Characterization and polyanion-binding properties of purified recombinant prion protein.

Certain polysulphated polyanions have been shown to have prophylactic effects on the progression of transmissible spongiform encephalopathy disease, presumably because they bind to prion protein (PrP). Until now, the difficulty of obtaining large quantities of native PrP has precluded detailed studies of these interactions. We have over-expressed murine recombinant PrP (recPrP), lacking its glycophosphoinositol membrane anchor, in modified mammalian cells. Milligram quantities of secreted, soluble and partially glycosylated protein were purified under non-denaturing conditions and the identities of mature-length aglycosyl recPrP and two cleavage fragments were determined by electrospray MS. Binding was assessed by surface plasmon resonance techniques using both direct and competitive ligand-binding approaches. recPrP binding to immobilized polyanions was enhanced by divalent metal ions. Polyanion binding was strong and showed complex association and dissociation kinetics that were consistent with ligand-directed recPrP aggregation. The differences in the binding strengths of recPrP to pentosan polysulphate and to other sulphated polyanions were found to parallel their in vivo anti-scrapie and in vitro anti-scrapie-specific PrP formation potencies. When recPrP was immobilized by capture on metal-ion chelates it was found, contrary to expectation, that the addition of polyanions promoted the dissociation of the protein.

Plate assay for simultaneous detection of alginate lyases and determination of substrate specificity.

A plate assay to detect the presence of alginate lyases (EC 4.2.2.3) has been developed. The simultaneous use of specific alginate block structures of defined composition allows the substrate specificity of the enzymes to be determined. Clearing zones in the alginate-containing media are visualized with either cetyl pyridinium chloride or ruthenium red.

Alkali-labile luminol derivatives as labels for quantitative binding studies with polyionic macromolecules.

Acylated derivatives of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) bearing a carboxyl or amino group can be linked by amide bonds to a macromolecule requiring labelling. Though themselves of low quantum yield these compounds are alkali-labile and can be detected at a similar level of sensitivity to the parent compound luminol. These cheap, readily accessible compounds are less hydrophobic than other currently employed chemiluminescent labels. They also lack a positively charged nitrogen atom which could complicate their covalent linkage to polyanionic compounds. They thus appear well suited for labelling heparin and other macromolecules which interact with the luminal surface of blood vessels.

Simulation of vascular surfaces: differential saturation with glycosaminoglycans and their quantitative analysis.

Surface layers of sulphated glycosaminoglycan can be quantified by X-ray microanalysis using the local mass-fraction of the element sulphur. As a calibration standard radiolabelled chondroitin sulphate has been attached covalently to a nylon surface at various densities to the point where the molecules are packed as close as the radius of gyration permits.

Chondroitin sulphate at the endothelial lumen of pig aorta: assay by radiolabelling and by X-ray microanalysis.

Trypsin-releasable glycosaminoglycans from the luminal surface of intact pig aorta were measured following metabolic labelling with [35S]sulphate. Chondroitin sulphate was found to be present at a surface density equal to that already established for heparan sulphate (5 X 10(11) chains per cm2). This result was confirmed by X-ray microanalysis of the luminal sulphur content before and after treatment with specific glycosaminoglycan-degrading enzymes. This result implies that approximately half of the luminal surface is occupied by sulphated glycosaminoglycans.

Glycosaminoglycan: cell interactions; their role in lipoprotein lipase secretion from isolated cardiac muscle cells.

When cardiac muscle cells from mature rats were incubated in vitro in the presence of heparin (8.7 nmole ml-1) lipoprotein lipase activity appeared in the incubation medium. The intracellular activity of the enzyme remained unchanged. Other glycosaminoglycans (heparan sulphate, dermatan sulphate, keratan sulphate and chrondroitin 6-sulphate) at the same or higher concentrations were totally ineffective in producing any enzyme redistribution between cells and medium. The release seen in the presence of heparin was blocked by the presence of cycloheximide. Cycloheximide by contrast had no effect on the release observed in the presence of dexamethasone, The action of endogenous glycosaminoglycans are unlikely therefore to have a significant role to play in the movement of lipoprotein lipase in heart tissue in vivo.

Carrier detection in Hunter syndrome.

We have studied the carrier state of the Hunter syndrome using a series of obligate carriers, females at high genetic risk, and normal control women. Specific odds of a female being a carrier of Hunter syndrome were based on serum levels of iduronate 2-sulphate sulphatase activity. These, together with the prior genetic odds, may be used in calculating the overall odds of a woman being a carrier. Iduronate 2-sulphate sulphatase levels were found to increase significantly with age. Obligate carriers from families of severe cases had significantly lower enzyme levels compared with those from families of mild cases. In contrast, enzyme levels in sera of mild and severe cases were not significantly different. With the accumulation of more data the effect of age of the potential carrier and the severity of the disease may have to be taken into consideration in the risk calculation. Hair-root analysis was more reliable in the detection of carriers than estimation of serum enzyme levels, but some individuals could not be classified with confidence by hair-root analysis alone. Carrier detection was most reliable when hair-root analysis and serum enzyme levels were taken together.

Heparan sulphate and the binding of lipoprotein lipase to porcine thoracic aorta endothelium.

Purified bovine milk lipoprotein lipase was shown to bind to intact porcine aortic endothelium in a specific, saturable fashion. The binding was reversed by exogenous heparin. A single class of binding sites was involved and at saturation 1.24 x 10(11) molecules of lipoprotein lipase/cm2 were bound. This represents 0.51 x 10(6) enzyme molecules per endothelial cell at a density of 1.2 x 10(3) molecules/micrometers 2. The enzyme binding was reduced by prior trypsinisation of the endothelial surface under conditions that removed cell surface glycosaminoglycan chains. The porcine endothelium was shown to have available at its surface 5.4 x 10(11) chains of heparan sulphate plus heparin-like glycosaminoglycans/cm2. Such an excess suggests that lipoprotein lipase may interact with approximately one in four of the available heparan sulphate chains.

Multiple forms of iduronate 2-sulphate sulphatase in human tissues and body fluids.

Iduronate 2-sulphate sulphatase (EC 3.1.6.-) was found in human placenta in three forms which could be separated by elution from DEAE-cellulose using an NaCl gradient. Form C, most firmly bound to DEAE-cellulose, was 40% larger than the other two (forms A and B in order of ease of elution from the ion exchanger). Forms B and C contained sialic acid which could be removed by neuraminidase digestion. After removal of sialic acid form B became indistinguishable from form A. The enzyme forms found in placenta were compared with those from other human tissues and fluids by means of DEAE-cellulose chromatography and gel chromatography. Serum and amniotic fluid contained only form C, urine and cultured fibroblasts contained the less-anionic forms as well, and kidney contained appreciable amounts only of form A. Pre- and post-natal diagnosis of the Hunter syndrome both involve measurements on the enzyme which is present in form C. This is not accompanied by less-anionic forms which constitute the bulk of the enzyme as it is isolated from easily available sources such as urine.

An improved assay for iduronate 2-sulphate sulphatase in serum and its use in the detection of carriers of the Hunter syndrome.

A more sensitive assay procedure has been developed for the enzyme iduronate 2-sulphate sulphatase which is deficient in the Hunter syndrome. The substrate is the same as previously described by Lim et al. [1], O-(alpha-L-idopyranosyluronic acid 2-sulphate)-(1leads to 4)-2,5 anhydro-D-[3H-1]mannitol 6-sulphate, but, after incubation, it is separated from the product by ion-exchange chromatography on a micro-column of Dowex 1 x 2 (Cl-1) instead of high voltage electrophoresis or ECTEOLA cellulose chromatography. Since the blank correction is then much smaller, a shorter incubation time can be used and conversion of the substrate reduced from approximately 50% down to levels where complications resulting from substrate depletion and product inhibition are minimal. Using whole serum the apparent Km for the substrate is 0.2 mmol/l. With an incubation time of 20 min, sera from heterozygotes exhibited approximately 35% of the normal levels of iduronate 2-sulphate sulphatase (0.11-0.61, mean 0.34 nmol.h-1.mg-1 protein for carriers; 0.24-2.35, mean 0.94 nmol.h-1.mg-1 protein for 37 normal females). Serum analyses can thus be used to supplement those on hair roots in the detection of carriers of the Hunter syndrome.

Degradation of [3H]chondroitin 4-sulphate and re-utilization of the [3H]hexosamine component by the isolated perfused rat liver.

Radiolabelled chondroitin 4-sulphate was isolated after incubation of rat rib cartilage with N-acetyl-D-[6-3H]galactosamine. After proteolytic digestion of the tissue with either papain or trypsin the released [3H]chondroitin 4-sulphate was added to an isolated perfused rat liver system. Analysis of perfusate after several hours perfusion showed that radiolabelled amino sugars were secreted by the liver in a low-molecular-weight form and as components of glycoproteins.

Utilization by the isolated perfused rat liver of N-acetyl-D-[1-14C]galactosamine and N-[3H]acetyl-D-galactosamine for the biosynthesis of glycoproteins.

The isolated perfused rat liver system has been used to monitor the utilization of N-[3H]acetyl-D-galactosamine and N-acetyl-D-[1-14C]galactosamine for the biosynthesis of radiolabelled glycoproteins, which are subsequently secreted into the plasma. Both radiolabels appear in a number of different glycoproteins, predominantly as sialic acid and N-acetylglucosamine. The ratio of labelled sialic acid to labelled N-acetylglucosamine varies for different glycoproteins, but the bulk of N-acetyl-D-galactosamine is incorporated without deacetylation.

The catabolism of intravenously injected heparan N-[35S] sulphate in the rat.

The metabolic fate of heparan N-[(35)S]sulphate was studied in rats. Heparan sulphate was obtained from either bovine aorta or lung and labelled with (35)S by desulphation and subsequent resulphation in vitro. Experiments in which heparan N-[(35)S]sulphate was administered intravenously to either free-range or wholly anaesthetized rats with ureter cannulae established that substantial desulphation occurs in vivo, with elimination of inorganic [(35)S]sulphate in urine. Oligosaccharides labelled with (35)S, possible intermediates in heparan sulphate degradation, could not be detected in urine or blood. The general distribution of radioactivity after administration of heparan N-[(35)S]sulphate, as demonstrated by whole-body radioautography, suggested that desulphation was not restricted to one organ in particular. Support for this view was obtained in experiments in which heparan N-[(35)S]sulphate was administered to animals after the removal of kidneys, liver, spleen, pancreas or gastrointestinal tract. In all cases inorganic [(35)S]sulphate was still produced. The ability of rats of desulphate heparan N-[(35)S]sulphate was progressively impaired by increasing concentrations of heparin administered simultaneously. It was concluded that heparan sulphate is metabolized at a number of sites in the body by a sequence of degradative events leading to the formation of inorganic sulphate. It is also concluded that at least some of these events are common to heparan sulphate and heparin.

Hyaluronic acid in elastic cartilage.

Bovine ear cartilage contains more hyaluronic acid than do hyaline cartilages of the same animal. Most of it is in the elastin-rich residue not extractable by 4 M guanidinium chloride where it is associated with chondroitin sulphate in low relative concentration and of lower molecular weight than in non-elastic cartilage residue.

The metabolic fate of intravenously injected peptide-bound chondroitin sulphate in the rat.

The degradation of intravenously administered chondroitin sulphate-peptide, obtained by trypsin digestion of rat cartilage preparations labelled in vitro with 35S (and, in some cases, with 3H), was studied in rats. As with free chains of chondroitin sulphate, the major site of accumulation and degradation in the body was the liver, although peptide-linked chains were taken up more rapidly than free chains. In the first 2h after intravenous injection of a chondroitin sulphate-peptide fraction, labelled macromolecular components were excreted in the urine. These were shown to be chondroitin sulphate-peptide of the same degree of sulphation but of smaller average size than the injected material. A similar observation was made when free chains of chondroitin sulphate from the same source were administered intravenously. An isolated perfused rat kidney failed to de-sulphate circulating chondroitin sulphate-peptide, but a component of lower average molecular weight was excreted in the urine. When a chondroitin sulphate-peptide fraction of relatively larger hydrodynamic volume was administered, very little chondroitin sulphate appeared in the urine in the first 2h. It was concluded that, depending on size and/or peptide content, the chondroitin sulphate-peptide released from connective tissues into the circulation would probably be subjected to one of two alternative fates. The smaller fragments are more likely to be excreted in the urine, whereas the larger ones are taken up by the liver and there degraded to inorganic sulphate and undefined carbohydrate components.

The composition of massive lesions in coal miners.

The nature of the material forming the massive lesions in the lungs of coal workers has never been demonstrated. The concept that it was in fact massive fibrosis, implying that it consisted of collagen impregnated with coal dust, has been challenged only during the last ten years. It was agreed that the best chance of obtaining more definite information was from a combined study of the biochemical, pathological, ultrastructural, and immunological features of a number of lungs containing these lesions. Six cases which were found to contain suitable material were studied. The preliminary results obtained suggest that collagen is present in the capsule of these lesions but that at the centre it is replaced by another insoluble protein or proteins which is probably stabilized by some form of cross-linking. This protein complex accounts for one-third of the weight of the lesions, the remaining two-thirds consisting of approximately equal amounts of mineral dusts and calcium phosphate. Serum proteins were also observed but their association with the lesions has yet to be determined.

The linkage region in the polypeptide of pig costal cartilage proteoglycan.

Proteoglycan from pig costal cartilage and fragments obtained by proteolytic digestion were characterized by equilibrium ultracentrifugation and amino acid analysis. The proteoglycan extractable in 4 M guanidinium chloride yielded, after proteolytic digestion with trypsin and chymotrypsin, a chondroitin sulfate peptide containing four chains of polysaccharide. The unextractable residue yielded chondroitin sulfate peptide containing only two chains. The amino acid composition indicated a fairly uniform spacing between all four chains with an average of eight amino acid residues between the serine residues involved in linkage. Following the alkaline sulfite elimination-addition reaction, free peptide was isolated and found to contain one unsubstituted serine residue for every two linked glycosidically. Glycine and glutamic acid were the only two amino acids sufficiently abundant to be part of an invariant sequence near to serine residues destined to be glycosylated. The linkage region of the polypeptide also contains some substituted serine residues which do not carry a full chondroitin sulfate chain.