Intermolecular cross-linking of vitelline envelope polypeptides predominates in the hardened sea urchin fertilization envelope

At fertilization, the sea urchin egg vitelline envelope (VE) elevates, and a subset of released cortical granule proteins, paracrystalline protein fraction (PCF), associates with the VE to form the fertilization envelope (FE). Cortical granule peroxidase cross-links FE polypeptides by phenolic coupling of tyrosyl residues. We have used an immunological approach to determine which polypeptides are linked together in the hardened FE of Strongylocentrotus purpuratus. Soluble polypeptides were extracted from hardened FEs, and antibodies were prepared in rabbits against the insoluble envelope matrix (FE ghost). Whole immune serum and purified IgGs each reacted with FE ghosts when using an enzyme-linked immunosorbent assay. VEs isolated by means of three published procedures cross-reacted with the immune serum and purified IgGs. Soluble FE polypeptides also cross-reacted with whole immune serum and IgGs owing to the presence of VE polypeptides. Hyalin, a protein not found in FEs, and PCF did not cross-react with antiserum against FE ghosts. To determine which VE polypeptides were cross-linked in the hardened FE, VE polypeptides were immunoblotted by using antiserum against FE ghosts. Most of the VE polypeptides that ranged from 68,000 to 283,000 molecular weight cross-reacted with the antibody.     

A comparison of fertilization envelope development in three species of Strongylocentrotus (S. franciscanus, S. droebachiensis, and S. purpuratus)

The ultrastructure of fertilization envelope (FE) development and the polypeptide spectra of Strongylocentrotus franciscanus and S. droebachiensis envelopes were compared to S. purpuratus. In S. franciscanus, the FE reached its maximum thickness of 67 nm by 3 minutes postinsemination (PI), and final structuralization was observed by 40 minutes PI. The fully formed FE did not have microvillar impressions (casts) and was symmetrical, with outer double laminar elements surrounding an amorphous central region. Isolated S. franciscanus FEs were soluble in reducing and denaturing solvents and the same set of 33 polypeptides ranging from 18.5 to 260 kD was detected in FEs isolated from 10 to 180 minutes PI. The S. droebachiensis FE retained microvillar casts, assumed its definitive form by 3 minutes PI, and was 70 nm thick between microvillar impressions. Isolated S. droebachiensis FEs were partially soluble in reducing and denaturing solvents, and the polypeptide spectra of FEs isolated between 10 and 60 minutes PI were identical and showed 14 polypeptides from 18.5 to 265 kD. Antisera against extracted FEs and the FE extract from S. purpuratus were immunologically cross-reactive (using an enzyme-linked immunosorbent assay) with S. franciscanus and S. droebachiensis FE preparations; immunoblots identified 13 and 5 cross-reactive polypeptides, respectively. Most of the cross-reactive polypeptides were of slightly different molecular weight. Based on comparative ultrastructural, solubility, and electrophoretic data, we suggest that S. droebachiensis FE development is most like that observed in S. purpuratus.     

The Primary Egg Envelope of the Anuran Lepidobatrachus laevis: Physicochemical and Macromolecular Alterations During Development

The coelomic egg envelope (CE) of the frog Lepidobatrachus laevis has a network of fibrillar bundles which disperse after transit through the oviduct. Following oviposition, the egg vitelline envelope (VE) has an additional amorphous zone on the exterior surface. The fertilization envelope (FE) formed after fertilization, appears to be very similar to the VE. The CEs, VEs, FEs and hatched envelopes (FE_h) were manually isolated. The CE, VE and FE were solubilized at 100° using denaturing conditions, but were only partially solubilized in phosphate buffer, pH 7.0. All envelopes and several purified polypeptides from the VE and FE were analyzed using gel electrophoresis and one-dimensional peptide mapping. Each of the envelopes contained 9 major polypeptides ranging from 118.5 to 22 kD and 8–12 minor polypeptides. Several envelope components were added/removed in the conversions based on the results of experiments in which preparations were incubated with activated egg exudate and crude hatching enzyme; some of these transformations were mimicked by tryptic and chymotryptic digestions. Therefore, serine proteases may be involved in envelope processing in vivo. Lepidobatrachus CE polypeptides and several major components from the VE, FE and FE_h were crossreactive with antibodies against Xenopus VE^*.     

Peptide fragment studies on the folding elements of dihydrofolate reductase from Escherichia coli

One of the necessary conditions for a protein to be foldable is the presence of a complete set of “folding elements” (FEs) that are short, contiguous peptide segments distributed over an amino acid sequence. The FE‐assembly model of protein folding has been proposed, in which the FEs play a role in guiding structure formation through FE–FE interactions early in folding. However, two major issues remain to be clarified regarding the roles of the FEs in determining protein foldability. Are the FEs AFUs that can form nativelike structures in isolation? Is the presence of only the FEs without mutual connections a sufficient condition for a protein to be foldable? Here, we address these questions using peptide fragments corresponding to the FEs of DHFR from Escherichia coli. We show by CD measurement that the FE peptides are unfolded under the native conditions, and some of them have the propensities toward non‐native helices. MD simulations also show the non‐native helical propensities of the peptides, and the helix contents estimated from the simulations are well correlated with those estimated from the CD in TFE. Thus, the FEs of DHFR are not AFUs, suggesting the importance of the FEs in nonlocal interactions. We also show that equimolar mixtures of the FE peptides do not induce any structural formation. Therefore, mutual connections between the FEs, which should strengthen the nonlocal FE–FE interactions, are also one of the necessary conditions for a protein to be foldable. Proteins 2006. © 2005 Wiley‐Liss, Inc.     

Immunological evidence that a 305-kilodalton vitelline envelope polypeptide isolated from sea urchin eggs is a sperm receptor

Direct isolation of the sea urchin egg vitelline envelope with intact sperm receptors is difficult because the envelope is firmly attached to the egg plasma membrane. We now report a method for producing an inseminated egg preparation in Strongylocentrotus purpuratus (using soybean trypsin inhibitor [STI] and Ca²⁺, Mg²⁺-free seawater) that contains an elevated vitelline envelope (VE*-STI). The VE*-STI is devoid of cortical granule material, and supernumerary sperm do not detach postinsemination, suggesting that the VE*-STI contains active sperm receptors. VE*-STIs contain a 305-kD polypeptide and additional components that range from 225 to 31 kD, whereas the 305-kD polypeptide was considerably reduced in VE*s. Electrophoresis of sperm receptor hydrolase digests of VE*-STIs showed that the 305-kD polypeptide and several other envelope polypeptides are protease substrates. Univalent Fab fragments against VE*s, VE*-STIs, and 305 and 225-kD polypeptides blocked sperm binding and fertilization in an Fab concentration-dependent manner. The 305 and 225-kD polypeptides were localized in the VE*-STI using indirect immunofluorescence. Enzyme-linked immunosorbent assays showed that the 305 and 225-kD polypeptides share determinants, suggesting that the 225-kD polypeptide may be derived from the 305-kD polypeptide by the proteolysis that occurs at the cell surface during fertilization. Fab fragments against S purpuratus VE*-STI antigens neither bound to nor blocked homologous sperm binding and fertilization of Lytechinus variegatus eggs. Cross fertilizability occurred to the extent of 5% or less between L variegatus and S purpuratus, therefore, we conclude that the 305 kD-polypeptide isolated from S purpuratus is a species-specific vitelline envelope sperm receptor.     

Sea urchin embryo fertilization envelope: immunological evidence that soluble envelope proteins are derived from cortical granule secretions

Although structural studies support the hypothesis that the sea urchin embryo fertilization envelope is derived from the preexisting vitelline envelope template and structural proteins secreted during the cortical reaction, biochemical evidence is minimal. We used an immunological approach to determine the subcellular origin of proteins which were extracted from the fertilization envelope. Fertilization envelopes were isolated from Stronglyocentrotus purpuratus embryos 30 min postinsemination and extracted with 6.0 M urea-0.15 M 2-mercaptoethanol, pH 10.5, for 10 min at 80°C. Extracted proteins were exhaustively dialyzed against 0.015 M 2-mercaptoethanol-0.100 M Tris-HCl at pH 8.6 and mixed with Fruend's complete adjuvant prior to injection into female New Zealand white rabbits. The antiserum which was prepared contained antibodies to six major and two minor polypeptides in the soluble fertilization envelope fraction based on two-dimensional sodium dodecyl sulfate immunoelectrophoresis. Extracts of vitelline envelopes and extracts of unfertilized egg surfaces which are known to contain viteline envelope proteins did not form immunoprecipitates with antiserum against soluble fertilization envelope polypeptides. Extracts of isolated cortical granules and the secreted paracystalline protein fraction formed four and three immunoprecipitates, respectively, which showed complete identity with the soluble fertilization envelope polypeptides based on rocket-line immunoelectrophoresis. Two-dimensional sodium dodecyl sulfate immunoelectrophoresis of cortical granule extract and the secreted paracrystalline protein fraction showed a complex pattern of immunoprecipitates, but a major finding was that cortical granules contain a 193,000-dalton polypeptide which was not found in the paracrystalline protein fraction. These results suggest that proteolytic processing of a cortical granule precursor of the paracrystalline protein fraction occurs during fertilization and that not all of the cortical granule polypeptides are incorporated into the fertilization envelope by means of di- and trityrosine crosslinks with the vitelline envelope proteins.     

Fertilization envelope assembly in sea urchin eggs inseminated in Cl− deficient sea water: I. Morphological effects

Elevation and hardening of the fertilization envelope (FE) occur within 15 min following insemination of the sea urchin egg. When chloride ions were replaced in the media with various anion substitutes, including methyl sulfonate, nitrates, bromide, and isethionate, the fertilization envelope failed to harden and collapsed back to the surface of the egg of Lytechinus variegatus, L. pictus, and Strongylocentrotus purpuratus. At the light microscopy level, the collapse of the envelope was accompanied by a decrease in birefringence, compared with controls. When examined with electron microscopy, the FEs of eggs inseminated in reduced Cl^? solutions failed to transform from an amorphous layer into the more robust laminar structure observed around eggs incubated in normal sea water. Furthermore, in the case of S. purpuratus, the I-T transformation of the FE did not occur. When transfer of the inseminated eggs from the Cl^?-deficient sea water to normal sea water was carried out before 10 min elapsed, the envelope did not collapse, and the birefringence of the envelope was similar to that of controls. Partial envelope collapse was also observed in a dose-dependent manner, varying with the concentration of the Cl^? in the sea water solution. The results suggest that lack of Cl^? in the media may interfere with proper fertilization envelope assembly. Possible mechanisms, including proper incorporation of the cortical granule exudate into the nascent envelope structure, are discussed.     

THE ORIGIN OF ECHINOID HATCHING ENZYME MESSENGER RNA*

To determine if echinoid hatching enzyme messenger RNA is newly synthesized from embryonic chromatin or is a maternal mRNA stored in the unfertilized egg, hybrid andromerogones have been constructed containing a sea urchin (Strongylocentrotus purpuratus) genome in sand dollar (Dendraster excentricus) cytoplasm. Such hybrid andromerogones developed at a normal rate to the blastula stage but failed to hatch. Diploid hybrids or merogones containing at least one complement of sand dollar genome hatched on the normal maternal schedule. Since the sea urchin hatching enzyme is not able to digest the sand dollar fertilization membrane, this failure to hatch is evidence that new mRNA synthesis from embryonic chromatin is required before hatching enzyme can be synthesized.     

Polypeptides of the Maize Amyloplast Stroma : Stromal Localization of Starch-Biosynthetic Enzymes and Identification of an 81-Kilodalton Amyloplast Stromal Heat-Shock Cognate

In the developing endosperm of monocotyledonous plants, starch granules are synthesized and deposited within the amyloplast. A soluble stromal fraction was isolated from amyloplasts of immature maize (Zea mays L.) endosperm and analyzed for enzyme activities and polypeptide content. Specific activities of starch synthase and starch-branching enzyme (SBE), but not the cytosolic marker alcohol dehydrogenase, were strongly enhanced in soluble amyloplast stromal fractions relative to soluble extracts obtained from homogenized kernels or endosperms. Immunoblot analysis demonstrated that starch synthase I, SBEIIb, and sugary1, the putative starch-debranching enzyme, were each highly enriched in the amyloplast stroma, providing direct evidence for the localization of starch-biosynthetic enzymes within this compartment. Analysis of maize mutants shows the deficiency of the 85-kD SBEIIb polypeptide in the stroma of amylose extender cultivars and that the dull mutant lacks a >220-kD stromal polypeptide. The stromal fraction is distinguished by differential enrichment of a characteristic group of previously undocumented polypeptides. N-terminal sequence analysis revealed that an abundant 81-kD stromal polypeptide is a member of the Hsp70 family of stress-related proteins. Moreover, the 81-kD stromal polypeptide is strongly recognized by antibodies specific for an Hsp70 of the chloroplast stroma. These findings are discussed in light of implications for the correct folding and assembly of soluble, partially soluble, and granule-bound starch-biosynthetic enzymes during import into the amyloplast.     

A MAJOR POLYPEPTIDE OF CHLOROPLAST MEMBRANES OF CHLAMYDOMONAS REINHARDI : Evidence for Synthesis in the Cytoplasm as a Soluble Component

Electrophoresis of thylakoid membrane polypeptides from Chlamydomonas reinhardi revealed two major polypeptide fractions. But electrophoresis of the total protein of green cells showed that these membrane polypeptides were not major components of the cell. However, a polypeptide fraction whose characteristics are those of fraction c (a designation used for reference in this paper), one of the two major polypeptides of thylakoid membranes, was resolved in the electrophoretic pattern of total protein of green cells. This polypeptide could not be detected in dark-grown, etiolated cells. Synthesis of the polypeptide occurred during greening of etiolated cells exposed to light. When chloramphenicol (final concentration, 200 µg/ml) was added to the medium during greening to inhibit chloroplastic protein synthesis, synthesis of chlorophyll and formation of thylakoid membranes were also inhibited to an extent resulting in levels of chlorophyll and membranes 20–25% of those found in control cells. However, synthesis of fraction c was not affected by the drug. This polypeptide appeared in the soluble fraction of the cell under these conditions, indicating that this protein was synthesized in the cytoplasm as a soluble component. When normally greening cells were transferred from light to dark, synthesis of the major membrane polypeptides decreased. Also, it was found that synthesis of both subunits of ribulose 1, 5-diphosphate carboxylase was inhibited by chloramphenicol, and that synthesis of this enzyme stopped when cells were transferred from light to dark.     

‘Phytoantibodies’: a general vector for the expression of immunoglobulin domains in transgenic plants

Sequences encoding the immunoglobulin heavy-chain variable (VH) domains were engineered in a new general purpose vector to transform plants via Agrobacterium. The expression of an isolated VH domain (IVD) after introduction into the plant genome has been monitored by northern, western and immuno-histochemical analysis. Immunoblotting showed that the polypeptide was stably expressed and accounted for up to 1% of the soluble protein fraction. It is therefore proposed that single immunoglobulin domains of suitable specificity expressed in plants may constitute an effective system to inhibit the activity of molecules involved in plant pathology or plant development.     

Pullulanase in mung bean cotyledons. Purification, some properties and developmental pattern during and following germination

Starch debranching enzyme was purified from mung bean ( Vigna radiata ) cotyledons to investigate its properties and developmental pattern during and following germination. A debranching enzyme was purified up to the step where only a doublet of polypeptides with molecular masses of 99 and 101 kDa, respectively, was detected by SDS-PAGE. The enzyme is thought to be a single chain monomer, as the molecular mass of the enzyme determined by gel filtration was 72 kDa. Monoclonal antibodies raised against the purified preparation recognized the doublet, indicating that the two polypeptides have immunological homology to each other. The enzyme preparation showed a high activity with pullulan as a substrate, low activity with soluble starch and amylopectin, and no activity with glycogen. These substrate specificities indicate that the debranching enzyme from mung bean cotyledons is of the pullulanase type. Immunoblotting profiles revealed that the enzyme is present in dry seeds and decreases gradually after imbibition, suggesting the possibility that the pullulanase plays a role in developing mung bean cotyledons.     

Characterization of vacuolar polypeptides of barley mesophyll cells by two-dimensional gel electrophoresis and by their affinity to lectins

Vacuoles were isolated from primary leaves of barley (Hordeum vulgare L.) by mechanical breakage of protoplasts, and their polypeptide composition analyzed by two-dimensional gel electrophoresis. Vacuoplasts which consist of the vacuole, a portion of the plasmalemma and of the cytoplasma were prepared from protoplasts by ultracentrifugation. By comparing the vacuolar polypeptide pattern with polypeptide patterns of isolated chloroplasts and of vacuoplasts, vacuolar polypeptides could clearly be distinguished from polypeptides derived from cross-contaminating cell compartments. At least 14 polypeptides of apparent molecular mass between 12 and 76 kilodaltons and an isoelectric point between 4.5 and 7.6 could be attributed to the tonoplast fraction of the vacuole, and 35 polypeptides to the soluble fraction of the vacuole. Several lectins with different specificity were employed to characterize the degree and nature of glycosylation of vacuolar polypeptides. Concanavalin A bound to a large number of polypeptides. Three out of the 14 tonoplast polypeptides exhibited detectable carbohydrate moieties and almost two-thirds of the surveyed soluble polypeptides were glycosylated.Abbreviations IEF isoelectric focussing - kDa kilodalton - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Co-localization of receptor and transducer proteins in the glycosphingolipid-enriched, low density, detergent-insoluble membrane fraction of sea urchin sperm

The low density, detergent-insoluble membrane fraction (LD-DIM), where gangliosides are likely to be highly enriched, was prepared from sperm of two sea urchin species, Hemicentrotus pulcherrimus and Strongylocentrotus purpuratus. Immunoblotting showed the presence in the LD-DIM of two receptors for egg ligands, a glycosylphosphatidylinositol (GPI)-anchored protein, and four proteins which may be involved in signal transduction. Co-immunoprecipitation revealed that at least three proteins, the speract receptor, the 63[emsp4 ]kDa GPI-anchored protein and the subunit of a heterotrimeric Gs protein, are localized in the LD-DIM. This suggests that the LD-DIM fraction may be a membrane microdomain for speract–speract receptor interaction, as well as the subsequent signal transduction pathway involved in induction of sperm respiration, motility and possibly the acrosome reaction.     

Proteins as Morphogenetic Markers in Callus Cultures of Buckwheat <Emphasis Type="Italic">Fagopyrum tataricum</Emphasis> (L.) Gaertn with Different Morphogenetic Potential

The content of soluble proteins and individual polypeptides was studied in calluses of buckwheat Fagopyrum tataricum (L.) Gaertn with different morphogenic potential. The morphogenic callus had a higher content of soluble proteins and cyclic pattern of changes in this index during passaging, which seems to be due to formation of proembryogenic cell complexes. Comparison of the protein patterns of the calluses demonstrated differences in composition and content of individual components. Morphogenic (35 and 73 kDa) and non-morphogenic callus-specific polypeptides (16 and 62 kDa) have been revealed.__________Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 3, 2005, pp. 306–310.Original Russian Text Copyright © 2005 by Maksyutova, Galeeva, Rumyantseva, Viktorova.     

Glycosylated polypeptides of soybean root endomembranes

Summary Endoplasmic reticulum, Golgi apparatus, plasma membrane and mitochondria vesicles were isolated from the roots of four-day-old dark-grown soybean [Glycine max (L.) Merr. cv. Wells II] seedlings and characterized by marker enzyme analyses. Glycoproteins of enriched membrane fractions were identified by concanavalin A (con A)-peroxidase staining of polypeptides separated by two-dimensional IEF-SDS-PAGE and transferred to nitrocellulose.Con A bound to many polypeptides in each endomembrane-enriched fraction with several glycopolypeptides common to all fractions. The mitochondria-enriched fraction possessed few glycopolypeptides and those appeared to be highly glycosylated contaminants of endomembrane origin. Comparison of the endomembrane con A-binding patterns revealed changes in relative stain intensity, molecular weight and isoelectric point of several membrane glycopolypeptides suggestive of processing reactions of the endomembrane complex.Abbreviations con A concanavalin A - PM plasma membrane - GA Golgi apparatus - ER endoplasmic reticulum     

Fertilization envelope assembly in sea urchin eggs inseminated in chloride-deficient sea water: II. Biochemical effects

Eggs of the sea urchin Strongylocentrotus purpuratus were fertilized in normal and in several chloride-deficient sea waters ([ Cl-]: normal greater than isethionate greater than methyl sulfonate greater than bromide). The fertilization envelopes (FE) were thinner and failed to harden, and the characteristic I-T transition did not occur. The permeability of the experimental FEs, as determined by release of protein from the perivitelline space, increased in the order of decreasing [Cl-]. Release of the enzymes beta-1,3-glucanase and cortical granule protease were not significantly altered. On the other hand, release of ovoperoxidase was increased three to four times in bromide sea water. Furthermore, a dose-response was observed in varying concentrations of bromide-normal sea water. With decreasing chloride (increasing bromide) concentration, more ovoperoxidase activity was observed. Cytochemical localization of ovoperoxidase activity with diaminobenzidine revealed almost a total lack of staining of FEs from bromide-substituted sea water. The results suggest that in chloride-deficient sea waters protein incorporation into the nascent FE is impaired. At least in the case of bromide, the incorporation of ovoperoxidase into the nascent FE was also inhibited.     

Probing the interactions between the folding elements early in the folding of Escherichia coli dihydrofolate reductase by systematic sequence perturbation analysis

One of the necessary conditions for a protein to be foldable is the presence of a complete set of folding elements (FEs) that are short contiguous peptide segments distributed over an amino acid sequence. Previous studies indicated the FE assembly model of protein folding, in which the FEs interact with each other and coalesce to form an intermediate(s) early in the folding reaction. This suggests that a clue to the understanding of the determinants of protein foldability can be found by investigating how the FEs interact with each other early in the folding and thereby elucidating roles of the FEs in protein folding. To reveal the formation process of FE-FE interactions, we studied the early folding events of Escherichia coli dihydrofolate reductase (DHFR) utilizing systematic sequence perturbation analysis. Here, systematic single amino acid substitutions were introduced inside of the FEs (W30X in FE2, V40X in FE3, N59X in FE4, and I155X in FE10; X refers to various amino acid residues), and their kinetic refolding reactions were measured by stopped-flow circular dichroism and fluorescence. We show that the interactions around Trp30 and Ile155 are formed in the burst phase intermediate, while those around Val40 and Asn59 are formed in the transition state of the subsequent folding phase (tau5-phase) and in much later processes, respectively. These and previous results suggest that FE2 and FE10, and also FE1 and FE7, involved in the loop subdomain of DHFR, interact with each other within a millisecond time range, while the stable FE3-FE4 interactions are formed in the later processes. This may highlight the important roles of the FEs mainly inside of the loop subdomain in formation of the burst phase intermediate having a hydrophobic cluster and native-like overall topology and in acquisition of the foldability of DHFR.     

Cloning and expression of the Clostridium thermohydrosulfuricum alpha-amylase-pullulanase gene in Escherichia coli

An alpha-amylase-pullulanase gene from Clostridium thermohydrosulfuricum DSM 3783 was cloned in Escherichia coli on a 7.0 kb EcoRI fragment using a lambda vector. The gene produced, from an indigenous promoter, active thermostable alpha-amylase-pullulanase, seemingly mostly a soluble intracellular enzyme in E. coli. Gel filtration separated the active enzyme produced into three peaks, each having both alpha-amylase and pullulanase activities. Immunoblotting after SDS-PAGE revealed more than ten alpha-amylase-pullulanase specific polypeptides; the biggest of these had an Mr of about 165,000, whereas the smallest enzymically active polypeptide had an Mr of about 100,000. Despite the marked degeneration of its constituent polypeptides, the apparent temperature optimum of the enzyme (80-85 degrees C) was only some 5 degrees C lower and the heat stability the same as that of the extracellular alpha-amylase-pullulanase produced by the native host. Oligonucleotide probes prepared according to the NH2-terminal amino acid sequences of the enzyme and its satellite polypeptide (a polypeptide associated with the extracellular enzyme of the native host) hybridized to different regions of the 7.0 kb DNA insert.     

Characterization of endothelin converting enzyme activities in soluble fraction of bovine cultured endothelial cells

Endothelin converting enzyme activities in the soluble fraction of cultured bovine aortic endothelial cells were characterized. The two major endothelin converting enzyme activities were eluted from a hydrophobic chromatography column and the elution profile of the endothelin converting enzyme activities was the same as that of cathepsin D activities. These activities had a same pH optimum at pH 3.5 and were effectively inhibited by pepstatin A. Furthermore, anti-cathepsin D antiserum absorbed these activities as well as cathepsin D activity. Immunoblotting analysis using the antiserum showed the major active fractions have immunostainable components of identical molecular weights with cathepsin D. From these results, we concluded that the major endothelin converting activities in the soluble fraction of endothelial cells are due to cathepsin D. In addition to these cathepsin D activities, a minor endothelin converting enzyme activity with an optimum pH at 3.5 was found, which does not have angiotensin I generating (cathepsin D) activity from renin substrate and needs much higher concentrations of pepstatin A to inhibit the activity than cathepsin D.