S Suzuki

Micromethods have been developed for the measurement of as little as 3 µg of chondroitin sulfate A, B, or C in mixtures with other mucopolysaccharides by the use of chondroitinase-ABC, chondroitinase-AC, chondro-4-sulfatase, and chondro-6-sulfatase. With these methods, 35S-labeled chondroitin sulfates A, B, and C in a given mixture have been precisely and rapidly determined by measuring radioactivity alone. The methods have also been utilized to determine chondroitin sulfates A, B, and C in as little as 4 ml of normal urine.

Abstract 1. An enzyme, has been purified to apparent homogeneity from extracts of Proteus vulgaris, NCTC 4636, which was adapted on a medium containing chondroitin sulfate C. It has the following properties. (a) At pH 8, it degrades chondroitin sulfates A, B, and C at greater rates than chondroitin and hyaluronic acid. It does not attack keratosulfate, heparin, or heparitin sulfate. (b) It carries out an elimination reaction, yielding Δ4,5-unsaturated disaccharides. (c) In the crude extract it is accompanied by two different types of sulfatase, which are removed during purification. 2. The two sulfatases, chondro-4-sulfatase and chondro-6-sulfatase, have been separated from chondroitinase-ABC and from each other; both are required for the hydrolytic desulfation of chondroitinase products, Δ4,5-unsaturated disaccharide sulfates. They do not attack polymer chondroitin sulfates, hexa-, penta-, tetra-, or trisaccharides derived from chondroitin sulfates A and C by digestion with crude testicular hyaluronidase, or acetylgalactosamine 4-and 6-sulfates. One of these enzymes, chondro-4-sulfatase, catalyzes the conversion of Δ4,5-unsaturated disaccharide 4-sulfate (i.e. the product from the degradation of chondroitin sulfate A or B by chondroitinase-ABC) and its saturated analogue (i.e. acetylchondrosin 4-sulfate) to the corresponding nonsulfated disaccharides and inorganic sulfate, but does not attack Δ4,5-unsaturated disaccharide 6-sulfate (i.e. the product from the degradation of chondroitin sulfate C by chondroitinase-ABC) or its saturated analogue (i.e. acetylchondrosin 6-sulfate). In contrast, chondro-6-sulfatase carries out the desulfation of the disaccharide 6-sulfates and acetylgalactosamine 4,6-disulfate at position 6 while it does not attack the disaccharide 4-sulfate isomers. 3. Another type of chondroitinase, chondroitinase-AC, has been purified also to apparent homogeneity from extracts of Flavobacterium heparinum, ATCC 13125, which was adapted on a medium containing chondroitin sulfate C. Its properties have been compared with those of chondroitinase-ABC from P. vulgaris. (a) Unlike chondroitinase-ABC, it has no measurable activity with chondroitin sulfate B; like chondroitinase-ABC it carries out essentially the same reactions with chondroitin sulfates A and C, chondroitin, and hyaluronic acid. (b) In the crude extract it is accompanied by an enzyme similar to chondroitinase-ABC, an enzyme similar to chondro-4-sulfatase, and a glucuronidase which hydrolyzes the β-glucuronidic bond of unsaturated disaccharides but not the bond of saturated disaccharides. All these accompanying enzymes are removed during purification.

Abstract By means of enzymatic digestion with chondroitinase-ABC, a novel unsaturated disaccharide bearing two sulfate residues has been derived from chondroitin sulfate of squid cartilage. Various data, particularly the formation of 2-acetamido-2-deoxy-3-O-(β-d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose and its 6-O-sulfate isomer by hydrolysis with chondro-6-sulfatase and chondro-4-sulfatase, respectively, and the formation of 2-acetamido-2-deoxy-4,6-di-O-sulfo-d-galactose by mild acid hydrolysis, indicate that the two sulfate residues are located at positions 4 and 6 of the hexosamine moiety. A distinct unsaturated disaccharide also bearing two sulfate residues has been obtained from chondroitin sulfate B preparations of bovine lung and pig skin. One of the sulfate residues has been shown to be substituted at position 4 of the hexosamine moiety. The resistance of the second residue to both chondro-4-sulfatase and chondro-6-sulfatase and of the uronic acid moiety to Flavobacterium heparinum glucuronidase suggests that the sulfate residue is substituted at position 2 or 3 of the uronic acid moiety. These disulfated disaccharides are, therefore, isomers of 2-acetamido-2-deoxy-3-O-(2- or 3-O-sulfo-β-d-gluco-4-ene-pyranosyluronic acid)-6-O-sulfo-d-galactose, the compound previously found in the digest of chondroitin sulfate from shark cartilage. The three isomeric disulfated disaccharides were separated from nonsulfated and monosulfated homologues and from one another by paper chromatography in 1-butyric acid-0.5 n ammonia (5:3). The separation of disaccharides from chondroitinase digests by paper chromatography permits comparison of chondroitinase digests from different chondroitin sulfate preparations and detection of slight variations in the types of sulfate linkage.

A glycosidase which attacks corneal keratan sulfate was purified from extracts of Pseudomonas sp. IFO-13309. When corneal keratan sulfate was degraded by the purified enzyme, Sephadex G-50 chromatography indicated the presence of a number of oligosaccharides differing in size and sulfate content. The characterization of two major fractions of the oligosaccharides indicated that the point of enzyme attack is limited to the endo-beta-D-galactoside bonds in which nonsulfated D-galactose residues participate. The enzyme, unlike ordinary exo-beta-D-galactosidases, did not catalyze the hydrolysis of phenyl beta-D-galactoside. Moreover, beta-D-galactosyl-(1 leads to 3)-2-acetamido-2-deoxy-beta-D-glucosyl-(1 leads to 3)-beta-D-galactosyl-(1 leads to 4)-D-glucose ("lacto-N-tetraose") was completely refractory to the action of this enzyme, suggesting that a structure of the type, X-(1 leads to 3)-beta-D-galactosyl-(1 leads to 4)-Y, is not the only specificity-determining factor, i.e. neighboring sugars, X and Y, or even larger portions of substrate molecule must have an important effect. Compared with corneal keratan sulfate, keratan sulfates from human nucleus pulposus and shark cartilage were attacked at lower rates with a resultant production of oligosaccharides of relatively large size. The result is in agreement with the view that considerable variations exist in the structure of keratan sulfates of different origin, and further suggests that the enzyme may serve as a useful reagent in studying these variations.

Access to abortion services in the United States has become increasingly limited because of a decrease in rural hospital providers and a growing shortage of clinicians willing to offer this service. As of 1988, 83% of United States counties had no identified provider. The deficit in numbers of clinicians stems from the current imbalance between incentives and disincentives. The single most powerful incentive appears to be altruism. On the other hand, disincentives include poor pay, frequent harassment, low prestige, suboptimal working conditions, and tedium. In 1990 a symposium on abortion provision was held, sponsored by the National Abortion Federation and ACOG. Among the remedies suggested by the attendees were increasing the integration of abortion training into the mainstream of residency education, improving the pay and work environments for clinicians, and where feasible expanding the capacity of physician providers by using midlevel practitioners working under physician supervision.