Bernard Weissmann

The hyaluronidase of rat skin

Abstract A hyaluronidase demonstrated in rat skin has been purified 75-fold from extracts. The enzyme is an endo-β-acetylhexosaminidase, acts on chondroitin 4- and 6-sulfate at a lesser rate than on hyaluronate, and catalyzes transglycosylation as well as hydrolysis. Optimal activity with hyaluronate occurs at pH 3.7. Smaller hyaluronate oligosaccharides are poor substrates. Enzyme fractions showing some small catalytic differences are separable from skin extracts by ion-exchange chromatography. This finding is extended by electrofocusing experiments, which partially resolve a number of isozymes. These differ by as much as 1.5 pH units in isoelectric point. Viral neuraminidase apparently effects a partial conversion of the more acidic to the less acidic isozymes. Colorimetric assays for hyaluronidase have been modified to overcome interference by β-glucuronidase and β-acetylglucosaminidase, both present in crude tissue extracts. The amount of hyaluronidase that occurs in skin is amply sufficient in itself to account for the rate of catabolism reported for hyaluronate in this tissue.

α-L-iduronidase in lysosomal extracts

Summary Use of phenyl α-L-iduronide as a test substrate now makes it possible to show occurrence in rat liver lysosomes of an α-L-iduronidase. The enzyme can be shown to be distinct from the well studied β-glucuronidase. With the phenyl glycoside, measurements can be made of this relatively weak mammalian activity, whose occurrence could previously be inferred only indirectly from the slow degradation of dermatan sulfate derivatives by tissue extracts. Trials with appropriate aryl glycosides indicate absence of detectible α-D-glucuronidase, α-D-mannuronidase, α-D-galacturonidase, or β-L-iduronidase activities from lysosomal extracts.

The phenyl α- and β-L-idopyranosiduronic acids and some other aryl glycopyranosiduronic acids

Abstract Condensation of 1,2,3,4,6-penta- O -acetyl-a- l -idopyranose ( 1 ) with phenol yielded phenyl 2,3,4,6-tetra- O -acetyl-α- ( 2 ) and α- l -idopyranoside ( 4 ). Deacetylation of 2 and 4 afforded phenyl α and β- l -idopyranosides ( 3 and 5 ), respectively, the structures of which were verified by periodate oxidation studies. A platinum-catalyzed oxidation of 3 and 5 produced the amorphous phenyl α- and β- L -idopyranosiduronic acids ( 9 and 11 ), respectively, which were isolated as the crystalline cyclohexylammonium salts. Phenyl β- and α- d -glucopyranosiduronic acids are apparent minor byproducts of the catalytic oxidations, resulting from an inversion at C-5. p -Nitrophenyl α- d -mannopyranosiduronic acid and p -nitrophenyl α- and β- d -galactopyranosiduronic acids are also described.

Preparation and characterization of tetrasaccharides from beef-lung heparin

Abstract Partial N -desulfation of beef-lung heparin prior to degradative deamination with butyl nitrite and reduction with sodium borotritide yielded many large fragments. From these, a tetrasaccharide tetra- O -sulfate (II-4NH; 8% yield from heparin) and a mixture of tetrasaccharide tri- O -sulfates (II-3NHh; 6% yield) were isolated by sequential chromatography on Sephadex G-25 and DEAE-Sephadex. For these and the other tetrasaccharide preparations, the radioactive disaccharides produced by deamination, with and without subsequent relabelling with sodium borotritide, have been quantitatively determined by the methodology described in the preceding paper. In most cases, the results permit a unique reconstruction of the relative proportions of monosaccharide components and of their sequences in the compounds present. Tetrasaccharide II-4NH appeared homogeneous and has the structure (IdoA-SO 4 )(GN-O-SO 4 )(IdoA-SO 4 )(anhMan-SO 4 ). In tetrasaccharide preparation II-3NHh, the preponderant species (57%) lacks ester sulfate at the terminal l -iduronic residue in the structure just mentioned, and five other species are present. By treatment of the tetra- O -sulfate with mild acid, tetrasaccharide preparations with 3, 2, 1, and no ester sulfate were produced and could be isolated. The isomeric tetrasaccharide tri- O -sulfate species have been partially resolved. Composition and sequence data are given for all of the preparations. The resolution of numerous small fractions suggests minor irregularities in the fine structure of heparin. Ion-exchange electrophoresis was applied to the acidic oligosaccharides and was found to be a useful technique.

Concerted Action of β–Glucuronidase and β–Acetylglucosaminidase on Hyaluronodextrins

A kinetic analysis of the stepwise alternating action of β–glucuronidase and β–acetylglucosaminidase on oligosaccharides and dextrins derived from hyaluronic acid was undertaken, for better definition of the contribution of this process to hyaluronate catabolism. Production of monosaccharide from larger dextrins by action of either enzyme is powerfully inhibited by electrolyts. In the study, as in mammalian tissues, β–glucuronidase is present in excess so that the concentration of β–acetylgucosaminidase is rate controlling in the action on dextrin substrates. For this action, Vmax shows limited variation with ionic strength or molecular weight of substrate. At ionic strength 0. 03, but not 0. 18, Km decreases some 100–fold for increase of molecular weight from 2, 000 to 15, 000. It is specifically this decrease in Km that accounts for the prominent electrolyte inhibition observed with larger dextrins. The extremely low values of Km are attributed to multiple ionic enzyme–substrate interactions at sites re...

Some chemical, enzymic, and physical properties of di-saccharides from beef-lung heparin

Abstract A study is reported of the reactivities of the disaccharides isolated after deamination of beef-lung heparin and reduction of the products by sodium borotritide: 2,5-anhydro-O-(α- l -idopyranosyluronic acid sulfate)- d -mannitol sulfate, SIMS; 2,5-anhydro-O-(α- l -idopyranosyluronic acid)- d -mannitol sulfate, IMS; 2,5-anhydro-O-(α- l -idopyranosyluronic acid sulfate)- d -mannitol, SIM; and 2,5-anhydro-O-(β- d -glucopyranosyluronic acid)- d -mannitol sulfate, GMS. Results for the non-sulfated disaccharides IM and GM, prepared by desulfation of SIMS and GMS, are also reported. SIMS and SIM were inert to purified α- l -iduronidase, showed unexpected resistance to periodate oxidation, and lost sulfate rapidly in 50m m hydrochloric acid at 100°. Hydrolysis of IM and of IMS was catalyzed by α- l -iduronidase, and of GM and GMS by β- d -glucuronidase; the radioactive products were identified as 2,5-anhydro- d -mannitol (aM) and its sulfate (aMS). The products SIM and IMS obtained by deamination of heparin and desulfation of SIMS (the major deamination product) are apparently identical. In heparin partially desulfated by methanolic hydrogen chloride, residual sulfate groups were mostly linked to l -iduronic acid residues. Chemical, chromatographic, and electrophoretic methods that are valuable for separation and characterization of the disaccharides are described.

Preparation of (aryl α-l-idopyranosid)uronic acids

Abstract The earlier preparation of cyclohexylammonium (phenyl α - l -idopyranosid)-uronate has been improved, and (4-methylumbelliferyl α - l -idopyranosid)uronic acid ( 14 ), a more sensitive substrate for α - l -iduronidase, has been synthesized by an analogous route. Zinc chloride-catalyzed condensation of 4-methylumbelliferone with 1,2,3,4,6-penta- O -acetyl- α - l -idopyranose (4) in 1,2-ethanediol diacetate gave crystalline 4-methylumbelliferyl 2,3,4,6-tetra- O -acetyl- α - l -idopyranoside (7). O -Deacetylation and catalytic oxidation gave 14 , characterized as a cyclohexylammonium salt. The starting material 4 was prepared, in 21 % yield from l -glucose, by conversion of the intermediate 1,2,3,4,6-penta- O -acetyl- β - l -glucopyranose to 2,3,4,6-tetra- O -acetyl- β - l -glucopyranosyl chloride and acetoxonium ion rearrangement, as described for the D-series.

A colorimetric method for α-naphthol and its application to assay of hydrolases

Abstract Coupling of α-naphthol with diazotized Aniline Yellow in sodium carbonate solution produces a water-soluble dye (λ max 580 mμ). The reaction serves as the basis of a sensitive estimation of α-naphthol. The general applicability of this colorimetry to assay of hydrolases, using naphthyl esters and glycosides as substrates, is illustrated by an assay of β-acetylglucosaminidase.

Characterization of Reference Disaccharides from Nitrous Acid Deamination of Beef Lung Heparin

Publisher Summary This chapter explains the characterization of reference disaccharides from nitrous acid deamination of beef lung heparin. In a study described in the chapter, uronosyl (IdoA or GlcA) anhydromannitol (AM) disaccharides were prepared as markers and as model compounds for structural studies of heparin and, particularly, of its oligosaccharides, which were required as enzyme substrates. The chapter explains the sensitivity of disaccharide to sodium metaperiodate (8mM) and HCl (0.05M). It also explains the hydrolysis of disaccharides with β-D-glucuronidase and α-L-iduronidase, which showed that disaccharides not sulfated at the uronosyl residue were appropriately cleaved by the same. The chapter also illustrates the anion exchange electrophoresis patterns at pH 6 of crude tetrasaccharide mixture from lung heparin, tetrasaccharide A, tetrasaccharide B, and a digest of tetrasaccharide B with rat spleen homogenate.

Determination of uronic acid in uronides by application of a new microgram-scale isotope dilution method for carbon dioxide.

Conditions studied earlier by Tracey [(1948) Biochem. J.43, 185] are used for acid decarboxylation in sealed tubes of uronide samples supplemented with 6-14C-labeled uronic acid. The specific activity of the CO2 evolved is measured as the ratio of radioactivity to area of the CO2 peak obtained in a gas chromatogram. By appropriate standardization, samples containing some 60 nmol of uronic acid can be analyzed with reproducibility and apparent accuracy of about ±2% (mean deviation). The techniques developed for uronic acid analysis should apply with minor modification to any problem requiring accurate measurement of CO2 in small amounts.