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Biomedical subjects

B G Hudson

Publications and source records attributed to B G Hudson.

At least 91 records · Page 5Linked to original sources
PubMed1984

Goodpasture antigen of the glomerular basement membrane: localization to noncollagenous regions of type IV collagen.

The glomerular basement membrane antigen in Goodpasture syndrome is a collagenase-resistant molecule with a monomer molecular weight of about 26,000. Type IV collagen isolated from glomerular basement membrane contains collagenase-resistant sequences within its structure. Polyacrylamide gel electrophoresis, enzyme-linked immunosorbent assay, and chemical analysis were used to demonstrate that the collagenase-resistant sequences of type IV collagen contain Goodpasture antigen.

Anti-Glomerular Basement Membrane Disease↗
PubMed1983

Isolation of type IV procollagen-like polypeptides from glomerular basement membrane. Characterization of pro-alpha 1(IV).

Type IV procollagen-like constituents of glomerular basement membrane were solubilized by reduction and alkylation of disulfide bonds under denaturing conditions. Four polypeptides were observed with apparent Mr = 185,000, 175,000, 164,000, and 152,000. The two largest chains correspond to pro-alpha 1(IV) and pro-alpha 2(IV), described in model systems which secrete a basement membrane-like matrix, while the smaller chains appear to be shortened forms of these polypeptides. Fractionation of the four polypeptides into two groups was achieved by ion exchange chromatography. Pro-alpha 1(IV) and 164,000 polypeptide are relatively acidic with respect to pro-alpha 2(IV) and 152,000 polypeptide, which is due in part to a relatively high content of arginine in the latter. Based on amino acid analysis of the collagenase-sensitive regions of these polypeptides, pro-alpha 1(IV) is the parent molecule from which alpha 1(IV) is derived on pepsin digestion of basement membranes and pro-alpha 2(IV) is the parent molecule of alpha 2(IV). Pro-alpha 1(IV) was isolated by gel filtration and ion exchange chromatography and characterized. It has a molecular weight of 194,000 as determined by sedimentation equilibrium. The polypeptide contains 14% carbohydrate in the form of both disaccharide, glucosylgalactosylhydroxylysine, and heteropolysaccharide units. The polypeptide backbone mass is calculated to be 167,000 daltons. Digestion of pro-alpha 1(IV) with bacterial collagenase resulted in two resistant segments of mass = 31,000 and 33,000 dalton, which make up approximately 30% of the polypeptide.

Amino Acids↗
PubMed1981

Detergent-prepared glomerular basement membrane is composed of a heterogeneous group of polypeptides.

Glomerular basement membrane was prepared from rat, bovine, and human kidneys using a detergent solubilization procedure, and the polypeptide composition of the membranes was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Reduced rat glomerular basement membrane resolves into 15 polypeptide components ranging in molecular weight from 25,000 to greater than 300,000. Rat, bovine, and human membrane preparations all yield a similar heterogeneous distribution of polypeptides. The same group of polypeptides was observed for membrane prepared with or without the use of proteolytic inhibitors during the isolation procedure. The detergent solubilization method was compared with the more commonly used sonication method on preparations of rat, humans, and bovine glomeruli. Some differences in the number and relative concentrations of polypeptide components was observed. However, a heterogeneous group of polypeptides was present in both types of preparations. These results contradict the recent claim of Cohen and Surma ((1980) J. Biol. Chem. 255, 1767-1770) that glomerular basement membrane prepared by the detergent method using protease inhibitors does not exhibit polypeptide heterogeneity. Therefore, it is concluded that polypeptide heterogeneity is a characteristic feature of glomerular basement membrane irrespective of the method of isolation, detergent or sonication, with or without the use of proteolytic inhibitors.

Animals↗
PubMed1981

Bovine glomerular basement membrane. Characterization of an alpha-size collagenous polypeptide.

The collagenase domain of bovine glomerular basement membrane was isolated in soluble form after limited digestion with pepsin. Gel filtration chromatography of the domain under denaturing conditions revealed that most of the polypeptide constituents exhibit apparent molecular weights greater than the type I collagen beta-chain, while approximately 15% are similar in size to that of alpha-chain. Carboxymethyl cellulose chromatography of the alpha-size region revealed that 70% of the protein was polypeptide XIV, as previously designated (West, T. W., Fox, J. W., Jodlowski, M., Freytag, J. W., and Hudson, B. G. (1980) J. Biol. Chem. 255, 10451-10459). This polypeptide exhibits an apparent molecular weight of 102,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An absolute molecular weight value of 86,000 was determined by sedimentation equilibrium ultracentrifugation in 6 M guanidine hydrochloride. About 15% of the mass is carbohydrate which exists in the form of glucosylgalactosylhydroxylysine. Thus, the polypeptide backbone has a molecular weight of 73,000, a value which is considerably smaller than the alpha-chains of classical collagen. The amino acid and carbohydrate composition and cyanogen bromide patterns indicate that polypeptide XIV has a structure similar to that of C-chain or alpha 1 (IV) collagen which has been identified in other tissues. In addition, the cyanogen bromide pattern of the entire collagenous domain is similar to that of polypeptide XIV, suggesting that the latter is a structural segment of many of the higher molecular weight components.

Amino Acids↗
PubMed1981

Characterization of the collagenous domain of tubular basement membrane.

The collagenous domain of rabbit renal tubular basement membrane was solubilized by controlled pepsin digestion. Complete solubilization occurred within 24 h at 4 degrees C, and the sodium chloride precipitable polypeptides remained resistant to further degradation for at least 72 h. These products, which represent 35 to 40% of the basement membrane, possess a composition similar to that od the collagenous domain of bovine glomerular basement membrane. Agarose gel electrophoresis in the presence of sodium dodecyl sulfate resolved 11 components, several of which comprise the majority of the protein in the system. Components I (Mr approximately 1,500,000) and II (Mr approximately 1,200,000), together with a minor component, III, comprise 45% of the Coomassie blue-staining material. An additional 25% is represented by component IV (Mr approximately 375,000) plus a minor component, V. The remaining components (VI through XI) span a molecular weight range from 34,000 to 200,000. Two-dimensional gel electrophoresis was used to establish which of these components contain disulfide cross-linked polypeptides and the number and molecular weight of such polypeptides. Components I, II, and IV each contained intensely staining polypeptides of Mr = 160,000, 140,000, and 80,000 plus lightly staining polypeptides of Mr = 43,000 and 34,000. Lightly staining polypeptides of Mr = 390,000, 180,000, and 125,000 were also detected among the reduction products of components I and II. In addition, evidence for the presence of aldehyde-derived cross-linked polypeptides within the structures of components I and II was observed in the form of reduction products whose molecular weight occurs in multiples of the smaller monomeric polypeptides.

Amino Acids↗
PubMed1980

The structure of the asparagine-linked carbohydrate unit of rat alpha-lactalbumin.

Rat alpha-lactalbumin is unique in contrast to alpha-lactalbumin isolated from other species in that it exists in three charge forms. Each form contains carbohydrate and is active in the lactose synthetase reaction. Form II comprises about 80% of the total alpha-lactalbumin and contains a single heteropolysaccharide unit which is attached to the polypeptide chain at Asn45. The detailed structure of this unit was ascertained using specific exoglycosidases, endoglycosidases, and methylation analysis. The following structure is proposed for the heteropolysaccharide unit: (formula: see text).

Animals↗
PubMed1980

Characterization of the collagenous domain of bovine glomerular basement membrane.

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 greater than 10(6). 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 a larger polypeptides to three lower molecular weight peptides, two of which exhibit an electrophoretic migration identical to alpha 1- and alpha 2-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 alpha-size segments within their structure.

Animals↗
PubMed1980

Comparison of the chemical and polypeptide composition of tubular and glomerular basement membranes.

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.

Animals↗
PubMed1979

Interaction of bovine platelets with bovine glomerular basement membrane.

The interaction of bovine platelets with bovine glomerular basement membrane has been studied by aggregometry, transmission and scanning electron microscopy and measurement of [3H] serotonin release. In the absence of added calcium platelets adhere to basement membrane but fail to undergo the release reaction or aggregation. In the presence of 0.2-0.5 mM calcium release of serotonin and complete aggregation of the platelets are observed when sufficient basement membrane is present. Platelets were strongly adhered to the basement membrane surface, the platelet surface in the aggregates closely following the surface of the basement membrane. Platelet morphology in aggregates with basement membrane closely resembled that of platelets from collagen-induced aggregates. Basement membrane differed from collagen in its requirement for calcium for the aggregation and release reactions. In addition purified basement membrane was 1.5-3 fold less active on a weight basis than bovine tendon collagen in promoting aggregation.

Animals↗
PubMed1979

Resolution of the charge forms and amino acid sequence and location of a tryptic glycopeptide in rat alpha-lactalbumin.

Three charge forms of rat alpha-lactalbumin were separated by ion exchange chromatography on DEAE-cellulose. The amino acid composition of each form was similar but they differed in carbohydrate composition. Each form contained a tryptic glycopeptide having a common polypeptide and heteropolysaccharide unit. The tryptic glycopeptide was sequenced and positioned in rat alpha-lactalbumin, which was partially sequenced from residues 1 to 50. The carbohydrate attachment site was at Asn45. Secondary structure calculations predicted that Asn45 is in a beta bend conformation whereas Asn45 in bovine alpha-lactalbumin, a poorly glycosylated protein, is not in a bend conformation.

Amino Acid Sequence↗
PubMed1979

Rabbit tubular basement membrane. Isolation and analysis of polypeptides.

Renal tubules from rabbit kidneys were isolated from thin shavings of the kidney surface. Basement membrane was then prepared following sonication of the isolated tubules. To insure preservation of the integrity of the basement membrane polypeptides, the protease inhibitors, diisopropyl fluorophosphate, ethylenediaminetetraacetic acid, N-ethylmaleimide, and epsilon-amino-caproic acid were used at all stages of the preparations. The optimal conditions of sonication and centrifugation were established and the chemical composition of basement membrane prepared under these conditions was examined in detail. Glycine, hydroxyproline, and hydroxylysine were found in concentrations of 206, 65, and 18 residues per thousand, respectively, in basement membrane from young kidneys. About 38% of the basement membrane was found to be soluble in sodium dodecyl sulfate upon incubation at 90 degrees C, and to possess relatively low amounts of the amino acids characteristic of collagen. Electrophoretic analysis of this fraction revealed that the major subunits ranged in approximate molecular weight from 18,500 to greater than 10(6). When analyzed with disulfide bonds reduced, a molecular weight range from 31,000 to 275,000 was observed for this fraction. The sodium dodecyl sulfate-insoluble fraction could be dissolved upon reduction and alkylation and its composition was enriched in the amino acids characteristic of collagen. Polypeptides from this fraction were analyzed by electrophoresis in agarose and in agarose-acrylamide gels. The approximate molecular weight of the smallest component was 164,000. Additional polypeptides were observed whose molecular weights occurred in multimers of this component, up to 1.1 x 10(6), possibly indicating covalent cross-linked multimers of a basic collagen-like polypeptide(s).

Amino Acids↗