Chemical composition and solubility of human glomerular and tubular basement membranes of adult and senescent men

The effect of aging on the composition of human renal basement membranes was studied in persons aged 22-90 years. The relative proportion of cortex in kidney appears to decrease with aging. Twenty pairs of GBM and TBM preparations were isolated using the detergent method. Protein content of the basement membrane preparations amounts to about 66% and is independent of type of membrane or age. Amino acid and carbohydrate analyses of both GBM and TBM revealed that the extents of hydroxylation of proline at the C4 position and of lysine decrease with aging. In the case of lysine this occurred from the seventh of life onwards. The decreases are probably not caused by a change of the collagen content. The extent of glycosylation of hydroxylysine is similar for all basement membrane preparations. An adapted protein assay is presented for solutions containing SDS and dithiothreitol. In the presence of these two compounds, solubility of GBM and TBM from adult and aged persons is similar and amounts to about 55 and 85% after 10 and 60 min, respectively, of heating at 95 degrees C. In SDS-polyacrylamide gels, no differences were observed for the major peptide bands between the preparations, irrespective of basement membrane type or age.     

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.     

A new method for the isolation of renal basement membranes.

A method is described for the isolation of basement membranes from rabbit renal cortex in which the detergent N-lauroyl sarcosine is used as the disruptive agent. The isolated membranes have been compared with membranes prepared using ultrasonication and they were comparable both in terms of purity and gross chemical composition. Glomerular and tubular basement membranes were isolated by first separating glomeruli from tubules by density gradient centrifugation followed by detergent treatment of the separated tissues. The detergent method has the advantage that the basement membranes retained their native structure to a large degree, whereas sonicated membranes were severely fragmented. Collagen fibres were a significant contaminant in both preparations and were revealed more clearly by negative staining than by examination of thin sections. Studies with the detergent-treated membrane revealed that a few proteins, which seemed to be membrane components, were extracted with 1 M NaCl and that these proteins were lost from the basement membranes during sonication used in the conventional isolation procedure.     

A new method for the isolation of renal basement membranes

A method is described for the isolation of basement membranes from rabbit renal cortex in which the detergent $\text{N-}\text{lauroyl}$ sarcosine is used as the disruptive agent. The isolated membranes have been compared with membranes prepared using ultrasonication and they were comparable both in terms of purity and gross chemical composition. Glomerular and tubular basement membranes were isolated by first separating glomeruli from tubules by density gradient centrifugation followed by detergent treatment of the separated tissues.The detergent method has the advantage that the basement membranes retained their native structure to a large degree, whereas sonicated membranes were severely fragmented. Collagen fibres were a significant contaminant in both preparations and were revealed more clearly by negative staining than by examination of thin sections. Studies with the detergent-treated membrane revealed that a few proteins, which seemed to be membrane components, were extracted with 1 M NaCl and that these proteins were lost from the basement membranes during sonication used in the conventional isolation procedure.     

A novel 53-kDa polypeptide from chicken embryo.

We have isolated from chicken embryos a novel 53-kDa protein possessing properties which are similar, but not identical to the 55-kDa PDI polypeptide from chicken embryos. The novel 53-kDa polypeptide copurifies with PDI, but is separated by ion-exchange chromatography. The novel 53-kDa polypeptide cross-reacts strongly with antibodies specific for bovine PDI and cross-reacts to varying degrees with six different preparations of antibodies specific for chicken PDI which is identical to the beta-subunit of chicken prolyl 4-hydroxylase. Anti-bovine PDI immunoglobulins selected by the purified 53-kDa polypeptide react with bovine PDI but not with the beta-subunit of prolyl 4-hydroxylase, suggesting that the 53-kDa polypeptide shares epitopes with bovine PDI but not with the chicken prolyl 4-hydroxylase beta-subunit. Amino acid compositional analysis of the purified polypeptide yielded unique data when compared to PDI and other PDI-like polypeptides. Edman degradation from the N terminus of the 53-kDa polypeptide yields a sequence very different from the N terminus of PDI. This sequence is unique when compared to all entries in available databases. A 20-residue sequence of an internal cyanogen bromide fragment of the 53-kDa polypeptide gives a nearly identical match with human beta-endorphin. The 53-kDa polypeptide is capable of cleaving the disulphides of insulin under conditions where PDI is active. The periodic acid-Schiff assay failed to detect bound carbohydrate. These observations support evidence for a family of PDI-like proteins in chicken embryo and suggest that PDI activity is not confined to only one protein.     

Preparation and characterization of the inner coat material associated with fat globule membranes from bovine and human milk

Milk fat globules of many species are characterized by a dense 10–50 nm thick layer sandwiched between the milk fat globule membrane (MFGM) and the outer shell of the fat droplet. This coat material is tightly associated with the membrane and survives isolation and extensive washing of the isolated MFGM. We have prepared these MFGM-associated coat structures from bovine and human milk by removal of membrane and loosely associated material using extractions in low and high salt buffers, non-ionic detergents such as Triton X-100, and/or solutions of lithium diiodosalicylate. Residual fractions obtained after such treatments are devoid of identifiable membrane structures but are enriched in MFGM coat material which appears in the form of densely stained plaques of a finely filamentous texture. MFGM fractions are enriched in some polypeptide bands seen after electrophoresis two of which are especially prominent in both species (band 3, apparent mol. wt 155 000; band 12, apparent mol. wt 67 000). Human and bovine MFGM coat fractions and isolated bovine band 12 polypeptide material separated after dissociation in sodium dodecylsulfate (SDS) by gel filtration, chromatography on hydroxylapatite or preparative electrophoresis in SDS-polyacrylamide gels are intimately associated with small amounts of phospholipids and gangliosides of a pattern different from that of total MFGM, contain carbohydrates (relatively high contents of mannose, glucosamine, galactose, and galactosamine; low levels of fucose and sialic acids) and show similar amino acid compositions. The relationship of band 12 polypeptide to components of MFGM coat preparations from various other species and to components present in other membrane fractions has been examined by immunodiffusion techniques and immunofluorescence microscopy using rabbit, mouse and guinea pig antibodies against purified band 12 polypeptide. Evidence is presented for the occurrence of related polypeptides in MFGM coat preparations from different species. The unusual structure and resistance of the MFGM coat material, especially the occurrence of glycopeptides in association with the cytoplasmic side of a membrane structure, are discussed in relation to the stabilization of the emulsified state of milk fat and the process of milk fat globule budding as well as a general model for local differentiation of membrane character.     

Ultrastructural analysis of major basement membrane types in rhesus monkey Macaca mulatta acellular renal cortex

Increasing interest in animal models of human nephropathies have led to a number of renal studies in nonhuman primates. In the current investigation, sequential detergent extraction of cellular elements was carried out on renal cortical tissue blocks from rhesus monkey in an effort to demonstrate clearly the morphological features of major basement membrane (BM) types and their associated extracellular matrix (ECM). LM and TEM views of acellular tissue blocks demonstrate planar arrangements of ECM components, while SEM studies provide striking three-dimensional images of their surface characteristics. All major BM types maintain their in vivo histoarchitectures despite the absence of cells. We propose that the intrinsic structural rigidity of tubular (TBM), Bowman's capsule (BCBM) and peritubular capillary BM (PTCBM) may be related to to their close external association with collagenous fibrils, while glomerular BM (GBM) may be internally supported by a network of mesangial matrix (MM) plates and trabeculae which extend onto internal surfaces of peripheral GBM loops. Thicknesses of rhesus monkey renal BMs show that they are similar to those seen in the laboratory rat and, in general, BCBM greater than TBM greater than GBM greater than PTCBM. We conclude that rhesus monkey renal BMs closely resemble those described by us in the human [J. Ultrastruct. Res. 82: 96-110, 1983] and that this species offers an attractive model for studies of renal diseases of BM origin-notably diabetes mellitus.     

Identification of flagellar and associated polypeptides of Helicobacter (formerly Campylobacter) pylori

Flagella of Helicobacter pylori were isolated from intact organisms by shearing and differential centrifugation. Treatment of the flagella with the detergent Triton X-100 removed the flagellar sheath, which was confirmed by electron microscopy, and the remaining naked flagella were shown by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to consist primarily of a single 54 kilodalton (kDa) polypeptide. This was confirmed by immunogold labelling and electron microscopy of detergent treated whole organisms, using a mouse antiserum specific for the 54 kDa polypeptide. Polypeptides solubilised from crude flagellar preparations by detergent treatment were found to have molecular weights of 26, 30, 58, 62, 66 and 80 kDa. These polypeptides are possible components of the flagellar sheath and they may represent outer membrane proteins, based on the assumption that the flagellar sheath is related in composition to the outer membrane of the organism. Analysis and definition of these components of the surface structures of the organism are important in understanding the interaction between the organism and its host in pathogenesis.     

Microtubule-membrane interactions in cilia. I. Isolation and characterization of ciliary membranes from Tetrahymena pyriformis

Tetrahymena ciliary membranes were prepared by four different techniques, and their protein composition was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electron microscopy, and two-dimensional thin-layer peptide mapping. Extraction of the isolated cilia by nonionic detergent solubilized the ciliary membranes but left the axonemal microtubules and dyneine arms intact, as determined by quantitative electron microscopy. The proteins solubilized by detergent included a major 55,000-dalton protein, 1-3 high molecular weight proteins that comigrated, on SDS-PAGE, with the axonemal dynein, as well as several other proteins of 45,000-50,000 daltons. Each of the major proteins contained a small amount of carbohydrate, as determined by PAS-staining; no PAS-positive material was detected in the detergent-extracted axonemes. The major 55,000- dalton protein has proteins quite similar to those of tubulin, based on SDS-PAGE using three different buffer systems as well as two- dimensional maps of tryptic peptides from the isolated 55,000-dalton protein. To determine whether this tubulin-like protein was associated with the membrane or whether it was an axonemal or matrix protein released by detergent treatment, three different methods to isolate ciliary membrane vesicles were developed. The protein composition of each of these differetn vesicle preparations was the same as that of the detergent-solubilized material. These results suggest that a major ciliary membrane protein has properties similar to those of tubulin.     

An immunologic probe for a defined region of the myelin proteolipid

Antiserum has been prepared against an isolated polypeptide fragment, designated BPS4, which comprises residues 181-211 of the bovine myelin proteolipid. The antiserum recognizes the intact bovine proteolipid protein but not several other polypeptide fragments within the molecule, nor the myelin basic protein, thus demonstrating specificity of the antiserum. In a competitive enzyme-linked immunosorbent assay, both the major proteolipid and the DM 20 bands observed on sodium dodecyl sulfate-polyacrylamide gels reacted equally well with the antiserum, indicating that the BPS4 segment is present in both molecular species. The rat myelin proteolipid protein cross-reacted with antiserum against the intact bovine protein but showed minimal cross-reactivity with the antiserum against the bovine BPS4 fragment. This was demonstrated in parallel experiments using three types of preparations, namely, sodium dodecyl sulfate-solubilized myelin, delipidated myelin, and isolated proteolipid apoprotein. The difference between the bovine and rat proteins, which presumably reflects amino acid sequence differences, is thus detectable by the antiserum against the polypeptide fragment but not by the antiserum against the intact protein. Isolated bovine myelin membranes did not bind the antiserum in the absence of detergent or without delipidation. On the other hand, in vesicles reconstituted with the intact bovine apoprotein, the BPS4 segment was oriented on the exterior face of the liposome where it was capable of binding antibody and was susceptible to Pronase digestion.     

Glial fibrillary acidic protein from bovine and rat brain. Degradation in tissues and homogenates.

Compared with human material glial fibrillary acidic protein isolated from bovine, rat and mouse brain was remarkably homogeneous and migrated as a single band at 54 000 mol. wt. on sodium dodecyl sulfate gel electrophoresis. The protein was extremely susceptible to proteolysis and lower molecular weight components were invariably isolated together with the major species when the brain was not rapidly frozen. Further degradation of the 54 000 mol wt. polypeptide in bovine tissues incubated at 24 degrees C resulted in preparations essentially identical to those previously isolated from human autopsy material and separating into a series of immunologically active polypeptides ranging in molecular weight from 54 000 to approximately 40 500. The gel band pattern obtained after progressively longer periods of autolysis suggested that small fragments were cleaved from the original polypeptide in successive steps of degradation. As in human brain, the lower molecular weight products in the 45 000-40 500 range were more resistant to proteolysis and still present after prolonged periods of tissue autolysis. The effect of the pH and of proteinase inhibitors on degradation was studied in homogenates of bovine brain stem incubated at 37 degrees C. At pH 8.0 PROTEOLYSIS OF The glial fibrillary acidic protein followed essentially the same pattern as in tissue. Cleavage of the major species was not prevented by the addition of proteinase inhibitors. At pH 6.0 and 6.5 a different type of degradation was observed, with rapid breakdown of the protein and loss of immunological activity. Increased solubility in buffer solutions was another effect of autolysis. Compared with cerebral cortex and brain stem, where most of the protein was water soluble, only a small fraction was extracted with buffer from bovine white matter. However, the solubility markedly increased following incubation and comparable amounts were extracted in buffer and in 6 M urea.     

Characterization of nucleoside diphosphatase in the onion root tip. III. Solubilization and activation of membrane-bound enzyme(s)

The latency of nucleoside diphosphatase (NDPase) in onions root homogenates has been examined by comparing the activation of NDPase activity resulting from detergent treatment with that due to storage of homogenates for several days in the cold. Both detergent treatment and cold storage activated NDPase approximately two-fold. In both cases this activation was paralleled by the loss of enzyme activity from the membrane fractions and its appearance in the supernatants. Electrophoresis of these supernatants revealed an identifical isoenzyme pattern of 5 NDPase bands for both preparations. Enzyme kinetic studies demonstrated that NDPase from the detergent-treated homogenate and the homogenate stored in the cold as well as NDPase from the membrane and supernatant fractions from each of the homogenates all had the same Km value. These data suggest that latency of NDPase is the result of a breakdown of cellular membranes and subsequent release of NDPase. Abbreviations: DOC, deoxycholate; NDPase, nucleoside diphosphatase; IDP, inosine 5'-diphosphate; UDP, uridine 5'-diphosphate; GDP, guanosine 5'-diphosphate.     

The size and shape of human and bovine antithrombin III.

Human and bovine antithrombin, purified by affinity chromatography on heparin-agarose, have been characterized with regard to chemical composition, size, shape and conformation. Both preparations were found to contain several active components of identical or similar size but different electrical charge. Amino acids and carbohydrate analyses revealed striking similarities between human and bovine antithrombin, while immunological analyses failed to demonstrate any cross-reactivity. The molecular weights were determined by sedimentation equilibrium to be 58 000 for human and 56 000 for bovine antithrombin. The small molecular weight difference suggested by these values was verified by several empirical methods of molecular weight estimation. Hydrodynamic measurements indicated that the two proteins have similar molecular shapes, both of which are slightly more extended that that of typical globular proteins. The internal folding of the two polypeptide chains is also similar, as evidenced by the identity of the far-ultraviolet circular dichroism spectra. Specifically, these analyses suggested a low alpha-helix content of both proteins. In conclusion, the marked structural similarity of human and bovine antithrombin indicates that the two proteins may also exhibit extensive functional similarities in the binding of heparin and the inhibition of various coagulation factors.     

The complete amino acid sequence of bovine skeletal muscle acylphosphatase

The primary structure of bovine skeletal muscle acylphosphatase was determined by performing the sequence analyses of the complete series of tryptic peptides. The amino acid composition of the entire series of peptic peptides was used to reconstruct the sequence by the overlapping method. The proposed structure is further confirmed by analogy with known amino acid sequences of acylphosphatase from skeletal muscle of other vertebrate species. The length of the polypeptide chain is 98 residues, identical to the length of the enzymes from other known mammalian species, but different from that found in turkey. The enzyme is NH2-acetylated and a comparison with the analogous molecular forms from other vertebrate species indicates that there are several long polypeptide stretches strictly conserved (93-97% identical position among mammals, and about 80% between calf and turkey enzymes).     

Ultrastructural examination of cytoskeletal linkage of L-selectin and comparison of L-selectin cytoskeletal association to that of other human and bovine lymphocyte surface antigens

L-selectin is constitutively expressed on most leukocytes and is responsible for the initial events in cell trafficking termed tethering and rolling. Recently, L-selectin has been shown to associate with the actin-based cytoskeleton under a variety of conditions. In an effort to better understand L-selectin cytoskeletal association and the ultrastructural nature of the cytoskeleton itself, we provide a comparison of the cytoskeletal association of various human and bovine surface proteins in relation to L-selectin. Electron microscopic examination of the cytoskeleton provided further data on the ultrastructure of freshly isolated peripheral lymphocytes as well as demonstrated L-selectin localization to the periphery of the cytoskeleton following low dose detergent treatment of the cell. Clusters of colloidal-gold-stained L-selectin were found on the surface of the detergent-treated lymphocytes, even though these particles completely lacked microvilli. By flow cytometry, we have defined three distinct patterns of cytoskeletal association; constitutive, inductive, and mAb crosslink-induced, and assigned human and bovine CD2, CD3, CD4, CD5, CD8, CD18, CD19, CD44, CD45RA, CD45RO, alphabeta TCR, gammadelta TCR, E-selectin ligands, and L-selectin surface antigens to one of these respective patterns. SDS-PAGE analyses confirmed most of the flow cytometry results. Depending upon its conformation, L-selectin fell into the inductive or mAb crosslink-induced pattern of association, similar to E-selectin ligand(s). Our data provide additional insight into the functional role of L-selectin and the cytoskeleton in immunological events.     

Compositional relationships among electrophoretic isolates from cottonseed protein bodies

Minor differences exist among polypeptides isolated from cottonseed storage globulins with respect to nutritionally-limiting amino acids, although the composition of most isolates closely resembles that of the entire storage globulin fraction. Glutamate, arginine and aspartate are the most abundant amino acids; cysteine and methionine are the least abundant. In contrast to the 10 polypeptide bands in the SDS-PAGE pattern, isoelectric focusing produced a pattern of 49 components within a range of pH 4.5–9.5. A two-dimensional electrophoretic separation indicated that most of the polypeptides resolved by SDS-PAGE are heterogeneous with respect to charge. The high ratio of isoelectric to molecular weight forms may prove advantageous for efforts to genetically improve the quality of cottonseed protein.     

N-terminal amino acid sequences and amino acid compositions of the Spirochaeta aurantia flagellar filament polypeptides.

The amino-terminal sequences and amino acid compositions of the three major and two minor polypeptides constituting the filaments of Spirochaeta aurantia periplasmic flagella were determined. The amino-terminal sequence of the major 37.5-kDa outer layer polypeptide is identical to the sequence downstream of the proposed signal peptide of the protein encoded by the S. aurantia flaA gene. However, the amino acid composition of the 37.5-kDa polypeptide is not in agreement with that inferred from the sequence of flaA. The 34- and 31.5-kDa major filament core polypeptides and the 33- and 32-kDa minor core polypeptides show a striking similarity to each other, and the amino-terminal sequences of these core polypeptides show extensive identity with homologous proteins from members of other genera of spirochetes. An additional 36-kDa minor polypeptide that occurs occasionally in preparations of S. aurantia periplasmic flagella appears to be mixed with the 37.5-kDa outer layer polypeptide or a degradation product of this polypeptide.     

STRUCTURAL AND IMMUNOLOGICAL PROPERTIES OF NEURON SPECIFIC PROTEIN (NSP) FROM RAT, CAT AND HUMAN BRAIN: COMPARISON TO BOVINE 14-3-2

Abstract— Neuron specific protein (NSP) has been isolated from cat (NSP-C) and human (NSP-H) brain utilizing the purification procedure described for rat brain 14-3-2 (M arangos et al. , 1975a,b,c), a protein which is now designated NSP-R. The protein as isolated from cat and human brain has a molecular weight of approx 80,000 as determined by sedimentation equilibrium. Sedimentation studies done in the presence of 6mg-HCl and 0.2%β mercaptoethanol yields a protomer M.W. of approx 40,000 for both preparations establishing the dimeric nature of each. The subunits appear identical in each case since one band is observed upon electrophoresis of either preparation in the presence of 8 M-urea. NSP-C and NSP-H have identical isoelectric points of 4.7 making them slightly more acidic than NSP-R (pi = 5.0).
Comparison of NSP-C and NSP-H with NSP-R and bovine 14-3-2 by electrophoretic and immunological criteria revealed that the cat, human and bovine proteins were very similar. NSP-R can be distinguished from the other three preparations electrophoretically and immunologically. The protomer unit of NSP-R differs in amino acid composition from that of the cat, human or bovine proteins since the former can be completely resolved from any of the latter three preparations on 8 M-urea polyacrylamide gels. The data indicate that NSP and bovine 14-3-2 are probably homologous proteins, and establish the general structural properties of NSP.     

Stable preparation of yeast mitochondria and mitoplasts synthesizing specific polypeptides

Yeast mitochondria isolated in the presence of 0.6 M sorbitol and 0.5% bovine serum albumin can be stored in liquid nitrogen without loss of translational activity. Frozen mitochondria retain the respiratory control and the mutant pattern of polypeptide synthesis identical to those detected for fresh preparations. Stored mitochondria may be efficiently transformed into a stable preparation of mitoplasts actively synthesizing mitochondrial polypeptides.