Shiro Hayashi

Determination of acetylcholine in human blood

Acetylcholine levels of whole blood from 80 healthy subjects were determined by pyrolysis-gas chromatography-mass fragmentography. Acetylcholine was extracted from 1.2 ml of venous blood in the presence of eserine, demethylated by pyrolysis, and assayed by selected ion-current monitoring using butyrylcholine as an internal standard. Inhibition of blood cholinesterase activity by eserine was essential to accurate measurement. The overall recovery of acetylcholine was 45%. The blood acetylcholine levels of healthy subjects varied over a wide range with a geometric mean of 0.49 mumole/liter, 90% of the levels falling into the range of 0.20 to 1.31 mumole/liter. There was no correlation between the blood acetylcholine level and age or sex.

The structures of N- and O-glycosidic carbohydrate chains of a chondroitin sulfate proteoglycan isolated from the media of the human aorta

A large Mr chondroitin sulfate proteoglycan was extracted from the media of human aorta under dissociative conditions and purified by density-gradient centrifugation, ion-exchange chromatography, and gel filtration chromatography. Removal of a contaminating dermatan sulfate proteoglycan was accomplished by reduction, alkylation and rechromatography on the gel filtration column. After chondroitinase ABC treatment, the proteoglycan core was separated from a residual heparan sulfate proteoglycan by a third gel filtration chromatography step. As assessed by radioimmunoassay, the isolated proteoglycan core was free of link protein, but possessed epitopes that were recognized by antisera against the hyaluronic acid binding region of bovine cartilage proteoglycan as well as those that were weakly recognized by anti-keratan sulfate antisera. Following beta-elimination of the protein core, the liberated low Mr oligosaccharides were partially resolved by Sephadex G-50 chromatography, and their primary structure was determined by 500-MHz1H NMR spectroscopy in combination with compositional sugar analysis. The N-glycosidic carbohydrate chains, which were obtained as glycopeptides, were all biantennary glycans containing NeuAc and Fuc; microheterogeneity in the NeuAc----Gal linkage was detected in one of the branches. The N-glycosidic glycans have the following overall structure: (Formula: see text). The majority of the O-glycosidic carbohydrate chains bound to the protein core were found to be of the mucin type. They were obtained as glycopeptides and oligosaccharide alditols, and possessed the following structures: NeuAc alpha(2----3)Gal beta(1----3)GalNAc-ol, [NeuAc alpha(2----3)Gal beta(1----3)[NeuAc alpha(2----6)]GalNAc-ol, and NeuAc alpha-(2----3) Gal beta(1----3)[NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)] GalNAc-ol. The remainder of the O-glycosidic carbohydrate chains bound to the isolated proteoglycan were the hexasaccharide link regions of the chondroitin sulfate chains that remained after chondroitinase ABC treatment of the native molecule. These latter glycans, which were obtained as oligosaccharide alditols, had the following structure (with GalNAc free of sulfate or containing sulfate bound at either C-4 or C-6): delta 4,5GlcUA beta(1----3)GalNAc beta(1----4)GlcUA beta(1----3)Gal beta(1----3)Gal beta(1----4)Xyl-ol.

On the Mode of Participation of Hyaluronidase and Exoglycosidases in the Degradation of Hyaluronic Acid and Chondroitin 4-Sulfate with Canine Liver Lysosomes

Publisher Summary This chapter outlines several biochemical attempts to clarify the degradation pathway of acidic glycosaminoglycans. It describes a study to determine the contribution of hyaluronidase, β-glucuronidase, 3-N acetylhexosaminidase, and sulfatase to the degradation of acidic glycosaminoglycans(AGAG) as well as to the degradation of the oligosaccharides derived from AGAG were examined on the basis of the characterization and the identification of the digestion products. Initially, HA was degraded into even-numbered oligosaccharides, mostly with release of a trace-amount of free glucuronic acid. However, in the prolonged digestion study, an increase of odd-numbered oligosaccharides, such as tri- and penta-saccharides, was observed in proportion to an increase of free glucuronic acid. The octasaccharide was not degraded into the heptasaccharide by the action of β-glucuronidase but was degraded into a mixture of di-, tetra-, and hexa-saccharides by hyaluronidase.