ISSN:1420-4096    

DOI:10.1159/000172732

出版商:S. Karger AG    

年代:1980

数据来源: Karger

摘要:

Glomerular basement membrane (GBM) preparations were enzymatically glucosylated and applied to proteolytic degradation by several enzymes. The split products were then characterized and quantitatively estimated by high pressure liquid chromatography. For this purpose, GBMs were isolated by a sieving and sonication method, incubated with glucose and digested with the proteases trypsin, chymotrypsin, papain, pepsin and a lysosomal preparation. Comparison of the concentrations of split products obtained by proteolytic degradation of normal and nonenzymatically glucosylated membranes showed a remarkable reduced susceptibility of the nonenzymatically glucosylated membranes, possibly due to steric hindrance or altered electrical charge of the glucosylated membrane proteins. This could be interpreted as an additional factor for accumulation of basement membrane material in the diabetic state, that not only increased basement membrane synthesis may occur but also reduced catabolism could possibly contribute to the diabetic changes.

ISSN:1420-4096    

DOI:10.1159/000172733

出版商:S. Karger AG    

年代:1980

数据来源: Karger

摘要:

Bovine glomerular basement membrane was subjected to limited pepsin digestion and the solubilized collagenous polypeptides were characterized. Several electrophoresis systems were used which enabled an examination over a molecular weight range from 20,000 to > 106. A 0.1% SDS-5% polyacrylamide gel system resolves the reduced digestion product into 17 polypeptides ranging in molecular weight from 78,000 to 340,000. The larger collagenous components were resolved on a 0.1 % SDS-2.5% agarose gel system. The nonreduced digestion product resolves into 13 components which vary in molecular weight from 85,000 to 5 million. Upon reduction, the majority of this material is converted to a 165,000 molecular weight component(s) and cross-linked (aldehyde derived) multimers of this component(s) containing as many as 6 cross-linked monomers. The digestion product was subjected to a second pepsin digestion after reduction and alkylation under nondenaturing conditions. This results in a conversion of the larger polypeptides to three lower molecular weight peptides, two of which exhibit an electrophoretic migration identical to α1-and α2-chains of collagen. The results indicate that the collagenous domain of glomerular basement membrane consists of various size collagen molecules connected by disulfide bonds and aldehyde-derived cross-links to form high molecular weight aggregates containing as many as 30 of these polypeptides, and that the larger collagenous polypeptides contain α-size segments within their structu

ISSN:1420-4096    

DOI:10.1159/000172734

出版商:S. Karger AG    

年代:1980

数据来源: Karger

摘要:

Renal tubular and glomerular basement membranes were isolated from rabbit, rat and mouse kidneys. Methods were developed to obtain the basement membrane from limited numbers of animals. Rabbit and mouse tubular basement membrane as well as rabbit and rat glomerular basement membrane were obtained from single animals in quantities sufficient for electrophoresis and chemical analysis. The chemical composition of all the basement membranes were compared and sodium dodecylsulfate-polyacrylamide gel electrophoresis was performed on rabbit tubular and glomerular basement membranes in order to compare their polypeptide composition. The chemical composition of the tubular basement membranes were similar, each species having nearly equal concentrations of glycine, hydroxyproline and hydroxylysine, respectively. Glomerular basement membranes were also similar to each other and close to tubular basement membranes in chemical composition. Within each species, the glycine, hydroxyproline and hydroxylysine values for tubular basement membrane were about 10% higher than for glomerular basement membrane. The polypeptide composition of a reduced, sodium dodecylsulfate-soluble fraction of the rabbit basement membranes appeared to be alike with two exceptions. A prominent band of Mr = 160,000 seen in gels of tubular basement membrane was present as a lightly staining band in glomerular basement membrane samples and a prominent band of Mr = 140,000 in gels of glomerular basement membrane was seen as a light band in samples of tubular basement membrane.

ISSN:1420-4096    

DOI:10.1159/000172736

出版商:S. Karger AG    

年代:1980

数据来源: Karger

摘要:

Native type IV collagen was purified from bovine kidney cortex homogenate by a method involving pepsin-solubilization and heat gelation. The near homogenous protein has a molecular weight of ∼ 380K and the chain composition C2D. The subunits are linked by disulfïde bridg

ISSN:1420-4096    

DOI:10.1159/000172737

出版商:S. Karger AG    

年代:1980

数据来源: Karger

摘要:

Primary cultures of rabbit corneal endothelial cells were labeled with proline and the medium proteins were fractionated by sedimentation through sucrose density gradients. A new collagen, endothelial cell (EC) collagen, was completely separated from type IV collagen without denaturation by this technique. Sodium dodecyl sulfate electrophoresis demonstrated EC collagen fragments of 180K, 120K, and 60K daltons. Prior pepsin treatment produced only the 60K fragment after electrophoresis. The cyanogen bromide peptide pattern of the 180K EC peptide demonstrated that EC was different from type IV. A preliminary structural model for the largest helical form (∼ 540K daltons) of EC collagen is presente

ISSN:1420-4096    

DOI:10.1159/000172738

出版商:S. Karger AG    

年代:1980

数据来源: Karger

摘要:

The biosynthesis of the collagenous components of the rat lens capsule and glomerular basement membrane (GBM) has been investigated. Intact lenses, isolated lens capsules and isolated renal glomeruli incorporate [3H]proline into 4-hydroxy[3H]proline containing polypeptides of approximate molecular weight 180,000 (analysed under reducing conditions). These polypeptides are deposited in the deoxycholate-insoluble basement membrane matrix and correspond to subunits of the intact, non-radioactive basement membranes. With time a proportion of the newly synthesised collagenous polypeptides become assembled into high molecular weight aggregates which are stabilized by lysine-and/or hydroxylysine-derived cross-links. Despite the similar patterns of collagen biosynthesis in the rat lens capsule and GBM, the latter has a more complex polypeptide composition the origin of which remains uncertain.

ISSN:1420-4096    

DOI:10.1159/000172739

出版商:S. Karger AG    

年代:1980

数据来源: Karger

摘要:

Glomerular basement membrane (GBM) was prepared from 2- to 3-month-old Sprague-Dawley rats by differential sieving and sonication. 80% of the membrane was soluble in 1% sodium dodecyl sulphate (SDS) and 1% 2-mercaptoethanol. The soluble fraction was resolved into 15 bands in the molecular weight range 30,000 to 300,000 by SDS-polyacrylamide gel electrophoresis. The major bands present had apparent molecular weights of 100,000 and 148,000. Treatment of GBM with SDS alone solubilised mainly low molecular weight components (45,000–150,000) but when the residue was treated with SDS and 2-mercaptoethanol higher molecular weight material was solubilised. Partial solubilisation of GBM was also achieved with pepsin. Digestion for 18 h at 4°C resulted in 20% of the membrane being solubilised but this was increased to 55% at 10 °C. The amino-acid composition of pepsin-soluble GBM was more collagen-like than the residue remaining after enzyme digestion. Although the residue was more polar than whole GBM it still contained significant amounts of glycine and hydroxyproline and could be further sub fractionated with SDS into a soluble fraction, the amino acid content of which was similar to whole GBM and a collagenous residue containing 317 residues/1,000 of glycine. When pepsin solubilised GBM was subjected to horizontal electrophoresis in SDS-polyacrylamide slab gels the principal bands migrated in the pro-α-chain region. This material was heterogeneous and in addition to the principal components, 6 components with apparent molecular weights less than 95,000 were present together with high molecular weight material in the γ and β regions of the gel. The band pattern of the pepsin-insoluble material was similar although the intensities of some individual bands varied significantly from that of the pepsin-soluble material. Rat GBM can therefore be fractionated by treatment with SDS alone, SDS together with 2-mercaptoethanol and pepsin digestion. The data reported is compatible with the presence of a mixture of collagen-like and polar regions rather than a major single collagenous component (type IV col

ISSN:1420-4096    

DOI:10.1159/000172740

出版商:S. Karger AG    

年代:1980

数据来源: Karger

摘要:

1-month-old Sprague-Dawley rats were fed synthetic diets containing 55% sucrose (SU) or starch (ST) as the sole source of carbohydrate for 2, 3 or 8 months. Both the ST and SU fed rats gained weight normally, but SU fed rats had enlarged kidneys. A higher yield of glomerular basement membrane (GBM) was recovered from 9-month-old SU rats. An increase in the hydroxylated amino acid content was found in GBM prepared from SU fed rats and the glycine content was also higher. The increase in the hydroxylation of lysine was accompanied by increased glycosylation and there was 30% more Glc-Gal-Hyl present in GBM from 9-month-old SU rats. GBM was solubilised with sodium dodecyl sulphate (SDS) and 2-mercaptoethanol and subjected to electrophoresis on 5% polyacrylamide gels. There was an apparent fall in the intensity of the bands with molecular weights greater than 200,000 and a concomitant rise in low molecular weight components (50,000–100,000) in GBM from 4-month-old SU rats. These differences between ST and SU membrane were accentuated when the membranes from 9-month-old rats were compared. No significant differences were found in the glucosyl transferase activities of renal cortical homogenates prepared from 3-month-old SU and ST rats, but the activities in SU rats were significantly higher at 4 and 9 months. The feeding of SU-rich diets to rats induces a number of biochemical changes in the kidney which are similar to those found in diabetes. The feeding of SU diets provides a useful animal model with which to study the effect of dietary carbohydrate on renal GBM. SU should not be included in diets fed to diabetic rats because of the similarity of some of its effects and those seen in chemically induced diabete

ISSN:1420-4096    

DOI:10.1159/000172741

出版商:S. Karger AG    

年代:1980

数据来源: Karger

摘要:

A graded sieving procedure was used to isolate both glomeruli and tubules from renal cortex of cattle, pig, sheep, horse, man and rat. In addition, tubules from rabbit renal cortex were isolated. Glomerular and tubular basement membranes (GBM and TBM, respectively) were obtained with a detergent procedure. Purity of preparations was established with light and electron microscopy and by estimating total phosphorus content. Amino acid and carbohydrate compositions of the basement membranes were determined and statistically evaluated. Chemical composition of the preparations of all species was typical for renal basement membranes. Significant species-dependent variations were found, however, for many chemical constituents. Comparison of the chemical composition of GBM with TBM as pairs from individuals showed as a common phenomenon in all species that GBM preparations contain more of the heteropolysaccharide constituents, especially twice as much neuraminic acids than TBM preparations. Observed differences may be related to variations in immunogenic properties of the different types of renal basement membranes.

ISSN:1420-4096    

DOI:10.1159/000172742

出版商:S. Karger AG    

年代:1980

数据来源: Karger