Heparan sulfate proteoglycan from human tubular basement membrane. Comparison with this component from the glomerular basement membrane

Heparan sulfate proteoglycan (HSPG) was extracted from human tubular basement membrane (TBM) with guanidine and purified by ion-exchange chromatography and gel filtration. The glycoconjugate was sensitive to heparitinase and resistant to chondroitinase ABC, had an apparent molecular mass of 200-400 kDa and consisted of 70% protein and 30% glycosaminoglycan. The amino acid composition was characterized by its high content of glycine, proline, alanine and glutamic acid. Hydrolysis with trifluoromethanesulfonic acid yielded core proteins of 160 and 110 kDa. The heparan sulfate (HS) chains obtained after alkaline NaBH4 treatment had a molecular mass of about 18 kDa. Results of heparitinase digestion and HNO2 treatment suggest a clustering of sulfate groups in the distal portion of the HS side chains. These chemical data are comparable to those obtained previously on glomerular basement membrane (GBM) HSPG (Van den Heuvel et al. (1989) Biochem. J. 264, 457-465). Peptide patterns obtained after trypsin, clostripain or V8 protease digestion of TBM and GBM HSPG preparations showed a large similarity. Polyclonal antisera and a panel of monoclonal antibodies raised against both HSPG preparations and directed against the core protein showed complete cross-reactivity in ELISA and on Western blots. They stained all basement membranes in an intense linear fashion in indirect immunofluorescence studies on human kidneys. Based on these biochemical and immunological data we conclude that HSPGs from human GBM and TBM are identical, or at least very closely related, proteins.     

Studies on collagen metabolism in rats. I. Age-related changes in turnover and amino acid composition of the collagen of glomerular basement membrane and tail tendon

To examine the effect of age on the collagen metabolism of glomerular basement membrane (GBM) and tail tendon, both structures were labeled in vivo by the injection of tracer amounts of radioactive proline. The analyses of the labeled proline and, especially, hydroxyproline revealed that synthesis and degradation are reduced in senescent rats. The calculated turnover rate for GBM-collagen is prolonged by about 40% with increased life span. The reduced catabolism is responsible for the age-related thickening of the GBM. In addition, compositional analyses of isolated GBM and tail tendon were performed. Substantial age changes were observed in the amino acid composition of both structures. The hydroxylated amino acids showed a progressive increase with age, while proline and lysine were found to decline.     

Collagen synthesis and secretion by isolated rat renal glomeruli.

Glomeruli were isolated from rat renal cortex and incubated with radioactive lysine to study in vitro collagen synthesis in these preparations. Glomerular basement membrane was obtained by sonication, and the appearance of [-14C]lysine and hydroxylysine in medium, membrane and intracellular proteins was determined. Total glomerular incorporation of [-14C]lysine into protein linearly increased for up to 2-h period, and membrane hydroxylysine content gradually rose during this time. Hydroxy[-14C]lysine was recovered in the 105 000 times g pellet, reaching a hydroxylysine content of 22 percent in this intracellular fraction after 90 min of incubation. 60 percent of the protein secreted into the medium, and about 75 percent of newly synthesized sonicated basement membrane was acetic acid soluble. Hydroxylysine content was 33 percent in the acetic acid-insoluble fraction of sonicated membrane, suggesting that basement-membrane collagen was a significant component of total collagen synthesized by these preparation, The ability of isolated glomeruli to synthesize and secrete basement-membrane protein will be useful for studies concerning control of glomerular collagen and basement-membrane synthesis.     

Changes in the glomerular filtration barrier during aging in rats

In this work, we analysed histochemical, biochemical and functional modifications of the glomerular basement membrane (GBM), occurring for aging, in the Rat. The results suggest an increase of collagenous components and a decrease of sulfated glucosaminoglycans as a function of age. In other respects, fixed anionic sites of the GBM, disclosed by colloidal iron, are almost exclusively restricted to the laminae rarae in one month-old rats, whereas the marker appears randomly scattered among the lamina densa in 12 month-old animals. These changes could be the cause of increased permeability of the GBM during aging.     

Chemical characterization of glomerular and tubular basement membranes of cattle of different ages

Glomerular and tubular basement membranes were isolated from fetal, neonatal, young and adult bovine kidneys.An isolation method with sieves for both glomeruli and tubules from the same kidney was developed. A detergent procedure appeared to give purer glomerular and tubular basement membrane preparations than the generally used sonication method. No large differences were found in the composition of glomerular and tubular basement membrane of adult animals.Glomerular and tubular basement membrane preparations of the four age groups showed an increase with age of hydroxylysine and both 3- and 4-hydroxyproline. The most marked increases appeared at different stages of development, that of tubular basement membrane being between fetal and neonatal stages and glomerular basement membrane between 18 weeks old and adult animals. The ratio of 3- to 4-hydroxyproline increased considerably during development. Total imino acid content was higher for both types of basement membrane from adult than from young animals, while total content of hydroxylysine plus lysine remained fairly constant.The increase in hydroxylation of lysine was accompanied by a corresponding change in glucose and galactose content so that the ratio of galactose to hydroxylysine or glucose to galactose remained constant. Fucose content of both types of basement membranes was the same for all age groups but content of aminosugars and mannose gradually increased with age.     

Glomerular extracellular matrices and anionic sites in aging ddY mice: a morphometric study

A morphometric study was undertaken to examine age-related changes in glomerular ultrastructure and anionic sites in ddY male mice at various ages. A progressive increase in glomerular extracellular matrices, including thickening of the glomerular basement membrane (GBM), formation of GBM nodules, and mesangial matrix increase, was found to be the primary age-related ultrastructural change in aging mice; there were also electron-dense deposits in mesangial and subepithelial regions. The extent of GBM thickening in mice was less than was reported in rats. Rather, the GBM nodules, which had the same electron density as the lamina densa (LD) and protruded on the subepithelial side of the GBM, were more striking. Quantitative evaluation showed that GBM thickness, number and size of GBM nodules, and the area of the mesangial matrix were significantly correlated with the age of the mice. The distribution of anionic sites in the glomeruli of aging animals was described for the first time. No statistically significant differences were noted between the number of glomerular anionic sites in the different age groups. These results indicate that the increase in glomerular extracellular matrices reported in aged rats was also present in aged mice, although the extent of various changes was different. The results also indicate that this increase in glomerular extracellular matrices with age was not accompanied by significant alteration in glomerular anionic sites.     

Post-embedding colloidal gold immunolocalization of laminin to the lamina rara interna, lamina densa, and lamina rara externa of renal glomerular basement membranes

Ultrastructural distribution of laminin within renal glomerular (GBM) and tubular basement membranes (TBM) was investigated using post-embedding immunolocalization with colloidal gold. Rat kidneys were fixed with 4% formaldehyde and embedded at 4 degrees C in Lowicryl K4M medium. Thin sections were then sequentially treated with affinity-purified rabbit anti-laminin IgG and anti-rabbit IgG conjugated to 10 nm diameter colloidal gold. Gold bound specifically to the GBM and TBM with particle densities of 690/micron2 and 731/micron2, respectively. In the GBM, the number of gold particles bound/micron2 of lamina densa greater than lamina rara externa greater than lamina rara interna. Closely similar binding patterns were found when kidneys were fixed with 0.5% glutaraldehyde plus 3% formaldehyde and embedded at 60 degrees C in L.R. White resin, but slightly less gold bound to sections overall than that seen with formaldehyde alone and Lowicryl. Taken together, these results illustrate that anti-laminin IgG, whether applied to fixed sections in vitro or introduced in vivo, bound to the lamina rara interna, lamina densa, and lamina rara externa of the GBM and throughout the TBM.     

Composition of renal basement membranes isolated under various conditions

GBM isolated from a surgical biopsy directly or after a 22 hr incubation period--to imitate the usual interval between death and isolation--appeared to be nearly identical in amino acid composition. Sonication and detergent procedures for isolation of GBM and TBM lead to preparations of different chemical composition. Phosphorus analysis and electron micrographs indicate the presence of material of supposedly cellular origin in sonicated but not in detergent-treated bovine and human GBM. Detergent-treated bovine and human GBM preparations are more enriched in the collagen-typical amino acids than sonicated samples. SDS-PAGE analyses show a nearly identical polypeptide pattern. Sonicated and detergent-treated bovine TBM preparations are free of cellular material. They show in SDS-PAGE a similar heterogeneous polypeptide pattern, but with lower intensities of three components with molecular weights between 30 and 60 kdalton. Sulfated GAG's are present in higher concentration in sonicated than in detergent-treated GBM and TBM. Collagen is not extracted from glomeruli and tubules by detergent treatment.     

Heparan sulfate proteoglycan from human and equine glomeruli and tubules

1. Proteoglycans were isolated from human and equine glomeruli or tubules by guanidine extraction and anion exchange chromatography. 2. These proteoglycan preparations contained about equal amounts of heparan sulfate and chondroitin sulfates. 3. During the preparation of glomerular or tubular basement membranes the main part of proteoglycans (greater than 50%) was extracted in the salt extract. Chondroitin sulfate proteoglycan was mainly found in the water and salt extracts of glomeruli and tubules, heparan sulfate proteoglycan in the deoxycholate extracts and the basement membranes. 4. The glomerular basement membrane (GBM) contains about 12% (human) or 20% (equine) of the proteoglycans of the total glomerulus. They consist of greater than 70% (equine) or 80% (human) of heparan sulfate. 5. Heparan sulfate proteoglycan was isolated from the proteoglycan preparations of human or equine glomeruli and tubules by additional treatment with nucleases and chondroitinase ABC followed by CsCl gradient centrifugation. 6. Protein accounts for about 40% (dry weight) of the heparan sulfate proteoglycans. Their amino acid composition is characterized by a high content of glycine, but 3-hydroxyproline, 4-hydroxyproline and hydroxylysine are lacking. 7. The biochemical characteristics of the heparan sulfate proteoglycan of human or equine glomeruli or tubules differ from that isolated from rat glomeruli by their higher protein content and their amino acid composition. The significance of these differences is discussed.     

Presence of lysosomal enzymes in the normal glomerular basement membrane matrix

Summary The question posed in the present study was: are there hydrolytic enzymes, including proteases, present in the extracellular matrix of the glomerular basement membrane? If these enzymes are present they may play a role in the catabolism of the glomerular basement membrane (GBM) and removal of macromolecular debris resulting from ultrafiltration. Enzymes, acid phosphatase - the marker for lysosomal enzymes - β-galactosidase, β-glucuronidase and acid protease (using albumin as substrate) were biochemically assayed in purified basement membrane preparations. It was found that all enzymes were present in significant amounts in the basement membrane. Compared to other enzymes, acid protease activity was present in much higher amounts. The pH optima of these enzymes were variable but all had significant activity at neutral pH. A method was developed to localize the marker enzyme, acid phosphatase, ultrastructurally in the basement membrane in order to substantiate the biochemical findings. Activity was shown by the presence of dense deposits of lead phosphate. Staining for acid phosphatase could also be shown on isolated, purified basement membrane. The demonstration of acid hydrolases in the GBM matrix argues for their role in (i) the extracellular turnover of basement membrane macromolecules, and (ii) clearance of debris of ultrafiltration which tend to clog the membrane pores.     

Phospholipid of the rat glomerular basement membrane in experimental nephrosis

Phospholipids were found to be a constant component of rat glomerular basement-membrane preparations. The concentration fell during preparation of basement membrane by sonication of whole glomeruli, but then remained constant despite continued sonication. The proportions of the individual phospholipids were different from those of whole renal tissue or of isolated glomeruli. The basement-membrane preparations had no (Na(+)+K(+))-activated adenosine triphosphatase activity, an enzyme that is bound to plasma membranes. The concentration of lipid P was decreased on exposure in vivo or in vitro to antiserum against basement membrane; 7 days after injection of antiserum there was a change in the phospholipid composition, with a relative increase in phosphatidylcholine and a decrease in sphingomyelin content. The metabolic turnover rate of the lipid P remaining in the membrane was normal, as determined by (32)P incorporation. The loss of phospholipid was associated with decreases in the relative concentrations of hydroxyproline, hydroxylysine and glycine, and relative increases in proline, lysine, serine, threonine and valine. Administration of aminonucleoside and daunomycin produced proteinuria but did not cause a decrease in lipid P. Anticollagen and anti-lymphocyte sera that attached to the basement membrane but failed to produce proteinuria, also failed to affect the phospholipid content.     

Selective immunoreactivities of kidney basement membranes to monoclonal antibodies against laminin: localization of the end of the long arm and the short arms to discrete microdomains

To examine the ultrastructural distribution of laminin within kidney basement membranes, we prepared rat anti-mouse laminin mAbs to use in immunolocalization experiments. Epitope domains for these mAbs were established by immunoprecipitation, immunoblotting, affinity chromatography, and rotary shadow EM. One mAb bound to the laminin A and B chains on blots and was located to a site approximately 15 nm from the long arm-terminal globular domain as shown by rotary shadowing. Conjugates of this long arm-specific mAb were coupled to horseradish peroxidase (HRP) and intravenously injected into mice. Kidney cortices were fixed for microscopy 3 h after injection. HRP reaction product was localized irregularly within the renal glomerular basement membrane (GBM) and throughout mesangial matrices. In addition, this mAb bound in linear patterns specifically to the laminae rarae of basement membranes of Bowman's capsule and proximal tubule. This indicates the presence of the long arm immediately beneath epithelial cells in these sites. The laminae densae of these basement membranes were negative by this protocol. In contrast, the lamina rara and densa of distal tubular basement membranes (TBM) were both heavily labeled with this mAb. A different ultrastructural binding pattern was seen with eight other mAbs, including two that mapped to different sites on the short arms by rotary shadowing and five that blotted to a large pepsin-resistant laminin fragment (P1). These latter mAbs bound weakly or not at all to GBM but all bound throughout mesangial matrices. In contrast, discrete spots of HRP reaction product were seen across all layers of Bowman's capsule BM and proximal TBM. These same mAbs, however, bound densely across the full width of distal TBM. Our findings therefore show that separate strata of different basement membranes are variably immunoreactive to these laminin mAbs. The molecular orientation or integration of laminin into the three dimensional BM meshwork therefore varies with location. Alternatively, there may be a family of distinct laminin-like molecules distributed within basement membranes.     

Studies on the rat glomerular basement membrane: Age-related changes in composition

To examine the effect of age on the glomerular basement membrane, compositional analyses were performed on membranes isolated in highly purified form from rats at different stages of their growth (35 to 200 days old). Substantial age-related changes were observed in the amino acid composition of the basement membranes. A significant correlation with age (P < 0.01) was evident in the contents of 3- and 4-hydroxyproline, threonine, serine, alanine, valine, half-cystine, hydroxylysine, and lysine. Of these amino acids, hydroxylysine and both isomers of hydroxyproline demonstrated a progressive increase with age, while the others were found to decline. The direct relationship of hydroxylysine content with age (P < 0.001) was associated with an inverse correlation of lysine with age (P < 0.001) so that the ratio of hydroxylysine to lysine increased in a highly significant manner from 0.92 at 35 days to 1.33 at 200 days. This elevation in the hydroxylysine content was accompanied by an augmentation in the number of saccharide units linked to it so that the percentage glycosylation of this amino acid was not significantly affected by age. The relative differences in the hydroxylysine and lysine levels between young and older rats were maintained in sodium dodecyl sulfateextracted membranes. These results suggest that the compositional changes observed during the aging process reflect an alteration in the subunit makeup of the basement membrane, possibly due to an increased synthesis or decreased degradation of the more collagen-like polypeptide components.     

Biosynthesis of basement membrane collagen by rabbit corneal endothelium in vitro.

The synthesis of collagen has been demonstrated in endothelial cells of Descemet's membrane isolated from rabbit cornea. Incorporation of [14C]proline and [14C]lysine into nondialyzable protein was measured in the medium and cell fraction after incubating Descemet's membrane for up to 5 hours. In the [14C]collagen synthesized by the endothelium, 15% of the hydroxy[14C]proline was present as the 3-isomer. About 98% of the hydroxy[14C]lysine in the 14C-labeled-protein found in the medium was glycosylated; 95% of the glycosylated hydroxy[14C]lysine was in the form of the disaccharide glucosyl-galactosyl-hydroxy[14C]lysine. Time course experiments with [14C]proline indicated that there was a delay of about 60 min before significant amounts of [14C]collagen were secreted into the medium. The initial polypeptides of [14C]collagen synthesized by the corneal endothelium had an apparent molecular weight of 155,000. The chemical and physical properties of the [14C]collagen synthesized by rabbit corneal endothelium are consistent with those of basement membrane collagen synthesized by other cell types.     

Glucosepane is a major protein cross-link of the senescent human extracellular matrix. Relationship with diabetes

The extracellular matrix in most tissues is characterized by progressive age-related stiffening and loss of proteolytic digestibility that are accelerated in diabetes and can be duplicated by the nonenzymatic reaction of reducing sugars and extracellular matrix proteins. However, most cross-links of the Maillard reaction described so far are present in quantities too low to account for these changes. Here we have determined in human skin and glomerular basement membrane (GBM) collagen the levels of the recently discovered lysine-arginine cross-links derived from glucose, methylglyoxal, glyoxal, and 3-deoxyglucosone, i.e. glucosepane, MODIC, GODIC, and DOGDIC, respectively. Insoluble preparations of skin collagen (n = 110) and glomerular basement membrane (GBM, n = 28) were enzymatically digested, and levels were measured by isotope dilution technique using liquid chromatography/mass spectrometry. In skin, all cross-links increased with age (p < 0.0001) except DOGDIC (p = 0.34). In nondiabetic controls, levels at 90 years were 2000, 30, and 15 pmol/mg for glucosepane, MODIC, and GODIC, respectively. Diabetes, but not renal failure, increased glucosepane to 5000 pmol/mg (p < 0.0001), and for all others, increased it to <60 pmol/mg (p < 0.01). In GBMs, glucosepane reached up to 500 pmol/mg of collagen and was increased in diabetes (p < 0.0001) but not old age. In conclusion, glucosepane is the single major cross-link of the senescent extracellular matrix discovered so far, accounting for up to >120 mole% of triple helical collagen modification in diabetes. Its presence in high quantities may contribute to a number of structural and cell matrix dysfunctions observed in aging and diabetes.     

Studies of the permeation properties of glomerular basement membrane: cross-linking renders glomerular basement membrane permeable to protein.

Cross-linking glomerular basement membrane (GBM) has been shown to render it more permeable to protein. Isolated pig GBM was cross-linked with dimethylmalonimidate which reacts selectively with lysine epsilon-NH2 groups or with glutaraldehyde, a less selective cross-linking agent. Studies of the ultrafiltration properties of these materials in vitro using cytochrome c, myoglobin, bovine serum albumin and immunoglobulin showed that cross-linking had markedly increased solvent and protein fluxes as compared with native membranes particularly at higher pressures. Filtration studies with serum demonstrated that the cross-linked membranes were more permeable to serum proteins. Thickness measurements under pressure indicated that cross-linked membrane was less compressed than native membrane as pressure was increased. Pore theory did not provide a suitable model for analysis of the results, but analysis of the results using the fibre-matrix hypothesis indicated that cross-linking had the effect of bundling together the fibres (type IV collagen) in the GBM matrix. The effect of cross-linking on filtration could be explained by a combination of contraction of the membrane, fibre bundling and increased rigidity compared with native membrane. Cross-linking of GBM might lead to long-term damage of the glomerular capillary wall in nephritis, so promoting proteinuria.     

Glycolipids from the cell walls of streptococci

A streptococcus, serologically defined as a Z₃III strain was compared with a mutant strain Z₃ lacking the type III polysaccharide antigen. The loss of type antigen represents a decrease in the carbohydrate content of the cell wall of the mutant and is accompanied by long-chain formation, increased sensitivity to streptomycin and agglutination in saline. Cell-wall preparations can be freed of membrane contamination by treatment with hot sodium dodecylsulfate (SDS). This resulted in a doubling of the ratio of muramic acid to lysine and in the disappearance of phospholipids. It could be shown that the membrane-free cell walls of these strains still contained appreciable amounts of glycolipids which could be identified as monoglucosyl glyceride and diglucosyl-diglyceride.     

Studies of the permeation properties of glomerular basement membrane: cross-linking renders glomerular basement membrane permeable to protein

Cross-linking glomerular basement membrane (GBM) has been shown to render it more permeable to protein. Isolated pig GBM was cross-linked with dimethylmalonimidate which reacts selectively with lysine ?-NH₂ groups or with glutaraldehyde, a less selective cross-linking agent. Studies of the ultrafiltration properties of these materials in vitro using cytochrome c, myoglobin, bovine serum albumin and immunoglobulin showed that cross-linking had markedly increased solvent and protein fluxes as compared with native membranes particularly at higher pressures. Filtration studies with serum demonstrated that the cross-linked membranes were more permeable to serum proteins. Thickness measurements under pressure indicated that cross-linked membrane was less compressed than native membrane as pressure was increased. Pore theory did not provide a suitable model for analysis of the results, but analysis of the results using the fibre-matrix hypothesis indicated that cross-linking had the effect of bundling together the fibres (type IV collagen) in the GBM matrix. The effect of cross-linking on filtration could be explained by a combination of contraction of the membrane, fibre bundling and increased rigidity compared with native membrane. Cross-linking of GBM might lead to long-term damage of the glomerular capillary wall in nephritis, so promoting proteinuria.     

Extralysosomal localisation of acid phosphatase in the rat kidney

 There is strong evidence that acid phosphatase (AcPase) plays an important role in the catabolism of the glomerular basement membrane (GBM) and the removal of macromolecular debris resulting from ultrafiltration. Recent enzyme histochemical investigations provide new evidence of the antithrombotic and anti-inflammatory function of ADPase and on the distribution of AcPase in mouse kidney tubule cells. By means of 3 mM cerium as the trapping agent and 1 mM p-nitrophenyl phosphate as the substrate, extralysosomal AcPase could be demonstrated at the ultrastructural level. Following a mild perfusion fixation (2% formaldehyde + 0.07% glutaraldehyde), an effective postfixation and short enzyme incubations (20 min) with microwave irradiation, highly specific enzyme histochemical reaction product and reasonable structural preservation were obtained. Extralysosomal, membrane-bound AcPase was observed along the endoplasmic reticulum, the trans-Golgi cisternae, the nuclear envelope, basal infoldings of the proximal and distal tubular cells and on glomerular profiles, e.g. cell membranes of podocytes, endothelium and basement membrane. Large amounts of extralysosomal AcPase were observed in the basement membrane of glomeruli, in contrast to no AcPase activity in the tubular and mesangial basement membrane. The observed difference in AcPase activity in the tubular epithelial basement membrane and the GBM supports the idea that AcPase in GBM specifically serves in the clearance of macromolecular debris to facilitate ultrafiltration. In the GBM a laminar distribution is observed, suggesting that both epithelial and endothelial cells are involved in the production of AcPase. Accepted: 16 September 1997     

The embryonic rat parietal yolk sac. The role of the parietal endoderm in the biosynthesis of basement membrane collagen and glycoprotein in vitro.

Basement membrane biosynthesis in vitro was studied in a rapidly growing embryonic tissue, the rat parietal yolk sac. This tissue consists of a thick, nonvascular basement membrane (Reichert's membrane) separating two cellular layers (parietal endoderm and trophoblast). Morphologically, Reichert's membrane appeared similar to other basement membranes. Previous analysis of the amino acid and carbohydrate composition of acellular Reichert's membrane showed it to be typical of basement membranes isolated from other tissues and species. Analysis of [14-C]proline incorporation and hydroxy [14-C]proline synthesis during the third quarter ogestation in vitro showed that basement membrane collagen synthesis in the parietal yolk sac was maximal around the 14th day of gestation. At this time, basement membrane collagen represented nearly 10% of the newly synthesized protein. The collagen synthesized in this system was characteristic of basement membrane collagen in that about 11% of the total hydroxy [14-C]proline was present as the 3-isomer. In addition, after incubation in the presence of [14-C]lysine, 83 to 94% of the hydroxy[14-C]lysine was glycosylated, with the predominant form being glucosylgalactosylhydroxy[14-C]lysine. When the parietal endoderm and trophoblast were incubated separately with [14-C]proline, it was determined that the former was solely responsible for the synthesis of basement membrane collagen since essentially all of the 4-hydroxy[14-C]proline was associated with this cell type. Autoradiographic experiments with [3-H]glucosamine also served to localize the synthesis of noncollagen basement membrane glycoprotein components to the parietal endoderm. As with the results reported for basement membrane collagen secretion in embryonic chick lens cells, there appeared to be approximately a 60-min delay between the incorporation of [14-C]proline into protein and the secretion of collagen as measured by the appearance of 4-hydroxy[14-C]proline in the culture medium. Experiments utilizing [3H]glucosamine to monitor glycoprotein synthesis did not show a delay between the incorporation of [3H]glucosamine and the secretion of nondialyzable 3-H into the medium. The results obtained using the parietal yolk sac system to study basement membrane biosynthesis were compared to those previously obtained using the kidney glomerular and embryonic chick lens systems. It was concluded that the parietal yolk sac system is superior for a number of reasons: (a) the extracellular matrix appeared to contain only basement membrane components; there was no contamination by acid mucopolysaccharides or other types of collagen; (b) only a single cell type appeared to be responsible for the synthesis of basement membrane components; and (c) a relatively large percentage of the newly synthesized protein was basement membrane collagen.