(4,5). AP remains in amyloid deposits for prolonged periods without protein catabolism (6) and might thereby contribute to the persistence which underlies their pathogenicity. However, the structure ofAP has not been well characterized. AP from only one individual has been completely sequenced at the protein level (7), and the two reports of the SAP cDNA sequence (8, 9), as well as preliminary reports of the glycostructure (10,11,37), are conflicting. We have therefore compared the complete covalent structures of SAP and AP, with particular emphasis on the oligosaccharide and its role in turnover of the SAP glycoprotein.MATERIALS AND METHODS Proteins. SAP was isolated from the serum (13) of two healthy subjects, from two large pools of normal serum containing 1-ml aliquots from each of >5000 different healthy volunteer blood donors, and from a pool of malignant effusion fluids containing material from >300 donors. AP was isolated from two spleens containing AA amyloid, two spleens and two livers with AL amyloid, one spleen containing [ArgW]apolipoprotein A-I amyloid, and the spleen, liver, heart, kidney, and adrenals of an individual patient with AA amyloidosis. Amyloidotic tissue was homogenized in 0.01 M Tris/0.14 M NaCl/0.01 M EDTA, pH 8.0, and the supernatant after centrifugation at 50,000 x g for 30 min was dialyzed extensively into 0.01 M Tris/0.14 M NaCl/0.002 M CaCl2, pH 8.0, before extraction of AP precisely as described for SAP from serum (13). All SAP/AP preparations were >991% pure in heavily overloaded SDS/polyacrylamide gradient gels (Excelgel, Pharmacia) run under reducing conditions and stained with brilliant blue R-350 or with silver.Glycan Structure Analysis. Oligosaccharides attached to SAP and AP were released by hydrazinolysis and radiolabeled with sodium borotritide at the reducing terminus, which incorporates a single label into each chain independently of its structure, so that the relative molar proportions of all glycans present can be estimated directly (14). These oligosaccharides were analyzed by high-voltage paper electrophoresis in pyridine/acetic acid water (3:1:387 by volume) at pH 5.4, 80 V/cm. They were then converted to neutral glycans by neuraminidase digestion and were analyzed on a highresolution 1.5 cm x 2 m Bio-Gel P-4 (<400 mesh) column by elution at 550C with water (14). The structure was determined by sequential exoglycosidase digestion in combination with permethylation and GS-MS (14). Heterogeneity of sialic acid linkage in the A-1 and A-2 structures was determined by digestion with neuraninidase from Newcastle disease virus, specific for a2-3 bonds, and neuraminidase from Arthrobacter ureafaciens, which cleaves a2-3(6) bonds, followed by selective exoglycosidase digestion and product analysis using permethylation and GC-MS (14). Oligosaccharide Modification. Bovine al acid glycoprotein (AGP) (Sigma) was desialylated by incubation at 800C for 1 hr at 2-5 mg/ml in 12.5 mM H2SO4 (16), resulting in the expected alteration in electrophoretic mobility in 1% agarose gel i...

Abdominal pain, laxative use, functional vomiting, and IBS were more common in uremic individuals than in controls without renal failure. Our data have not shown whether these differences are due to the uremic state itself, or, alternatively, its treatment.

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Amyloid fibrils were isolated by extraction in water from the livers and spleens of four patients who had died of monoclonal, light-chain (AL)-type, systemic amyloidosis and one with reactive systemic, amyloid A protein (AA)-type amyloidosis. Each fibril preparation contained 1-2% by weight of glycosaminoglycan (GAG) which was tightly associated with the fibrils and not just co-isolated from the tissues with them. After exhaustive digestion of the fibrils with papain and Pronase, the GAGs were specifically precipitated with cetylpyridinium chloride and were identified by cellulose acetate electrophoresis and selective susceptibility to specific glycosidases. All the preparations contained approximately equal amounts of heparan sulphate and dermatan sulphate. There was no evidence for the presence of chondroitin sulphate or other GAGs. Fine structural analysis by oligosaccharide mapping in gradient polyacrylamide gels, following partial digestion with specific glycosidases, showed very similar structures among the heparan sulphates and the dermatan sulphates, respectively. GAGs were also extracted by solubilizing amyloid fibrils in 4 M-guanidinium chloride followed by CsCl density-gradient ultracentrifugation. Although a minor proportion of the GAG material obtained in this way was apparently in the form of proteoglycan molecules, most of it was free GAG chains. The presence in amyloid fibrils of different types, in different organs and from different patients of particular GAG classes with similar structures supports the view that these molecules may be of pathogenic significance.