Isolation and culture of ram rete testis epithelial cells: structural and biochemical characteristics

Cultures of rete testis epithelial cell-enriched preparations from testes of adult rams have been investigated, and some of their properties have been determined. In monolayers, the cells form mosaic-like borders, and retain many ultrastructural features characteristic of rete epithelial cells in situ, including an indented nucleus with prominent heterochromatin clumps, short rod-shaped or round mitochondria that are easily distinguished from the elongated mitochondria of Sertoli cells, the presence of desmosomes, and few if any lipid droplets or vacuoles. Unlike Sertoli cell-enriched aggregates in culture, rete testis epithelial cell preparations do not form cytoplasmic extensions, and no associated germ cells are present. Rete cells in culture express cytokeratin and vimentin in the cytoskeleton, whereas Sertoli cells prepared from testes of adult rams contain vimentin but not cytokeratin. Both rete cells and Sertoli cells stain positively for laminin but not for fibronectin, Collagen Type I, or Collagen Type III. The rete cells synthesize and secrete several proteins into the culture medium, evident in gel electrophoresis patterns of radiolabeled proteins. This pattern is similar, but not identical, to that secreted by Sertoli cell-enriched preparations. Rete cells in culture in the presence of serum continue to undergo mitotic division, but Sertoli cells do not. A variety of criteria were employed to estimate the relative numbers of Sertoli cells present in the rete testis epithelial cell-enriched preparations from testes of adult rams, including morphological and ultrastructural differences between the two cell types, and the presence of desmosomal proteins and cytokeratin in rete cells but not in Sertoli cells. The relative number of fibroblast-like cells was determined by measuring the expression of fibronectin and Collagen Type I, and an immunocytochemical probe for the detection of Factor VIII was used to estimate the degree of contamination by vascular endothelial cells. Using these markers, we determined that the rete testis epithelial cell-enriched preparations were about 93% pure. Primary cultures under defined conditions contained relatively few Sertoli cells (0.4%), but were contaminated to a larger extent by fibroblast-like cells (approximately 4%) and by endothelial cells (about 3%). The possible functions of rete testis epithelial cells are discussed herein.     

Immunolocalization of clusterin in the ram testis, rete testis, and excurrent ducts

Clusterin, a glycoprotein that elicits cell aggregation, has previously been isolated from ram rete testis fluid, and has been partially characterized. In experiments reported, we have used monoclonal antibodies against clusterin in combination with indirect immunofluorescence microscopy to investigate the distribution of clusterin in the adult ram testis, rete testis, and excurrent ducts. Tissue blocks (5 mm3) were fixed in periodate/lysine/paraformaldehyde containing 0.1% glutaraldehyde and, after embedding, 5-microM sections were prepared for immunolocalization. In the testis, 2 basic patterns were observed: 1) strong to moderate staining for clusterin in the adluminal region with little staining in the basal region of the seminiferous epithelium and germinal cells; and 2) moderate staining throughout the seminiferous epithelium between germinal cells. In the rete testis, strong clusterin staining was localized intracellularly in the rete epithelial cells, most often associated with the luminal surface. In the epididymis, intracellular clusterin was localized in some principal cells of the caput epididymidis. The luminal surfaces and spermatozoa within the lumen were strongly positive. In the vas deferens, clusterin staining was associated with the luminal surface only. The presence of clusterin was clearly detected in unwashed isolated epididymal spermatozoa, but not in spermatozoa washed with phosphate-buffered saline containing 0.05% Tween 20.     

The absence of specific interactions of Sertoli-cell-secreted proteins with antibodies directed against H-Y antigen

Radiolabeled proteins secreted into the medium by rat Sertoli cells in primary culture have been examined for specific interactions with polyclonal and monoclonal antibodies directed against serologically detectable H-Y antigen(s). None of the proteins secreted by Sertoli cells reacted specifically with H-Y antibodies, as determined with immunoprecipitation procedures and immunoabsorbent affinity chromatography, followed by SDS gel electrophoresis. Radioactivity profiles of proteins obtained after reaction with H-Y antibodies were similar to those observed after treatment with nonimmune sera or with irrelevant antibodies. We obtained comparable findings with proteins secreted by the mouse cell line TM4, which is of presumptive Sertoli cell origin, and with proteins present in ram rete testis fluid. These and other findings presented do not support the contention that Sertoli cells secrete a protein having the properties of serologically detectable H-Y antigen as previously described.     

Rat clusterin isolated from primary Sertoli cell-enriched culture medium is sulfated glycoprotein-2 (SGP-2)

Clusterin, a glycoprotein originally isolated from ram rete testis fluid, is a dimer composed of monomers with non-identical NH2-terminal amino acid sequences. In view of its possible role in cell-cell interactions in the seminiferous epithelium, we sought to identify such a protein in the rat. Using the bioassay developed for the ovine protein, rat clusterin was purified to apparent homogeneity by HPLC from primary Sertoli cell-enriched culture media. This protein is also a heterodimer consisting of monomers of Mr 43,000 (alpha) and Mr 35,000 (beta). NH2-Terminal amino acid sequence analysis indicated that the alpha subunit has a sequence of NH2-SLMPLSHYGPLSFHNMFQPFFDMIHQAQQA and the beta subunit, NH2-EQEFSDNELQELSTQGSRYVNKEIQNAVQG. These two subunits show marked similarity with the corresponding subunits of ram clusterin isolated from rete testis fluid. Using an antibody against the alpha subunit of rat clusterin, a cDNA clone was isolated from a rat testicular lambda gt11 cDNA library. Analyses of the amino acid sequence derived from the isolated rat clusterin cDNA and of the NH2-terminal amino acid sequences indicate that rat clusterin is identical to a Sertoli cell glycoprotein previously designated sulfated glycoprotein-2.     

Structural analysis of clusterin and its subunits in ram rete testis fluid

Clusterin is a protein present in the rete testis fluid of the ram that elicits aggregation of erythrocytes and Sertoli cells in vitro. In view of its possible biologic function in relation to cell-cell interaction in the testis, we isolated this protein from ram rete testis fluid using sequential high-performance liquid chromatography columns and performed a detailed physicochemical characterization. This protein consists of two molecular variants designated form I and form II clusterin. Each form of clusterin consists of two subunits with an apparent molecular weight of 40,000. It is of note that the two subunits have no homology in their N-terminal amino acid sequences. However, the N-terminal amino acid pairs of the two subunits derived for the two forms of clusterin are identical. Using o-phthalaldehyde to block the Lys residue at the fourth amino acid pair from the N-terminus which leaves the Pro residue free for subsequent Edman degradation, we have deduced the N-terminal sequence of each of the two subunits for form I clusterin. Comparison of the NH2-terminal sequences of the two subunits of clusterin with the release 10.0 of the protein sequence data base of the Protein Identification Resource indicated no homology between either of the subunits of clusterin and any of the known proteins in the data base. A highly specific radioimmunoassay developed for clusterin was used to measure its concentrations in the fluids of the rete testis and cauda epididymis. Since a significant amount of immunoreactive clusterin was found in serum, the protein was partially purified from this source by immunoaffinity chromatography. Immunoreactive serum clusterin was smaller than the testicular clusterin (Mr 37,000 vs 40,000), but both proteins share common epitopes as demonstrated by radioimmunoassay and immunoblots. However, serum clusterin does not possess the biologic activity of the testicular clusterin in that it does not elicit cell aggregation in vitro. It is of note that deglycosylation of testicular clusterin can also eliminate this in vitro biologic activity, suggesting that the serum clusterin might be a deglycosylated form of the testicular protein and the carbohydrate core plays an important role in determining the cell aggregation activity. Studies on the distribution of this protein in the reproductive compartment indicate that it is highly concentrated in the rete testis and the cauda epididymal fluids. This suggests that this protein might have some important functions in the reproductive tract.     

青稞中β-1,3葡聚糖酶的纯化及部分性质研究

β-1,3-葡聚糖酶[EC．3．2．1．39]存在于大多数的高等植物中,它们由一个小的基因家族编码,在植物不同的生理活动中起着重要的作用。采用NaCl抽提、硫酸铵分部沉淀和2步离子交换法和分子筛从青稞的胚芽中提纯得到了一种β-1,3-葡聚糖酶。在非变性电泳中纯化的β-1,3-葡聚糖酶用银染只有一条蛋白带,运用底物进行的活性染色时在相同位置也只显示一条酶活性带。此活性染色可以直接在电泳胶板上快速鉴定和检验β-1,3-葡聚糖酶。纯化的β-1,3-葡聚糖酶在还原及非还原条件下的SDS-PAGE变性电泳中,呈现一条分子量为32kD的主要蛋白带及2条低分子量的弱带,表明此酶无链内二硫键存在。等电聚焦分析显示其等电点为8．1。上述结果表明纯化得到的是一种碱性β-1,3-葡聚糖酶。     

Sertoli cell synthesizes and secretes a protease inhibitor, alpha 2-macroglobulin.

The mechanism by which the seminiferous epithelium limits the damaging effects of proteases that are released from degenerating late spermatids does not depend upon protease inhibitors in the systemic circulation since these proteins are excluded from the seminiferous tubule by the blood-testis barrier. The purpose of this study was to identify the major protease inhibitor of the testis and determine its cellular origin. Sertoli cells, the major epithelial component of the seminiferous epithelium, release a protease inhibitor, testicular alpha 2-macroglobulin, in vitro. Immunoprecipitation using [35S]methionine and a monospecific polyclonal antibody prepared against purified testicular alpha 2-macroglobulin establishes that this protein is actively synthesized and secreted by Sertoli cells. Measurements of immunoreactive protease inhibitors in tubular and rete testis fluids collected by micropuncture suggest that alpha 2-macroglobulin rather than alpha 1-antitrypsin is the major protease inhibitor in the seminiferous tubules in vivo. The ability of alpha 2-macroglobulin to inactivate proteases and growth factors such as TGF-beta by a common mechanism suggests that this protein may have a dual function in the testis.     

The intratesticular excurrent ducts of the camel (Camelus dromedarius)

The histology and fine structure of the epithelial cells of the intratesticular excurrent ducts were studied in material collected from fourteen adult camels and fixed by perfusion. The intratesticular excurrent ducts consisted of a terminal segment of the seminiferous tubule, a tubulus rectus, and a rete testis. The terminal segment was lined with modified Sertoli cells which formed a plug-like structure in the receptacle. The tubulus rectus was subdivided into the receptacle, the narrow main part, and the wider distal part, and these parts were lined with different types of epithelium. The rete testis occupied an axial mediastinum testis, and the height of its epithelium varied quite considerably. Degenerated spermatozoa were seen engulfed by the epithelial cells of the entire intratesticular duct system. Light cells, lymphocytes, and macrophages were observed. The fine structure of the epithelium of the intratesticular ducts is discussed in relation to its possible functions.     

Neutral proteinase inhibitors in PMN leukocytes. II. Isolation and characterization of a neutral proteinase inhibitor from porcine PMN leukocytes

An inhibitor of neutral proteinases was purified from porcine PMN leukocytes by gel filtration on Sephadex G-75 superfine and ion-exchange chromatography on Mono S. Thus an inhibitor preparation with a specific inhibitory activity against chymotrypsin of 10 IU/mg was obtained. In dodecyl sulfate gel electrophoresis a single protein band with an apparent molecular mass of 40 kDa was found under reducing conditions. Under non-reducing conditions the inhibitor forms higher molecular mass aggregates. On isoelectric focusing several protein bands with isoelectric points between pH 7.0 and 7.5 could be separated. The amino-acid composition of the inhibitory protein was determined. The inhibition mechanism was studied and association rate constants (kon) were measured and calculated for the reaction with chymotrypsin as well as leukocyte and pancreatic elastase. In Western blot analysis and in enzyme immunoassay studies crossreactivity between antibodies directed against porcine leukocyte neutral proteinase inhibitor and the corresponding inhibitor of bovine PMN leukocytes could be demonstrated.     

Characterization of rat Leydig cell gonadotropin receptor structure by affinity cross-linking

The present study was intended to examine the structure of the rat Leydig cell gonadotropin receptor. Leydig cell suspensions were prepared by either collagenase digestion or mechanical disruption of the testes. The cells were incubated with 125I-human chorionic gonadotropin (hCG) following which the bound 125I-hCG was covalently cross-linked to the cell surface receptor using a cleavable (dithiobis(succinimidyl propionate] and a noncleavable (disuccinimidyl suberate) cross-linking reagent. The extracted cross-linked membrane proteins were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions and subjected to autoradiographic analysis. Under nonreducing conditions, three radiolabeled bands, in addition to intact hCG and its alpha-subunit, were detected with apparent molecular weights of 184,000, 136,000, and 103,000. However, under reducing conditions, three radiolabeled bands migrated on the gel corresponding to molecular weights of 144,000, 106,000, and 75,000. The binding of 125I-hCG to the receptor was inhibited by hCG and luteinizing hormone, but not by a number of other peptides or proteins. The radiolabeled bands were not detectable in hCG down-regulated Leydig cells. Furthermore, a similar autoradiographic pattern of 125I-hCG-linked complexes was seen when the 125I-linked receptor complex was subjected to immunoprecipitation with anti-hCG antibodies followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition, evidence was obtained indicating that these three labeled bands were derived from the same molecular species. The data suggests that the hCG receptor in Leydig cell is probably an oligomeric complex with a molecular weight of about 250,000, which is composed of three polypeptide chains of molecular weights 121,000, 83,000, and 52,000 held together through noncovalent forces. Additionally, collagenase treatment of Leydig cells does not appear to alter the autoradiographic pattern of the 125I-hCG-linked receptor.     

Differential expression of estrogen receptors alpha and beta in the reproductive tracts of adult male dogs and cats

Expression of estrogen receptors (ERs) in the reproductive tracts of adult male dogs and cats has not been reported. In the present study, ERalpha and ERbeta were localized by immunohistochemistry using ER-specific antibodies. ERalpha was found in interstitial cells and peritubular myoid cells in the dog testis, but only in interstitial cells of the cat. In rete testis of the dog, epithelial cells were positive for ERalpha staining, but in the cat, rete testis epithelium was only weakly positive. In efferent ductules of the dog, both ciliated and nonciliated cells stained intensely positive. In the cat, ciliated epithelial cells were less stained than nonciliated epithelial cells. Epithelial cells in dog epididymis and vas deferens were negative for ERalpha. In the cat, except for the initial region of caput epididymis, ERalpha staining was positive in the epithelial cells of epididymis and vas deferens. Multiple cell types of dog and cat testes stained positive for ERbeta. In rete testis and efferent ductules, epithelial cells were weakly positive for ERbeta. Most epithelial cells of the epididymis and vas deferens exhibited a strong positive staining in both species. In addition, double staining was used to demonstrate colocalization of both ERalpha and ERbeta in efferent ductules of both species. The specificity of antibodies was demonstrated by Western blot analysis. This study reveals a differential localization of ERalpha and ERbeta in male dog and cat reproductive tracts, demonstrating more intensive expression of ERbeta than ERalpha. However, as in other species, the efferent ductules remained the region of highest concentration of ERalpha.     

Tyrosine sulfation site is located in the C-terminal fibrin-binding domain in secreted fibronectin from rat embryo fibroblasts, line 3Y1

Tryptic fragments of [35S]sulfate-labeled 3Y1 secreted fibronectin were fractionated by hydroxylapatite column chromatography and examined using sodium dodecyl sulfate gel electrophoresis, followed by autoradiography. Radioactive bands containing tyrosine-O-[35S]sulfate were detected at 17- and 40-kDa positions under reducing conditions. Under nonreducing conditions, the 17-kDa band was no longer present and new bands at 57- and 80-kDa positions appeared, indicating a disulfide linkage between the two smaller fragments in the native state. These fragments exhibited binding affinity toward fibrin and could be immunoprecipitated by the monoclonal antifibronectin Fib-2 domain antibody. These results suggested that the tyrosine sulfation site in 3Y1 secreted fibronectin is located in the C-terminal fibrin-binding (Fib-2) domain, being within 17 kDa of the C-terminus.     

Purification and characterization of natural and recombinant human plasminogen activator inhibitor-1 (PAI-1)

Human plasminogen activator inhibitor-1 (PAI-1) was purified from the conditioned medium of endotoxin-stimulated umbilical vein endothelial cell cultures by combinations of zinc-chelate-Sepharose chromatography, gel filtration on Sephacryl S-300 and immunoadsorption on an insolubilized murine monoclonal antibody (MA-7D4). The final product was obtained with a recovery of approximately 20% from conditioned medium containing about 3 micrograms/ml PAI-1. The yield of PAI-1 was 15-100 micrograms/umbilical cord, depending on the culture and harvest conditions. SDS gel electrophoresis revealed a main band with Mr = 46,000 both under reducing and non-reducing conditions. On gel filtration on Sephacryl S-300, however, the material was separated in two fractions, one eluting at the void volume, which contains active PAI-1, and one with Mr = 46,000 containing inactive material that could be reactivated with 12 M urea. SDS gel electrophoresis of the isolated high-Mr fraction revealed several bands including a main 46,000-Mr component, which reacted with anti-(PAI-1) antibodies on immunoblotting and neutralized tissue-type plasminogen activator (t-PA). The active high-Mr fraction and the reactivated low-Mr fraction of PAI-1 inhibited t-PA very rapidly with an apparent second-order rate constant of (1.5-4) x 10(7) M-1 s-1. The cDNA of endothelial cell PAI-1 was cloned and expressed in Chinese hamster ovary cells. The translation product, purified from conditioned medium of transfected cells, also revealed a high-Mr and a low-Mr fraction on gel filtration, which were indistinguishable from the natural proteins by physicochemical, immunochemical and functional analysis. On reduced SDS gel electrophoresis, the high-Mr fraction was separated into the Mr-46,000 low-Mr PAI-1 and two other components with Mr 65,000 and one barely entering the gel. When reactivated low-Mr PAI-1 was added to plasma, PAI activity and PAI-1 antigen eluted with an apparent Mr greater than or equal to 300,000 on gel filtration, indicating that active PAI-1 complexes with one or more binding proteins in plasma.     

Post-translational modifications and binding properties of the apically secreted 80-kDa glycoprotein from Madin-Darby canine kidney cells: similarities to the C-terminal portion of the basolaterally secreted fibronectin

Apically secreted 80-kDa glycoprotein (gp 80) from Madin-Darby canine kidney cells was found to be immunoprecipitated by the polyclonal antiserum against fibronectin or a monoclonal antibody specific for the fibronectin C-terminal fibrin binding domain. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), gp 80 migrated as a doublet band under nonreducing conditions. Under reducing conditions, gp 80 was resolved into three distinct bands, respectively of 45-, 40-, and 35-kDa molecular mass. Analysis by two-dimensional SDS-PAGE revealed that gp 80 exists in two molecular forms: one consisting of a 45-kDa subunit and a 40-kDa subunit, and one consisting of a 45-kDa subunit and a 35-kDa subunit. V-8 protease mapping indicated the 40 and 35-kDa subunits as being of the same homologous group and also as bearing partial homology to the 45-kDa subunit. Radioactive labeling revealed that labeled gp 80 was subjected to covalent modifications by sulfation and phosphorylation. Sulfate analysis showed that [35S]sulfate-labeled gp 80 contained ca. 2.45 +/- 0.07% tyrosine-bound [35S]sulfate with the rest being presumably carbohydrate-bound. [32P]-Phosphate-labeled gp 80, on the other hand, was found to contain serine-O-phosphate as the predominant phosphorylated amino acid residue. Employing the affinity gel fractionation technique, it was shown that gp 80 exhibited binding affinities toward heparin and fibrin. Binding of gp 80 to heparin-agarose or fibrin-Sepharose, however, was inhibited in the presence of added fibronectin or the monoclonal antibody. Tryptic peptide mapping revealed common peptide spots between fibronectin and the three subunits of gp 80. Furthermore, Western blot analysis showed that fibronectin could be recognized and bound by anti-gp 80 antibodies. These results indicate that gp 80 bears both structural and functional similarities to the C-terminal portion of the fibronectin molecule.     

Role of epithelial cells of the male excurrent duct system of the rat in the endocytosis or secretion of sulfated glycoprotein-2 (clusterin)

The localization of sulfated glycoprotein-2 (clusterin; SGP-2) was investigated in the rete testis, efferent ducts, and epididymis of the rat using light (LM) and electron (EM) microscope immunocytochemistry. At the LM level, the epithelial cells of the rete testis and efferent ducts demonstrated an intense immunoperoxidase reaction over their apical and supranuclear regions, and sperm in the lumen of the efferent ducts were unreactive. In the EM, gold particles were found exclusively over the endocytic apparatus of these cells. In the proximal area of the epididymal initial segment, an insignificant immunostaining of epithelial cells and sperm was observed. However, the distal area of the initial segment showed a moderate staining over the epithelial principal cells and sperm, while in the intermediate zone of the epididymis a stronger reaction was observed over these cells. The strongest immunoperoxidase reaction was noted in the caput epididymidis, where it formed a distinct mottled pattern. Thus, while some principal cells were intensely stained, others were moderately or weakly stained; a few were completely unreactive. In the corpus and cauda epididymidis, the staining pattern was similar but not as intense. In the EM, only the secretory apparatus of these cells was found to be immunolabeled with gold particles. Sperm in the lumen of these different regions were also labeled. The epithelial clear cells were unreactive throughout the epididymis. Northern blot analysis substantiated these results and showed the presence of highest levels of SGP-2 mRNA in the caput epididymidis, especially in its proximal area, whereas increasingly lower levels were found in the corpus and cauda epididymidis. In summary, these results suggest that testicular SGP-2 dissociates from the sperm during passage through the rete testis and efferent ducts, where it is endocytosed by the epithelial cells lining these regions. In the epididymis, it is replaced by an epididymal SGP-2 that is secreted by the epithelial principal cells of the epididymis. Furthermore, in the epididymis, the principal cells appear to be in different functional states with respect to the secretion of epididymal SGP-2 within a given region of the duct as well as along the epididymal duct.     

Characterization of high mobility group protein levels during spermatogenesis in the rat

The distribution, quantitation, and synthesis of high mobility group (HMG) proteins during spermatogenesis in the rat have been determined. HMG1, -2, -14, and -17 were isolated from rat testes by Bio-Rex 70 chromatography combined with preparative gel electrophoresis. Amino acid analysis revealed that each rat testis HMG protein was similar to its calf thymus analogue. Tryptic peptide maps of somatic and testis HMG2 showed no differences and, therefore, failed to detect an HMG2 variant. Testis levels of HMG proteins, relative to DNA content, were equivalent to other tissues for HMG1 (13 micrograms/mg of DNA), HMG14 (3 micrograms/mg of DNA), and HMG17 (5 micrograms/mg of DNA). The testis was distinguished in that it contained a substantially higher level of HMG2 than any other rat tissue (32 micrograms/mg of DNA). HMG protein levels were determined from purified or enriched populations of testis cells representing the major stages of spermatogenesis; spermatogonia and early primary spermatocytes, pachytene spermatocytes, early spermatids, and late spermatids; and testicular somatic cells. High levels of HMG2 in the testis were due to pachytene spermatocytes and early spermatids (56 +/- 4 and 47 +/- 6 micrograms/mg of DNA, respectively). Mixtures of spermatogonia and early primary spermatocytes showed lower levels of HMG2 (12 +/- 3 micrograms/mg of DNA) similar to proliferating somatic tissues, whereas late spermatids had no detectable HMG proteins. The somatic cells of the testis, including isolated populations of Sertoli and Leydig cells, showed very low levels of HMG2 (2 micrograms/mg of DNA), similar to those in nonproliferating somatic tissues. HMG proteins were synthesized in spermatogonia and primary spermatocytes, but not in spermatids. Rat testis HMG2 exhibited two bands on acid-urea gels. A "slow" form comigrated with somatic cell HMG2, while the other "fast" band migrated ahead of the somatic form and appeared to be testis-specific. The "fast" form of HMG2 accounted for the large increase of HMG2 levels in rat testes. These results show that the very high level of HMG2 in testis is not associated with proliferative activity as previously hypothesized.     

Physicochemical characterization of photoaffinity-labeled angiotensin II receptors

Specific receptor sites for angiotensin II (AII) were analyzed in the adrenal cortex and other target tissues including liver, anterior pituitary gland, and smooth muscle, after covalent labeling with the radioactive photoaffinity analog 125I-[Sar1,(4-N3)Phe8]-AII. The photoreactive AII derivative retained high affinity for adrenal receptors and full steroidogenic activity in adrenal glomerulosa cells. In bovine adrenal cortex, covalent labeling of AII receptors by the photoreactive analog was specifically inhibited by [Sar1]AII with an IC50 of about 5 nM. The Mr of the covalent AII-receptor complex during polyacrylamide gel electrophoresis of the labeled protein under reducing conditions was 58,000. Under non-reducing conditions, a minor band with Mr of 105,000 was also observed. Two labeled species were also found during gel permeation chromatography of the detergent-solubilized complex, with Mrs of 64,000 and 86,000. The pl of the solubilized photolabeled complex was absorbed to wheat germ lectin Sepharose 6MB and could be eluted by N-acetylglucosamine. The Mrs of specific AII-binding sites in several target tissues, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed target tissues, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed significant differences within and between species. The most striking differences were between rat adrenal cortex (79,000) and both rat liver (60,000) and bovine adrenal cortex (58,000). After enzymatic deglycosylation, the Mr of the major component present in the bovine and rat adrenal cortex decreased by 40% and 55% to 35,000 and 34,000, respectively, suggesting that variations in carbohydrate content contribute to the physical heterogeneity of AII receptors in individual target tissues.     

Characterization of C1q-binding material released from the membranes of Raji and U937 cells by limited proteolysis with trypsin.

C1q-binding material was released, by limited proteolysis with trypsin, from the membranes of intact cells of the Raji lymphoblastoid cell line and the U937 monocytic cell line. The trypsin-digested C1q-binding material was purified from the supernatant of the trypsin-treated cells by affinity chromatography on C1q-Sepharose followed by gel filtration. On gel filtration in non-dissociating conditions this material behaved as a molecule of approx. Mr 65,000, while on SDS/polyacrylamide-gel electrophoresis two peptides of Mr 10,000 and Mr 15,000 were seen under both reducing and non-reducing conditions. Evidence for the synthesis of the C1q-binding material by both Raji and U937 cells was obtained by biosynthetic-labelling studies using [35S]cysteine and [35S]methionine.     

External labelling of glycoproteins from first-trimester human placental microvilli.

The brush-border glycoproteins of first-trimester human placentas were investigated by using two external labelling techniques: (1) sequential digestion with neuraminidase and galactose oxidase, followed by reduction with NaB3H4, which 3H-labels terminal galactose and galactosamine residues; and (2) sequential treatment with periodate and NaB3H4, which 3H-labels terminal sialic acid residues. The labelling procedures were performed on intact tissue so that the results would more closely approximate the topography of the brush border in vivo. The microvilli were isolated, subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and the [3H]glycoproteins detected by fluorography. Densitometer scans of the fluorograms of the [3H]galactoproteins showed that, under reducing conditions, 90% of the protein-associated radioactivity was incorporated into two glycoproteins. The major [3H]galactoprotein of early placental microvilli had an estimated molecular mass of 92 kDa (desialylated) and migrated as a diffuse band. A minor 180 kDa glycoprotein was less consistently labelled. No change in the apparent molecular mass of either component was detected in the absence of beta-mercaptoethanol, suggesting that the 180 kDa component was not a dimer of the 92 kDa glycoprotein. The remaining 10% the radioactivity was equally distributed among several minor membrane components. Densitometer scans of the fluorograms of the [3H]sialoproteins showed that, under either reducing or non-reducing conditions, 90% of the 3H was preferentially incorporated into the 92-110 kDa region of the gel. Although no distinct bands were visible, the higher-molecular-mass region of this area was always most heavily labelled. A minor 180 kDa glycoprotein was also 3H-labelled. The pattern of brushborder [3H]glycoproteins from first-trimester placentas differed markedly from that of term placental microvilli and from placental fibroblast plasma membranes that were 3H-labelled by identical external labelling techniques. These results indicate that: (1) the glycoprotein determinants of brush-border topography change during pregnancy; (2) within the placenta, the major 92 kDa (desialylated) determinant, which has not been previously described, is unique to the trophoblastic cells.