[117] Sialidase from Clostridium perfringens

Cassidy, James T.; Jourdian, George W.; Roseman, Saul

doi:10.1016/0076-6879(66)08123-0

Cited by 77 publications

(56 citation statements)

References 16 publications

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“…Cell Biology: Kanwar (21), and Pronase (4-6 units/ ml) in balanced salt solution (pH 7.4) for 10-20 min at 30-350C. In each case control specimens were perfused with buffer alone under the same conditions.…”

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confidence: 99%

Presence of heparan sulfate in the glomerular basement membrane

Kanwar

Farquhar

1979

Proc. Natl. Acad. Sci. U.S.A.

495

191

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The glomerular basement membrane was subjected to digestion with specific enzymes to determine the chemical nature (sialoglycoproteins, collagenous peptides, or glycosaminoglycans) of the anionic sites previously demonstrated in the laminae rarae. Enzyme digestion was carried out both in situ and in vitro. Kidneys were perfused in situ with enzyme solutions followed by perfusion with fixative containing the cationic dye, ruthenium red, to detect the anionic sites. Glomerular basement membranes were isolated by detergent treatment of glomeruli and incubated with enzyme solutions, followed by incubation with cationized ferritin (pI 7.3-7.5 Physiologic studies have established that the glomerular capillaries function as a size-and charge-selective barrier in the production of the glomerular filtrate and in the retention of plasma proteins in the circulation (1-3). By inference it has been assumed (1-3) that charge selectivity is due to the presence in the glomerulus of fixed negative charges, but their precise nature and location in the capillary wall have not been established. Since macromolecular compounds carrying sialic acid residues have been-up to recently-the only known polyanions present in the glomerulus, they have been considered the most likely candidates responsible for the charge barrier (1, 2, 4-7). Sialyl residues have been found in all three layers of the capillary wall. They have been detected in isolated glomerular basement membranes (GBM) (8-10) and in association with the cell membranes of both the endothelium (11-12) and epithelium (4-7, 11), where they are part of the sialic acid-rich, cell-surface coats. A colloidal iron-stainable, cell-coat material is particularly concentrated on the epithelium. Accordingly, it has been repeatedly suggested (1, 2, 4-7) that this "epithelial polyanion" is responsible for establishing the charge-selective properties of the glomerular filter, and, concomitantly, that its loss leads to proteinuria since loss of epithelial cell-coat staining occurs in several renal diseases (1, 2, 5). Materials. Ruthenium red was purchased from Ventron Corp. (Danvers, MA); horse-spleen ferritin (2X crystallized, cadmium free) from Calbiochem; and chondroitin sulfates (types A, B, and C) and heparin (grade I) from Sigma. Chondroitinase ABC (Proteus vulgaris) was obtained from Miles; testicular hyaluronidase from Sigma; leech hyaluronidase from Biotrics, Inc. (Boston, MA); neuraminidase (Clostridium perfringens, NEUA) from Worthington; collagenase form III (Cl. histolyticum) from Advance Biofactures (Lynbrook, NY); and Pronase (Streptomyces griseus, grade B) from Calbiochem. Crude heparinase and purified heparitinase (Flavobacterium heparinium) were the generous gift of Alfred Linker (17).* Neuraminidase and heparitinase were tested by the Azocoll assay (18) and were free of proteolytic activity. Cationized ferritin was prepared as described previously (16).Perfusion Experiments. The left kidney was exposed and perfused as described (16). Initially, the kidney was flush...

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confidence: 99%

Presence of heparan sulfate in the glomerular basement membrane

Kanwar

Farquhar

1979

Proc. Natl. Acad. Sci. U.S.A.

495

191

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“…Cassidy et al (1966) recommend that the assay of C. welchii neuraminidase with NAN-lactose as substrate should be performed in potassium-acetate buffer at pH 4-5. When the assay is performed with FVII substrate and sodium acetate buffer at pH 5-1, the results fall on the high plateau between the two peaks of activity; for assays performed in trismaleate buffer the pH should be pH 5.7 with this substrate.…”

Section: Discussionmentioning

confidence: 99%

Preparation of a Glycoprotein Fraction From Pooled Human Plasma and Its Evaluation as a Substrate for the Assay of Clostridium Welchii (C. Perfringens) Neuraminidase

Fraser

Smith

1975

Journal of Medical Microbiology

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“…It is then submitted to tryptic (EC 3.4.4.4) or chymotryptic (EC 3.4.4.5) digestion (E/S: 1/50, pH 8.2, at 37°C for 8 hr [8]. The enzymic digests were submitted to gel filtration (Biogel P-30) and the glycopeptide subfractions were further repurified by a free flow electrophoretic separation (Elphor Vap-II) which was carried out at pH 2.4 in 0.5 M acetic acid, at a potential gradient of 1700 V. The glycopeptide subfractions obtained by this method were digested with neuraminidase (EC 3.2.1.18) [9] and the desialyzed glycopeptides were isolated by preparative paper electrophoresis (pH 6.5 in pyridine-acetic acid-water 90:3:1400, 7 V/cm for 16 hr).…”

Section: Methodsmentioning

confidence: 99%