species. After dissociation of the AF by sodium dodecyl sulfate and 2-mercaptoethanol, followed by electrophoretical fractionation, a 47-kD protein was identified by immunoblotting which interacted with the McAbs: 5D2-D11. During this dissociation procedure, the sunburst structure of the AF was destroyed. In a second approach, the 47-kD protein was isolated by immunoprecipitation; 12 molecules of this protein species were calculated to be associated with the intact AF particle. The 47-kD AF fragment bound to dissociated Geodia cells with a high affinity (Ka of 7 X 10(8) M-1) even in the absence of Ca++ ions; the number of binding sites was approximately 4 X 10(6)/cell. This interaction was prevented by addition of the aggregation receptor to the 47-kD protein in the homologous cell system. Moreover, it was established that this binding occurs species-specifically. The 47-kD fragment of the AF was localized only extracellularly by indirect immunofluorescence staining in cryostat slices. These data suggest that the 47-kD protein is the cell binding molecule of the AF from Geodia.     

Sponge cell aggregation

Summary Dissociated sponge cell system has proved to be a useful model to study the process of cell aggregation both on cellular and subcellular level. The purpose of this review is to discuss recent results obtained from experiments with the marine sponge Geodia cydonium.Dissociated cells form functional aggregates during a process which can be sub-divided into three phases: first, formation of small primary aggregates in the presence of Ca²⁺; second, formation of secondary aggregates in the presence of an aggregation factor and third, reconstitution of a functional system of watercontaining channels by rearrangement in the secondary aggregates.On subcellular level a series of macromolecules are known which are involved in the control of aggregation and separation of sponge cells: Aggregation factor, aggregation receptor, anti-aggregation receptor, glucuronidase, ß-glucuronosyltransferase, ß- ß-galactosidase and a lectin. These components might be linked in the following sequence: (a) Activation of the aggregation receptor by its enzymic glucuronylation; (b) Adhesive recognition of the cells, mediated by the aggregation factor and the glucuronylated aggregation receptor; (c) Inactivation of the aggregation receptor by its deglucuronylation with the membrane-associated ß-glucuronidase; (d) Cell separation due to either the loss of the recognition site (glucuronic acid) of the aggregation receptor for the aggregation factor or to an inactivation of the aggregation factor by the anti-aggregation receptor. The activity of the anti-aggregation receptor is most likely controlled by the Geodia lectin.The events leading to cell-cell recognition cause a change in the following metabolic events: Increase of oxygen uptake, decrease of cyclic AMP level, increase of cyclic GMP level and stimulation of programmed syntheses.Abbreviations AF aggregation factor - CPP circular proteid particle - AR aggregation receptor - aAR anti-aggregation receptor - CMF calcium- and magnesium-free artificial sea water - ASW calcium- and magnesium-containing artificial sea water This paper is dedicated to PROF. DR. R. K. ZAHN on the occasion of his 60^th birthday.     

Evidence for an O-glycan sialylation system in brain. Characterization of a beta-galactoside alpha 2,3-sialyltransferase from rat brain regulating the expression of an alpha-N-acetylgalactosaminide alpha 2,6-sialyltransferase activity

We present evidence for the existence in rat brain of several sialyltransferases able to sialylate sequentially asialofetuin. [14C]Sialylated glycans of asialofetuin were analyzed by gel filtration. Three types of [14C]sialylated glycans were synthesized: N-glycans and monosialylated and disialylated O-glycans. The varying effects of N-ethylmaleimide, lysophosphatidylcholine (lysoPtdCho) and trypsin, were helpful in the identification of these different sialyltransferases. One of them, selectively inhibited by N-ethylmaleimide, was identified as the Neu5Ac alpha 2----3Gal beta 1----3GalNAc-R:alpha 2----6 sialyltransferase previously described [Baubichon-Cortay, H., Serres-Guillaumond, M., Louisot, P. and Broquet, P. (1986) Carbohydr. Res. 149, 209-223]. This enzyme was responsible for the synthesis of disialylated O-glycans. LysoPtdCho and trypsin selectively inhibited the enzyme responsible for the synthesis of monosialylated O-glycan. N-ethylmaleimide, lysoPtdCho and trypsin did not inhibit Neu5Ac transfer onto N-glycans, giving evidence for three different molecular species. To identify the enzyme responsible for monosialylated O-glycan synthesis, we used another substrate: Gal beta 1----3GalNAc--protein obtained after galactosylation of desialylated ovine mucin by a GalNAc-R:beta 1----3 galactosyltransferase from porcine submaxillary gland. This acceptor was devoid of N-glycans and of NeuAc in alpha 2----3 linkages on the galactose residue. When using N-ethylmaleimide we obtained the synthesis of only one product, a monosialylated structure. After structural analysis by HPLC on SAX and SiNH2 columns, we identified this product as Neu5Ac alpha 2----3Gal beta 1----3GalNAc. The enzyme leading to synthesis of this monosialylated O-glycan was identified as a Gal beta 1----3GalNAc-R:alpha 2----3 sialyltransferase. When using lysoPtdCho and trypsin, sialylation was completely abolished, although the Neu5Ac alpha 2----3Gal beta 1----3GalNAc-R:alpha 2----6 sialyltransferase was not inhibited. We provided thus evidence for the interpendence between the two enzymes, the alpha 2----3 sialyltransferase regulates the alpha 2----6 sialyltransferase activity since it synthesizes the alpha 2----6 sialyltransferase substrate.     

Species-specific aggregation factor in sponges. V. Influence on programmed syntheses.

Isolated cells from the siliceous sponge Geodia cydonium as well as small primary aggregates (diameter: 70 mum) consisting of them show no increase in rates of programmed syntheses and mitotic activity with time. After addition of a highly purified aggregation factor to a culture with primary aggregates which subsequently form secondary aggregates (diameter: larger than 1000 mum), a dramatic increase of DNA, RNA and protein synthesis occurs. Together with this increase, the cells show a high mitotic activity. The values for the mitotic coefficient reach a first maximum 8 h after the beginning of the secondary aggregation process. The stimulation of the mitotic activity of cells during the aggregation factor induced secondary aggregation process can be suppressed by inhibitors of RNA and protein synthesis as well as by a blocker of DNA synthesis. This finding may indicate that cells from the G0-population enter the proliferating cell pool via the G1-phase.     

Species-specific aggregation factor in sponges. XIII. Entire and core structure of the large circular proteid particle from Geodia cydonium

High-molecular weight particles have been isolated from the sponge Geodica cydonium. In the "native" from these particles consist of a spherical center and have 25-30 filaments attached to it. The core structure of the particles is assembled of a central circle and 25 radially-arranged filaments. The core structure is obtained from the entire structure by incubation in a medium, containing a non-ionic detergent and EDTA. The molecular weight of the enitre structure was in the range of 1.4 X 10(9) daltons or more and of the core structure 6.1 x 10(8) daltons. Two functional proteins are released from the "native" particles: the aggregation factor and the sialytransferase.     

Sialyltransferase activity in regenerating rat liver

Liver microsomal fractions catalyse the transfer of sialic acid from CMP-N-acetyl-neuraminic acid to various exogenous acceptors such as desialylated fetuin, desialylated human Tamm–Horsfall glycoprotein and desialylated bovine submaxillary-gland mucin. An increase in the rate of incorporation of sialic acid into desialylated glycoproteins was found after a lag period (7h) in regenerating liver. The increase was maximum 24h after partial hepatectomy for all acceptors tested. At later times after operation the sialyltransferase activity remained high only for desialylated fetuin. No soluble factors from liver or serum of partially hepatectomized animals influenced the activity of the sialyltransferases bound to the microsomal fraction. The sensitivity of sialyltransferases to activation by Triton X-100, added to the incubation medium, was unchanged in the microsomal preparation from animals 24h after sham operation or partial hepatectomy. The full activity of sialyltransferases towards the various desialylated acceptors showed some differences. Human Tamm–Horsfall glycoprotein was a good acceptor of sialic acid only when desialylated by mild acid hydrolysis. After this treatment, but not after enzymic hydrolysis, a decrease in molecular weight of human Tamm–Horsfall glycoprotein was observed. Further, the sialyltransferase activity as a function of incubation temperature gave different curves according to the acceptor used. The relationship between the biosynthesis of glycoproteins by regenerating liver and the sialyltransferase activity of microsomal fraction after partial hepatectomy is discussed.     

Specific phosphorylation of proteins in pore complex-laminae from the sponge Geodia cydonium by the homologous aggregation factor and phorbol ester. Role of protein kinase C in the phosphorylation of DNA topoisomerase II.

We have recently shown that the aggregation factor (AF) from the sponge Geodia cydonium stimulates DNA synthesis in quiescent, dissociated cells from the same organism; this event was correlated with the release of the two second messengers: inositol trisphosphate and diacylglycerol. Here we describe that after binding of the AF to the plasma membrane-bound aggregation receptor, a rapid and drastic increase in the incorporation of 32Pi into a series of proteins in the pore complex-lamina fraction occurs. Addition of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, to quiescent cells resulted in a similar stimulation of phosphorylation of nuclear proteins. Among them we have selected one protein with a polypeptide Mr of 170,000 (pp170) for detailed studies. By immunoblotting pp170 was identified as DNA topoisomerase II. In vitro studies with nuclei and purified, homogeneous protein kinase C together with the required activators of this enzyme also showed a phosphorylation of pp170. After phosphorylation, DNA topoisomerase II activity was found to be 2.5-fold that of the non-phosphorylated enzyme. From these data we conclude that protein kinase C is involved in AF induced transmembrane signalling, ultimately leading to an initiation of DNA synthesis.     

A brain sialyltransferase having a narrow specificity for O-glycosyl-linked oligosaccharide chains

The existence of a brain sialyltransferase catalyzing the specific transfer of NeuAc on native fetuin was demonstrated. This enzyme was not able to sialylate either asialofetuin or desialylated and nondesialylated orosomucoid, transferrin, and bovine submaxillary mucin. It required the presence of Mn2+ for optimal activity. Moreover, in fetuin, this activity was closely related to the proportion of NeuAc residues, but in liver tissue sialylation occurred only onto asialofetuin. In native fetuin, sialylation took place on O-glycan chains to give an O-disialyltetrasaccharidic structure. The Gal----GalNAc----protein was not an acceptor, but alpha-NeuAc-(2----3)-Gal----GalNAc----protein was, suggesting a specific transfer alpha-(2----6) to the GalNAc residue.     

Lipid requirements for the aggregation of CTP:phosphocholine cytidylyltransferase in rat liver cytosol.

Two forms of CTP:phosphocholine cytidylyltransferase were identified in rat liver cytosol by gel filtration chromatography. The low molecular weight form (L form) is the major form in fresh cytosol. The enzyme associates into a high molecular weight form (H form) upon storage of the cytosol at 4 degrees C. Aggregation of the purified L form of cytidylyltransferase is caused by total rat liver lipids, neutral lipids, diacylglycerol, or phosphatidylglycerol. Diacylglycerol was the only lipid isolated from the rat liver that caused aggregation of the purified enzyme. Although the addition of diacylglycerol to the cytosol did not change the amount of aggregation of the enzyme, a 2.5-fold increase in H form was observed in cytosol pretreated with phospholipase C, or in cytosol from rats fed a high cholesterol diet. In both of these cytosolic preparations, the concentration of diacylglycerol was elevated twofold. Phosphatidylglycerol did not seem to affect the association of the enzyme in cytosol since it is present in very low concentrations in the rat liver cytosol, and its degradation in cytosol by a specific phospholipase did not affect the rate of aggregation. The results suggest that diacylglycerol in an appropriate form is required for association of cytidylyltransferase in rat liver cytosol.     

Glycoprotein biosynthesis in calf kidney. Glycoprotein sialyltransferase activities towards serum glycoproteins and calf Tamm-Horsfall glycoprotein.

CMP-AcNeu:glycoprotein sialyltransltransltransltransltransferase of calf kidney cortex was characterized using serum glycoproteins and Tamm-Horsfall glycoprotein, obtained from calf urine, as acceptors. Native calf Tamm-Horsfall glycoprotein showed the best acceptor properties, followed by desialylated calf fetuin and desialylated human alpha 1-acid glycoprotein exhibiting V values of, respectively, 114, 63 and 41 nmol/h per g wet wt. of kidney cortex and Km values of 0.12, 0.16 and 0.26 mM glycoprotein acceptor. Desialylated ovine submaxillary mucine appeared to be a very poor acceptor. Tamm-Horsfall glycoprotein sialyltransferase could be distinguished from serum glycoprotein sialyltransferase by competition studies. In addition the two glycoprotein sialyltransferase activities showed different distributions over the three regions of the calf kidney: the ratios of the Tamm-Horsfall to serum glycoprotein sialyltransferase activities decreased from 3.3 in the cortex to 0.8 and 0.4 in the medulla and the papilla, respectively. It was concluded that in calf kidney at least two different sialyltransferases exist. The high cortical Tamm-Horsfall glycoprotein sialyltransferases activity corresponds markedly to the origin of the urinary Tamm-Horsfall glycoprotein, namely the distal part of the kidney tubule. Inactivation of glycoprotein sialyltransferase activity by preincubation at various temperatures and during storage at 0 degree C, could be reduced by the addition of CMP-AcNeu. The possible relevance towards the in vivo sialylation of this finding is discussed.     

The polypeptide part of human chorionic gonadotrophin affects the kinetics of alpha 6-sialylation of its N-linked glycans but does not alter the branch specificity of CMP-NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase.

Human chorionic gonadotrophin (hCG) is a heterodimeric glycoprotein hormone consisting of an alpha- and a beta-subunit, both containing two N-linked, complex-type glycans. Using this hormone as a model glycoprotein, the influence of its polypeptide part on the activity and specificity of bovine colostrum CMP-NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase (alpha 6-sialyltransferase) was investigated. Initial rates of sialic acid incorporation into the desialylated glycans of hCG alpha and hCG beta in the heterodimer were higher with the alpha-subunit. This appeared to be due to a higher V which, together with a slightly lowered affinity (higher Km), resulted in a higher kinetic efficiency of the sialyltransferase for the glycans of this subunit. By contrast, the kinetic parameters did not differ significantly when the subunits were in the free form, indicating that the differences in the kinetics of sialylation found for the subunits in the heterodimeric state were not caused by the differences in N-linked carbohydrate structures of the subunits. It is proposed that these effects are due to conformational constraints which the polypeptide moieties put on the glycan chains upon dimerization. Furthermore, it was investigated whether the polypeptide of hCG would interfere with the sialyltransferase so as to alter the branch specificity of the enzyme. 1H-NMR spectroscopy (400 MHz) of the glycan chains, alpha 6-sialylated in vitro, showed that the enzyme highly prefers the galactosyl residue at the Gal beta 1----4GlcNAc beta 1----2-Man alpha 1----3Man branch for attachment of the first mol of sialic acid into the diantennary glycans of desialylated hCG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of carbohydrates as multiple low affinity interaction sites in the self-association of the aggregation factor from the marine sponge Microciona prolifera

Cell aggregation in the marine sponge Microciona prolifera is mediated by a multimillion molecular weight aggregation factor (MAF) and is based on two functional properties, a Ca2+-independent cell binding activity and a Ca2+-dependent factor-factor self-interaction. Monoclonal antibodies were prepared against purified MAF, and one clone was characterized which selectively inhibited the MAF-MAF association activity. Binding of the blocking antibody (Block 1) to MAF demonstrated that this epitope structure was present in 1100 copies per one MAF molecule of Mr = 2 X 10(7). Such blocking antibodies precipitated a small molecular weight protein-free glycan fraction prepared from MAF by Pronase digestion, thus indicating that the highly repetitive epitope is located in the carbohydrate portion of the molecule. Since the inhibitory activity of the Block 1 antibody could only be achieved when most of the sites were occupied by Fab fragments of this antibody, the self-association of MAF seemed to be based on the polyvalency of the carbohydrate determinants. The affinity of the protein-free glycans was very low as shown by the fact that they did not display any measurable self or MAT binding activity in their monomeric form. After cross-linking them with glutaraldehyde into polymers of the size of MAF, however, the self-interaction could be reconstituted. Thus, the MAF-MAF association activity, which is needed for cell aggregation in sponges, seems to be based on multiple low affinity carbohydrate-carbohydrate interactions, which is different from most interactions mediated by adhesion molecules characterized so far.     

A unique enzyme from Saccharothrix sp. catalyzing d-amino acid transfer

A newly isolated actinomycete belonging to Saccharothrix sp. was found to produce a unique enzyme catalyzing d-amino acid transfer. The enzyme, which was tentatively named d-amino acid transferase, was purified 2600-fold to electrophoretic homogeneity and the molecular mass was 41 kDa. The enzyme was d-configuration specific and recognized aromatic d-amino acid esters to form oligo d-amino acid esters. d-Phenylalanine ester was favored as substrate over other d-amino acid esters. The optimum conditions for oligo d-phenylalanine ester formation by d-amino acid transferase were pH 7.0 and 40°C. The enzyme was inhibited by DAN, EPNP and DFP.     

Control of the aggregation factor-aggregation receptor interaction in sponges by protein kinase C

By means of immunobiochemical and immunocytological techniques it was found that the aggregation factor (AF) from the sponge Geodia cydonium is stored in vesicles of spherulous cells. During the reaggregation process of dissociated cells, the AF which is present extracellularly was determined to be bound to the cell-surface-associated aggregation receptor (AR) only during the initial phase (0-5 h after addition of the AF to the single cell suspension). At later stages (20 h), the AF colocalized with extracellular structures, e.g., collagen and glycoconjugates. Immobilized to nitrocellulose, the AR, a molecule with Mr of 43.5 kDa, displayed its binding affinity to the AF only if it was isolated from early aggregates (5 h). The transition of the AF-susceptible to the AF-deficient state of the plasma membrane was mimicked in vitro by incubation of plasma membranes from early aggregates with purified protein kinase C. This conversion to the AF-deficient state could be prevented by the protein kinase C inhibitor staurosporine. Together with earlier findings, which revealed that the AR is phosphorylated by protein kinase C, we propose that in the sponge system this enzyme controls intercellular processes involved in morphogenesis.     

Purification and characterization of a species-specific aggregation factor in sponges

The events during re-aggregation of sponge cells, dissociated in Ca²⁺- and Mg²⁺-free artificial sea water, containing trypsin, can be subdivided into three phases. The first event is the formation of primary aggregates with a diameter of 82 μm. It is Ca²⁺- and Mg²⁺-dependent and insensitive towards trypsin and puromycin even at high concentrations. The formation of secondary aggregates with a diameter greater than 1 000 μm, is initiated by an aggregation factor. This factor can be separated from the formed elements with calcium- and magnesium-free artificial sea water. Its action is Ca²⁺- and Mg²⁺-dependent, temperature-independent and insensitive to puromycin. The last event is the reconstitution of functional aquiferous systems in the aggregates.The aggregation factor which was found to be species-specific could be purified 158-fold. Its functional group seems to be a protein probably with polar amino acids in critical positions. Secondary aggregates generated with the aggregation factor show high viability. Some evidence is presented that the aggregation factor may be an annular particle with a circular contour length of 3 500 Å with about 25 filaments attached to it.