Zensaku Yosizawa

A partial modification of the carbazole method of Bitter and Muir for quantitation of hexuronic acids.

Abstract The carbazole method of T. Bitter and H. M. Muir (1962, Anal. Biochem. 4 , 330–334)for quantitation of hexuronic acids was modified partially. In this modification (procedure g), 0.2 m anhydrous sodium tetraborate was used instead of 0.025 m sodium tetraborate decahydrate. The color intensity of d -glucuronolactone was slightly diminished by this modification, however, that of l -iduronolactone significantly increased. Thus, the difference in color intensity between d -glucuronolactone and l -iduronolactone became very small. When evaluated by procedure g, the hexuronic acid content of dermatan sulfate was comparable to that of chondroitin sulfate A.

Werner's syndrome as "hyaluronuria".

Abstract Although excretion of acid glycosaminoglycans into urine of five patients with Werners syndrome were within normal limits, the quantity of hyaluronic acid increased in this disease. To this novel finding, a term “Hyaluronuria” was proposed.

Glycolipids isolated from porcine intestine

Abstract Preparative silicic acid-Hyflo Supercel column chromatography of crude lipid extracts of porcine intestine gave two glycolipid fractions. The larger fraction was separated into hematosides, porcine intestinal glycolipids I & II (PIGL-I & PIGL-II) and sphingomyelin by rechromatography on a silicic acid-Hyflo Supercel column and a DEAE-cellulose column. Final purification of these glycolipids was achieved by thinlayer chromatography. The amounts of PIGL-I & PIGL-II were found to be more than half of the total sphingoglycolipids containing hexosamine or sialic acid, although other glycolipids of this type, hematosides and globoside I, were also isolated in appreciable amounts. From the smaller fraction, cerebrosides (CMH), ceramide dihexosides (CDH), ceramide trihexoside (CTH), cerebroside sulfate (CMHS) and globoside I were isolated by means of rechromatography with silicic acid-Hyflo Supercel, and subsequently by column chromatography with DEAE-cellulose or Florisil. CDH, CTH and globoside I were finally separated by thin-layer chromatography. These ceramide hexosides also were found to be major glycolipids in porcine intestine. CMHS was however obtained in a small amount. These glycolipids were characterized further by qualitative and quantitative analyses as follows: ceramide glucoside & ceramide galactoside (CMH); 3 ceramide galactoside sulfate (CMHS); ceramide lactoside & ceramide digalactoside (CDH); digalactosylglucosyl-ceramide (CTH); N -acetylgalactosaminyldigalactosylglucosyl-ceramide (globosidel); N -acetyl- and N -glycolyl-neuraminylgalactosylglucosylce-ramides (hematosides); glycolipid containing glucose, galactose, N -acetylhexosamine (glucosamine: galactosamine = 1:1), fucose, and ceramide in the molar ratio of approximately 1:2:1:1:1 (PIGL-I); glycolipid consisting of glucose, galactose, N -acetylglu-cosamine, fucose and ceramide in the molar ratio of about 1:2:2:1:1 (PIGL-II). The glycolipids showed large variation in fatty acid composition. Fatty acid compositions of PIGL-I and PIGL-II resembled each other, showing a similarity to that of CDH, but differed remarkably from those of globoside I and hematosides. On the basis of column and thin-layer chromatography and of analytical data, PIGL-I and PIGL-II were shown to be novel glycolipids. From the present observations and comparison with previous reports, the glycolipid composition of porcine intestine is species and organ specific.

Purification of chondroitinase B and chondroitinase C using glycosaminoglycan-bound AH-Sepharose 4B.

Chondroitinase B and chondroitinase C were separated from an extract of Flavobacterium heparinum induced with chondroitin 6-sulfate by using column chromatography on hydroxylapatite. Chondroitinase C was eluted together with the activities of hyaluronidase, delta4,5glycosiduronase, and sulfatase. The latter two activities were eliminated exclusively by passing the crude chondroitinase C fraction through a phosphono-cellulose column pre-equilibrated with 0.07M sodium phosphate buffer (pH 6.8). Chondroitinase C was then purified by affinity chromatography using dermatan sulfate-bound AH-Sepharose 4B coated with the same glycosaminoglycan. Purification of the enzyme was achieved 18-fold and in 73% yield. On the other hand, the activities of delta4,5glycosiduronase and sulfatase were decreased to 50 and 60%, respectively, as compared with those in the crude chondroitinase B fraction, after passing the fraction through a column of phosphono-cellulose pre-equilibrated with 0.1M sodium phosphate buffer (pH 6.8). The remaining activities of these two enzymes were then eliminated from chondroitinase B by affinity chromatography with heparin-bound AH-Sepharose 4B coated with dermatan sulfate. In the affinity chromatography used in the present study, non-covalent coating of the glycosaminoglycan-bound (covalently) AH-Sepharose 4B with the same or another glycosaminoglycan was found to be important.

Hormonal effect on glycoproteins and glycosaminoglycans in rabbit uteri.

Abstract The effect of the female hormones on glycoproteins and glycosaminoglycans in uteri has been studied. The uteri taken from the ovariectomized rabbits treated with estrogen, estrogen plus progesterone, and sham administration (control) were incubated in vitro with [U- 14 C]glucose. Subsequently, the tissues were digested extensively with pronase, yielding crude glycan fractions. The amount and radioactivity of the crude glycan fraction increased by the treatment with estrogen, but reduced to certain level with progesterone. Separation of glycoproteins and glycosaminoglycans was achieved by stepwise elution from Dowex 1 (X2, chloride form) with increasing concentration of NaCl. The yield and radioactivity, together with the results of chemical, enzymatic, and electrophoretic studies on the resulting fractions indicated that the metabolism of a slightly acidic glycoprotein, hyaluronic acid, sulfated glycoproteins, low-sulf ated chondroitin sulfate, heparan sulfate, chondroitin 4-sulfate, and dermatan sulf ate were stimulated remarkably with estrogen, but the stimulation was restored to certain level with progesterone. The degree of the change with these hormones was, however, found to be different from each other. It was noticed that sulfated glycoproteins were the most sensitive to the hormones. On the other hand, the estrogenic stimulation of the metabolism of a neutral glycoprotein and oversulfated chondroitin sulfates was not restored with progesterone.

Purification and properties of sulfated sialopolysaccharides isolated from pig colonic mucosa

Abstract Mucopolysaccharide fractions were obtained from Pronase-digested pig colonic mucin. All mucopolysaccharide fractions were sulfated and acidic in nature. They were separated into two groups: sulfated sialopolysaccharides (MPS-I) and sulfated uronic acid-containing mucopolysaccharides (MPS-II). The former was much more abundant than the latter. MPS-I was fractionated into four subfractions and each fraction was purified to the highly homogeneous state. MPS-I consists of macromolecules with molecular weights of 200,000–300,000 and is composed of large polysaccharides and small peptides. The component sugars were galactose, fucose, glucosamine, galactosamine, and sialic acid. The position of the sulfate group was not determined but evidence to show that the sulfate group is the integral component of the polymer was given. Threonine, serine, and proline were predominant amino acids composing the peptide moiety.

Stability of the biological activities of heparins to mild acid treatments

Abstract The stabilities of the anticoagulant and lipolytic activities of bovine heparin (α-heparin) and whale heparin (ω-heparin) to mild acid treatments were investigated. The results showed that the anticoagulant property of α-heparin was more labile, and its lipolytic activity less labile, than that of ω-heparin. Release of approximately 7 to 8% of N -sulfate groups resulted in almost complete loss of the anticoagulant activity, in both α- and ω-heparin. Treatment with 40% acetic acid at 37° for 1 day almost completely deprived these heparins of their anticoagulant properties without causing any substantial decrease in their lipolytic activities.

A rapid method for separation and identification of several hexuronic acids and hexuronic acid-containing oligosaccharides

Abstract Five naturally occurring hexuronic acids and several hexuronic acid-containing oligosaccharides were separated and identified by high voltage paper electrophoresis, using one of the following buffers. (i) Pyridine-acetic acid-water (1:10:89, by volume), the pH of which was adjusted to 2.3–3.5 with 98% formic acid. (ii) Pyridine-water (1:90, by volume), the pH of which was adjusted to 3.5–4.0 with glacial acetic acid. The best separation of the five hexuronic acids and heparin disaccharides was observed at pH 2.7 after electrophoresis for 180 min at 100 V/cm. At pH 3 l -gulosyluronic acid- l -guluronic acid could be easily isolated from an acid hydrolysate of alginate by the present method.

Methods for analysis of urinary glycosaminoglycans

Methods for the analysis of urinary GAGs that can be used for or are applicable to routine assays are described. The most popular method for isolation of GAGs from a urine sample is CPC precipitation, in spite of the fact that it is time-consuming. To identify the different types of GAGs excreted, separation by one-dimensional cellulose acetate electrophoresis followed by staining with alcian blue or toluidine blue may suffice for routine purposes. Solvents such as barium acetate, calcium acetate, barbital buffer and pyridine-formic acid are used for the separation. However, the separation of the seven types of GAGs by conventional one-dimensional electrophoresis is difficult, and a discontinuous electrophoretic method with barium acetate buffer and barium acetate buffer containing ethanol has proved effective for the separation. HPLC separation methods are used for assaying the profiles of enzymatic digestion products of GAGs. Advanced HPLC methods for separating intact GAGs of different types are currently unavailable. Unsaturated disaccharides produced with heparitinase and/or heparinase from heparan sulphate and oligosaccharides produced by hyaluronidase digestion of hyaluronic acid can be separated by HPLC. For chondroitin sulphate isomers, unsaturated disaccharides produced by digestion of the samples with chondroitinase ABC or chondroitinase AC are separated by HPLC and determined by their UV absorbance or by fluorescence labelling. Highly sensitive quantitation of chondroitin sulphate isomers is possible by these methods, which are also efficient for the investigation of the constituents of GAG polymers. Some of these methods have been applied to urine samples from patients with, e.g., mucopolysaccharidoses.

Glycosaminoglycans and acidic glycoproteins in rabbit uterus under estrogenic conditions

Uterine slices obtained from the estrogen-treated rabbits were digested with pronase. Glycosaminoglycans and acidic glycopeptides were then isolated by Dowex 1 column chromatography and preparative electrophoresis on cellulose acetate membrane (Separax), in succession. Each subfraction thus obtained was identified by the mobility on Separax electrophoresis and the digestibility with mucopolysaccharidases (Streptomyces hyaluronidase, testicular hyaluronidase, chondroitinase AC, chondroitinase ABC and heparinase). The resulting data showed that each complex saccharide (hyaluronic acid, heparan sulfate, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, sulfated glycopeptide and sialoglycopeptide) was separated into 2-5 fractions, indicating charge and/or molecular heterogeneity of each complex saccharide.