Immunolocalization of a 25-kilodalton protein in mouse testis and epididymis.

We have recently observed that a polyclonal antibody raised against a mouse epididymal luminal fluid protein (MEP 9) recognizes a 25-kDa antigen in mouse testis and epididymis [Rankin et al., Biol Reprod 1992; 46:747-766]. This antigen was localized by light and electron microscopic immunohistochemistry. The immunoreactivity in the testis was found in the residual cytoplasm of the elongated spermatids, in the residual bodies, and in the cytoplasmic droplets of spermatozoa. In the epididymis, the epithelial principal cells were stained from the distal caput to the distal cauda. Immunogold labeling in the principal cells showed diffuse distribution without preferential accumulation in either the endocytic or the secretory apparatus of the cells. In the epididymal lumen, the immunoreactivity was restricted to the sperm cytoplasmic droplets. No membrane-specific labeling was observed in luminal spermatozoa, cytoplasmic droplets, or isolated sperm plasma membranes. Three weeks after hemicastration or severance of the efferent ducts, a normal distribution of the immunoreactive sites was found in the epididymis. Immunoreactivity, was also detected in the epididymal epithelium of immature mice as well as in that of XXSxr male mice having no spermatozoa in the epididymis. These results suggest that the immunoreactivity seen in the principal cells originates from synthesis rather than endocytosis of the testicular protein from disrupted cytoplasmic droplets. Furthermore, these results suggest that the 25-kDa protein is synthesized independently by both testis and epididymis.     

Germinal angiotensin I-converting enzyme is totally shed from the rodent sperm membrane during epididymal maturation

Acquisition of sperm fertilizing ability is due, in part, to the reorganization of plasma membrane proteins that occurs during epididymal sperm transit. Using polyclonal antibodies against angiotensin I-converting enzyme (ACE), we showed that this enzyme is immunolocalized mainly on the middle piece of rat and mouse testicular sperm and with less intensity along the initial part of the principal piece of the flagellum. In both species, only some sperm from the caput epididymis were still reactive, whereas no labeling was observed on cauda epididymal sperm. The 105- to 110-kDa germinal ACE was absent from the rat testicular fluid but appeared in the fluid of the anterior epididymis. Thereafter, its molecular weight shifted to 94 kDa in the corpus epididymal fluid and remained at this weight in the caudal region. The 105- to 110-kDa immunoreactive protein was present in testicular rat sperm extract but was completely absent from epididymal sperm extracts. Western blot analysis of testicular and epididymal tissue extracts from the rat and mouse also confirmed that the germinal enzyme was absent from the epididymal sperm cell. Our results demonstrated that the rodent germinal ACE is released from the testicular sperm membrane when sperm enter the epididymis, a process similar to that observed in domestic mammals. This result is discussed in view of the suggested role for this enzyme in sperm fertility.     

Isolation, immunolocalization, and sperm-association of three proteins of 18, 25, and 29 kilodaltons secreted by the mouse epididymis.

Three murine epididymal secretory proteins have been characterized by their site of synthesis, sperm association, and tissue localization by use of polyclonal antisera and immunochemistry. Mouse epididymal protein 7 (MEP 7) was localized initially within the supranuclear regions of some principal epithelial cells in the proximal corpus while other cells remained unstained. In the mid-proximal corpus, all principal cells and stereocilia were stained, and luminal staining increased from corpus to cauda. Some clear cells in the distal corpus and cauda also showed immunoperoxidase staining. Sequential extraction of caudal spermatozoa indicated that MEP 7 was predominantly loosely associated with spermatozoa and that only a small amount of MEP 7 required detergent to extract it from spermatozoa. Examination of other rodent caudal fluids revealed a related protein in rat caudal fluid of 32 kDa, and amino acid sequence analysis of MEP 7 showed a 68% sequence similarity with rat proteins AEG and D/E. MEP 9 immunolocalized within the cytoplasm of all principal cells of the distal caput. In a transition zone between the distal caput and the corpus, some principal cells were stained while others were not. Distal to the corpus, the principal cell staining gradually decreased. In the distal caput and proximal corpus, large heavily stained droplets associated with spermatozoa were seen in the lumen. The staining intensity of these droplets also decreased from corpus to cauda. The clear cells of the distal corpus and cauda did not stain with the antibody to MEP 9. Sequential extraction of caudal spermatozoa showed that some MEP 9 was extractable under low-salt conditions, whereas extraction with 0.1% Triton X-100 was required to remove all MEP 9, indicating it was firmly associated with spermatozoa. The antibody to MEP 9 cross-reacted with a 25-kDa protein present in rat caudal fluid. MEP 10 was localized within the cytoplasm of the principal cells, the stereocilia, and the lumen of the epididymis at the junction of the distal caput and corpus. In the distal corpus, a large number of clear cells were stained, but very few of these cells stained in the cauda. MEP 10 dissociated completely from caudal spermatozoa under low-salt conditions, indicating that it was not firmly bound to spermatozoa. The antiserum to MEP 10 cross-reacted with proteins present in rat and guinea pig caudal fluid. The related rat protein migrated at approximately 20 kDa. Amino acid sequence analysis of MEP 10 revealed an 86% sequence similarity with rat proteins B and C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Localization of a maturation-dependent epididymal sperm surface antigen recognized by a monoclonal antibody raised against a 135-kilodalton protein in porcine epididymal fluid.

A specific 135-kDa protein was purified from porcine cauda epididymal fluid. Analysis of its N-terminal amino acid sequence revealed it to be a new protein. Stable clones of hybridomas that produced monoclonal antibodies against the purified 135-kDa protein were established. A clone, B-11, reacting both with epididymal fluid and with sperm plasma membranes was selected and used in this study. Immunoblotting analysis showed that B-11 reacted only with a 135-kDa protein among epididymal fluid proteins. In contrast, B-11 did not recognize a similar 135-kDa sperm protein but did strongly react with a 27-kDa protein among sperm membrane proteins, extracted by NP-40 in the presence of protease inhibitors. B-11 also reacted only with a 27-kDa protein fragment among trypsin digests of the 135-kDa epididymal protein. The 135-kDa protein was first detected, by ELISA or immunoblotting analysis, at the beginning of the corpus epididymis. Maximal levels were reached in the distal corpus and levels were slightly decreased in the cauda epididymis. On the other hand, the surface of caput sperm were found to contain small amounts of antigen(s), the concentration of which gradually increased during epididymal transit. In immunocytochemical studies, the antigen was detectable in the epithelial cells from the initial segment to the corpus of the epididymis but not in the caudal cells. In the lumen, the presence of the 135 kDa protein was apparent in the corpus (at a maximum in the middle and distal corpus) and to a lesser degree in the caudal lumen. The 27-kDa protein was distributed all over the equatorial region of the acrosome of less than 10% of caput epididymal sperm. As sperm passed through the corpus epididymis, the percentage of immunoreactive cells increased and the protein was restricted to specific domains of the sperm head. Thus, on the mature sperm, antigen was localized in a crescent-shaped area of the equatorial segment just behind the anterior part of the acrosome and on the apical rim of the sperm head. This is the first observation of a sperm surface antigen derived from an epididymal protein as a proteolytic fragment that interacts with specific regions of the sperm membrane during the process of spermatozoa maturation.     

A 105- to 94-kilodalton protein in the epididymal fluids of domestic mammals is angiotensin I-converting enzyme (ACE); evidence that sperm are the source of this ACE

SDS-PAGE analysis of luminal fluid from the ram testis and epididymis revealed a protein of about 105 kDa in the fluid in the caput epididymal region. The molecular mass of this fluid protein shifted from 105 kDa to 94 kDa in the distal caput epididymidis and remained at 94 kDa in the lower regions of the epididymis. The possible sperm origin of this protein was suggested by the decrease in intensity of a 105-kDa compound on the sperm plasma membrane extract and by its total disappearance from the fluid of animals with impaired sperm production caused by scrotal heating. The 94-kDa protein was purified from ram cauda epididymal fluid, and a rabbit polyclonal antiserum was obtained. This antiserum showed that membranes of testicular sperm and sperm from the initial caput were positive for the presence of an immunologically related antigen. The protein was immunolocalized mainly on the flagellar intermediate piece, whereas in some corpus and caudal sperm, only the apical ridge of the acrosomal vesicle was labeled. The purified protein was microsequenced: its N-terminal was not found in the sequence database, but its tryptic fragments matched the sequence of the angiotensin I-converting enzyme (ACE). Indeed, the purified 94-kDa protein exhibited a carboxypeptidase activity inhibited by specific blockers of ACE. All the soluble seminal plasma ACE activity in the ram was attributable to the 94-kDa epididymal fluid ACE. The polyclonal antiserum also showed that a soluble form of ACE appeared specifically in the caput epididymal fluid of the boar, stallion, and bull. This soluble form was responsible for all the ACE activity observed in the fluid from the distal caput to the cauda epididymidis in these species. Our results strongly suggest that the epididymal fluid ACE derives from the germinal form of ACE that is liberated from the testicular sperm in a specific epididymal area.     

Gene and protein expression in the epididymis of infertile c-ros receptor tyrosine kinase-deficient mice

Transgenic male mice bearing inactive mutations of the receptor tyrosine kinase c-ros lack the initial segment of the epididymis and are infertile. Several techniques were applied to determine differences in gene expression in the epididymal caput of heterozygous fertile (HET) and infertile homozygous knockout (KO) males that may explain the infertility. Complementary DNA arrays, gene chips, Northern and Western blots, and immunohistochemistry indicated that some proteins were downregulated, including the initial segment/proximal caput-specific genes c-ros, cystatin-related epididymal-spermatogenic (CRES), and lipocalin mouse epididymal protein 17 (MEP17), whereas other caput-enriched genes (glutathione peroxidase 5, a disintegrin and metalloproteinase [ADAM7], bone morphogenetic proteins 7 and 8a, A-raf, CCAAT/enhancer binding protein beta, PEA3) were unchanged. Genes normally absent from the initial segment (gamma-glutamyltranspeptidase, prostaglandin D2 synthetase, alkaline phosphatase) were expressed in the undifferentiated proximal caput of the KO. More distally, lipocalin 2 (24p3), CRISP1 (formerly MEP7), PEBP (MEP9), and mE-RABP (MEP10) were unchanged in expression. Immunohistochemistry and Western blots confirmed the absence of CRES in epididymal tissue and fluid and the continued presence of CRES in spermatozoa of the KO mouse. The glutamate transporters EAAC1 (EAAT3) and EAAT5 were downregulated and upregulated, respectively. The genes of over 70 transporters, channels, and pores were detected in the caput epididymidis, but in the KO, only three were downregulated and six upregulated. The changes in these genes could affect sperm function by modifying the composition of epididymal fluid and explain the infertility of the KO males. These genes may be targets for a posttesticular contraceptive.     

Biochemical characterization of two ram cauda epididymal maturation-dependent sperm glycoproteins

Rabbit polyclonal antibodies were raised against ram cauda epididymal sperm proteins solubilized by N-octyl-beta-D-glucopy-ranoside (anti-CESP) and against proteins of the fluid obtained from the cauda epididymidis (anti-CEF). The anti-CESP polyclonal antibody reacted with several bands from 17 to 111 kDa with different regionalization throughout the epididymis. The strongest epitopes at 17 kDa and 23 kDa were restricted to the cauda epididymidis. The anti-CEF polyclonal antibody reacted mainly with a 17-kDa and a 23-kDa compound in the cauda sperm extract. These cauda epididymal 17- and 23-kDa proteins disappeared after orchidectomy, but they reappeared in the same regions after testosterone supplementation, indicating that they were secreted by the epithelium. The fluid and membrane 17- and 23-kDa antigens had a low isoelectric point and were glycosylated. The fluid 17- and 23-kDa proteins had hydrophobic properties: they were highly enriched in the Triton X-114 detergent phase and could be extracted from the cauda epididymal fluid by a chloroform-methanol mixture. These proteins were further purified, and their N-terminal sequences did not match any protein in current databases. A polyclonal antibody against the fluid 17-kDa protein recognized the protein in the cauda epididymal sperm extract and immunolocalized it on the sperm flagellum membrane and at the luminal border of all cells in the cauda epididymal epithelium. These results indicated that secreted glycoproteins with hydrophobic properties could be directly integrated in a specific domain of the sperm plasma membrane.     

The 18-kDa mouse epididymal protein (MEP 10) binds retinoic acid.

Mouse epididymal protein (MEP) 10 has recently been characterized in our laboratory. Amino acid sequence analysis of the N-terminal of MEP 10 revealed an 86% similarity in sequence with rat proteins B and C, characterized by Brooks and Higgins [J Reprod Fertil 1980; 59:363-375]. Proteins B and C, have been recently shown to possess retinoic acid-binding ability [Newcomer ME, Ong DE. J Biol Chem 1990; 265:12876-12879; Ong DE, Chytil F. Arch Biochem Biophys 1988; 267:474-478]. Therefore, it was of interest to determine whether MEP 10 possessed the same ability to bind retinoic acid. Mouse caudal fluid was trace-labeled with 3H-retinoic acid and applied to a DEAE ion-exchange column. Analysis of the fractions for both the presence of radioactivity by scintillation counting and MEP 10 by ELISA revealed that the peak of radioactivity corresponded to the peak of MEP 10 immunoreactivity. These peak fractions were pooled and used for subsequent binding analysis and SDS-PAGE and Western blot analysis. SDS-PAGE and Western blot analysis revealed that the peak fractions were enriched for a protein of 18 kDa and that this protein was MEP 10. Competitive binding assays revealed that all-trans-retinoic acid was effective in inhibiting binding of labeled retinoic acid, but that the 13-cis isomer of retinoic acid was only moderately effective in inhibiting binding of the labeled ligand. All-trans-retinol was ineffective in the binding inhibition assay. Similar ligand specificity has also been described for the rat proteins B and C by Ong and Chytil [Arch Biochem Biophys' 1988; 267:474-478].(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in sperm surface membrane and luminal protein fluid content during epididymal transit in the boar

The surface membrane protein of boar sperm and the proteins in the fluid surrounding the gametes were analyzed during epididymal transit. The present study demonstrated that sequential dramatic changes occur in protein composition of the sperm membrane and epididymal fluid during epididymal transit. The maturation process of the boar sperm surface was characterized by a complex sequential evolution of the composition and orientation of macromolecules in the sperm membrane. Epididymal maturation resulted in the progressive disappearance of most of the surface testicular compounds, which were either renewed or masked by new permanent or transient low molecular weight polypeptides on the boar sperm surface membrane. In the fluid surrounding the spermatozoa, composition of the luminal proteins was altered throughout the epididymal transit and several new compounds were characterized. Very few proteins were correlated either with blood plasma or sperm surface compounds.     

Rat epididymis-specific sperm maturation antigens. I. Evidence that the 26 kD 4E9 antigen found on rat caudal epididymal sperm tail is derived from a protein secreted by the epididymis

Monoclonal antibody 4E9, which was raised against a partially purified detergent extract of rat caudal epididymal sperm, recognizes the tail of sperm from the cauda, but not from caput epididymidis, as well as epithelial cells in a restricted region of the distal caput/corpus epididymidis and proteins in epididymal fluid from corpus and cauda epididymidis. The antigen is apparently a glycoprotein, since it is retained on a Ricinus communis agglutinin l lectin column. Epididymal fluid antigens have apparent M_rS of 38–26 kD, whereas the memrane-associated form of the molecule has an M_r of 26 kD. Immunocytochemical data and Western immunoblot data suggest that the membrane antigen is derived from the fluid antigen, which, in turn, is secrteted by the epididymal epithelium. Characterization of the membrane antigen indicates that it is tightly associated with the sperm surface, behaving as though it is an integral membrane protein. The antigen persists on ejaculated sperm. © 1994 Wiley-Liss, Inc.     

Immunolocalization of CRES (Cystatin-related epididymal spermatogenic) protein in the acrosomes of mouse spermatozoa.

The CRES (cystatin-related epididymal spermatogenic) protein is a member of the cystatin superfamily of cysteine protease inhibitors and exhibits highly restricted expression in the reproductive tract. We have previously shown that CRES protein is present in elongating spermatids in the testis and is synthesized and secreted by the proximal caput epididymal epithelium. The presence of CRES protein in developing germ cells and in the luminal fluid surrounding maturing spermatozoa prompted us to examine whether CRES protein is associated with spermatozoa. In the studies presented, indirect immunofluorescence, immunogold electron microscopy, and Western blot analysis demonstrated that CRES protein is localized in sperm acrosomes and is released during the acrosome reaction. Interestingly, while the 19- and 14-kDa CRES proteins were present in testicular and proximal caput epididymal spermatozoa, the 14-kDa CRES protein was the predominant form present in mid-caput to cauda epididymal spermatozoa. Furthermore, following the ionophore-induced acrosome reaction, CRES protein localization was similar to that of proacrosin/acrosin in that it was detected in the soluble fraction as well as associated with the acrosome-reacted spermatozoa. The presence of CRES protein in the sperm acrosome, a site of high hydrolytic and proteolytic activity, suggests that CRES may play a role in the regulation of intraacrosomal protein processing or may be involved in fertilization.     

Biochemical and binding characteristics of boar epididymal fluid proteins

During the passage through the epididymis, testicular spermatozoa are directly exposed to epididymal fluid and undergo maturation. Proteins and glycoproteins of epididymal fluid may be adsorbed on the sperm surface and participate in the sperm maturation process, potentially in sperm capacitation, gamete recognition, binding and fusion. In present study, we separated proteins from boar epididymal fluid and tested their binding abilities. Boar epididymal fluid proteins were separated by size exclusion chromatography and by high-performance liquid chromatography with reverse phase (RP HPLC). The protein fractions were characterized by SDS-electrophoresis and the electrophoretic separated proteins after transfer to nitrocellulose membranes were tested for the interaction with biotin-labeled ligands: glycoproteins of zona pellucida (ZP), hyaluronic acid and heparin. Simultaneously, changes in the interaction of epididymal spermatozoa with biotin-labeled ligands after pre-incubation with epididymal fluid fractions were studied on microtiter plates by the ELBA (enzyme-linked binding assay) test. The affinity of some low-molecular-mass epididymal proteins (12-17 kDa and 23 kDa) to heparin and hyaluronic acid suggests their binding ability to oviductal proteoglycans of the porcine oviduct and a possible role during sperm capacitation. Epididymal proteins of 12-18 kDa interacted with ZP glycoproteins. One of them was identified as Crisp3-like protein. The method using microtiter plates showed the ability of epididymal fluid fractions to change the interaction of the epididymal sperm surface with biotin-labeled ligands (ZP glycoproteins, hyaluronic acid and heparin). These findings indicate that some epididymal fluid proteins are bound to the sperm surface during epididymal maturation and might play a role in the sperm capacitation or the sperm-zona pellucida binding.     

Organ-specific antigens of Clonorchis sinensis

This study was carried out to find out specific proteins from different organs of Clonorchis sinensis. Crude extract, organ-specific and excretory-secretory (ES) proteins were analyzed by immunoblot with infected human sera. The bands of 7- and 17-kDa were main component of intestinal fluid and ES protein and commonly found in all organspecific proteins. The 17-kDa protein was observed from ES antigen, intestinal fluid, eggs and sperms, 26- and 28-kDa proteins were from the uterus, vitellaria, and ovary, and 34-, 37-, 43- and 50-kDa proteins were mainly from the testis and sperms. Serum of mice immunized with sperms reacted to the 50-kDa protein by immunoblotting and immunohistochemical staining showed a positive reaction at the seminal receptacle and seminiferous tubule. The present results show that the 7-kDa protein is a common antigen of every part or organ of C. sinensis, but different organs express their specific antigenic protein bands.     

Primary structure, genomic organization and expression of the major secretory protein of murine epididymis, ME1

The mouse cDNA and its genomic clones encoding the epididymal secretory glycoprotein ME1 were identified. The Me1 gene spans 15kb with four exons and three introns. The deduced amino-acid sequence of the ME1 cDNA revealed that it consists of 149 amino acid residues, which contain a signal peptide characteristic of secretory proteins, six cysteine residues and a proline-rich region conserved in the orthologous proteins. Northern blot analysis revealed that 1.3kb ME1 mRNA is highly expressed in the mouse epididymis. The polyclonal antibodies generated against human HE1 (ME1 orthologous protein) expressed in bacteria reacted with approximately 17 to 25kDa components in mouse epididymis crude extract. The reduction of the molecular mass of the recombinant ME1 protein with the digestion of glycopeptidase A indicated that it is modified by Asn-linked glycosylation.     

Isolation and partial characterization of a glial hyaluronate-binding protein

A glial hyaluronate-binding protein (GHAP) with an isoelectric point of 4.3-4.4 was isolated from human brain white matter. The 60-kDa glycoprotein appeared to be quite resistant to proteolysis, and comparison with GHAP from a viable glioma removed at surgery showed that the protein isolated from autopsy material was not a degradation product resulting from postmortem autolysis. The protein was localized immunohistochemically with mouse monoclonal and rabbit polyclonal antibodies in cerebral white matter. Only small amounts could be found in the gray matter. After enzymatic deglycosylation, an immunoreactive 47-kDa polypeptide was obtained. Two amino acid sequences of GHAP showed a striking similarity (up to 89%) with a highly conserved region of cartilage proteins (bovine nasal cartilage proteoglycan and rat and chicken link protein). However, the amino acid composition and other amino acid sequences suggested that there are also differences between brain-specific GHAP and cartilage proteins.     

Proteins of demembraned mouse sperm heads. Characterization of a major sperm-unique component

A 15-kilodalton protein has been identified as a major component of the residual protein fraction of mouse epididymal/vas spermatozoal heads, demembraned by treatment with Triton X-100 and sequentially extracted with 1 M NaCl/2-mercaptoethanol/DNase I. Two-dimensional electrophoresis of that protein before and after treatment with alkaline phosphatase indicated that it is present in epididymal/vas spermatozoa as a series of five differentially phosphorylated molecules with pI 6.0-7.0. Cyto-immunofluorescence with an affinity-purified antibody to the 15-kDa protein localized that protein to a circumscribed region of the demembraned mouse sperm head mediad from the dorsal margin. By radioimmunoassay, the 15-kDa protein was shown to be sperm-unique and species-specific. The antibody was nonreactive with homogenates of meiotic spermatogenic cells and round spermatids (stages 1-11) but was reactive with a non-phosphorylated 15.5-kDa protein of elongating spermatids (stages 12-16) and testicular spermatozoa. Following alkaline phosphatase treatment, the spermatozoal 15-kDa protein migrated to the position of the spermatidal 15.5-kDa protein on a sodium dodecyl sulfate gel. Thus, we conclude that the 15-kDa protein of mouse spermatozoa is synthesized during the elongation phase of spermiogenesis (stages 12-16) and is phosphorylated in the terminal period of that phase and/or after excursion of spermatozoa from the seminiferous tubules.     

Purification and characterization of a new GTP-binding protein of Mr 24,000 in bovine brain membranes

A GTP-binding protein with an Mr of 24,000 was purified from a cholate extract of bovine brain membranes in addition to the previously reported alpha beta gamma-trimeric GTP-binding proteins (G proteins). Partial amino acid sequence analysis of the purified 24-kDa protein revealed that it was not identical to any of the low Mr GTP-binding proteins already reported, but similar to the rac-gene products serving as the substrate of an ADP-ribosyltransferase (C3) purified from the culture medium of Clostridium botulinum type C. However, the 24-kDa protein was not ADP-ribosylated by the botulinum C3 enzyme. The 24-kDa protein was purified as a nucleotide-free form and characterized by the following unique properties distinct from those of alpha beta gamma-trimeric G proteins. (1) Mg2+ was essentially required for nucleotide binding to the 24-kDa protein; there was a progressive increase in its binding affinity for nucleotides as the concentration of the divalent cation was increased. (2) Nucleotides previously bound to the 24-kDa protein were rapidly dissociated from the protein in Mg(2+)-free medium, in accord with the fact that the protein was indeed purified as a nucleotide-free form with Mg(2+)-free solutions. (3) The 24-kDa protein apparently exhibited much lower GTPase activity than do alpha beta gamma-trimeric G proteins because the product GDP was released from the 24-kDa protein in exchange for the substrate GTP only at a very low rate. Based on these findings, a possible role of the 24-kDa protein in cellular signalling is discussed in comparison with well characterized alpha beta gamma-trimeric G proteins.     

The protein components of the 12-nanometer microfibrils of elastic and nonelastic tissues

A procedure has been developed which is much more specific for the solubilization of the elastin-associated microfibrils from fetal bovine nuchal ligament using treatment with reductive saline in place of reductive guanidine hydrochloride buffer. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reductive saline extracts were shown to contain only five major protein bands with Mrs of 340,000, 78,000, 70,000, 31,000, and 25,000. The 31-kDa species was identified immunologically as the previously described macromolecule named microfibril-associated glycoprotein (MAGP) (Gibson, M. A., Hughes, J. L., Fanning, J. C., and Cleary, E. G. (1986) J. Biol. Chem. 261, 11429-11436). The proteins were purified by gel permeation, ion exchange, and affinity chromatography. Amino acid analyses showed that each protein had a profile which was distinct from that of MAGP although each was also high in acidic amino acids and cystine. The 340- and 78-kDa species were each demonstrated by immunoelectron microscopy with affinity-purified antibodies to be derived from the elastin-associated microfibris, and these were provisionally named microfibrillar protein 340 (MP340) and microfibrillar protein 78 (MP78), respectively. Each of the above antibodies gave a tissue distribution identical to that of anti-MAGP antibodies, and thus MP340 and MP78 also were identified with the 12-nm microfibrils of nonelastic tissues. MP340 was shown to absorb out completely the microfibrillar immunoreactivity of anti-(reductive guanidine hydrochloride extract) antibodies, indicating that MP340 was (a) the major microfibrillar constituent in these extracts and (b) the second unidentified microfibrillar antigen described previously. The relationship of the 70- and 25-kDa proteins to microfibrils is yet to be established. Immunoblot and immunoabsorption studies showed that MAGP and MP78 were immunologically related to MP340 but not to each other. Cyanogen bromide peptide mapping indicated that MAGP was structurally related to MP340. It is postulated that MAGP and MP78 are constituents of MP340 which in turn is the subunit of which the 12-nm microfibrils are composed.