Tsuneo Ozeki

Glycopeptides isolated from bovine submaxillary mucin

Abstract Extensive pronase digestion of the desialized bovine submaxillary major mucin (BSM) resulted in a mixture of glycopeptides. Separation of the glycopeptides into nine fractions, fractions a to i , was achieved by linear gradient elution at constant pH (2.6) of 0.05–0.2 m pyridine-formate buffer from Dowex 50-X2 (pyridinium form). Glycopeptide b 6 was isolated from fraction b by preparative high-voltage paper electrophoresis, and glycopeptides g 5 and e 7 from fractions g and e , respectively, by preparative high-voltage paper electrophoresis and preparative paper chromatography, in succession. Homogeneity of these glycopeptides was shown by high-voltage paper electrophoresis at pH 1.9, 3.7, and 6.5 and by paper chromatography. The results of the N-terminal analysis by dansylation method with or without sequential Edman degradation and the analytical data before and after a β-elimination reaction of glycopeptides b 6 , g 5 , and e 7 indicated the structures of these glycopeptides as follows: Glycopeptide b 6 , seryl-( O -α- N -acetylgalactosaminyl-)-serine; glycopeptide g 5 , glycyl-( O -α- N -acetylgalactosaminyl-)-serine; glycopeptide e 7 , seryl-( O -α- N -acetylgalactosaminyl-)-threonyl-glycyl-( O -α- N -acetylgalactosaminyl-)-serine.

The effect of diphenylamine derivatives on arachidonic acid metabolism in rat peritoneal macrophages

The effect of diphenylamine derivatives such as diclofenac sodium, mefenamic acid and lobenzarit disodium on arachidonic acid metabolism in rat peritoneal macrophages was examined. Lobenzarit disodium has no effect on prostaglandin E2 production as measured by radioimmunoassay although two other diphenylamine derivatives have a potent inhibitory activity. Three diphenylamine derivatives have no effect on Ca2+ ionophore-stimulated release of radioactivity from (3H)arachidonic acid-labeled macrophages. HPLC analysis revealed that lobenzarit disodium had no effect on the synthesis of lipoxygenase products as observed in diclofenac sodium and mefenamic acid. It is concluded that lobenzarit disodium, although its fundamental chemical structure resembles diclofenac sodium and mefenamic acid, has no inhibitory activity on arachidonic acid metabolism, suggesting that immunomodulatory activities of lobenzarit disodium are manifested without interfering with arachidonic acid metabolism.

Studies on severe hepatic damage induced by galactosamine

SummarySevere hepatic damage with submassive necrosis induced in rats by an intraperitoneal injection of a single dose of galactosamine hydrochloride was studied. In the severely damaged liver, the remarkable decreases of glycogen and UDPG in the damaged liver were seen. This means extreme decrease in the reserve power of glycolysis. Moreover, the activities of glucose-6-phosphatase and fructose-l, 6-diphosphatase decreased. Therefore, the glucose release from liver into the blood stream decreases and the inhibition of gluconeogenesis occurs. In the damaged liver, the decrease of UTP which is essential for the synthesis of sugar moiety of polysaccharide, was seen. Further, the activities of L-glutamine: D-fructose-6-phosphate amidotransferase and UDP N-acetylglucosamine 2’-epimerase which are two key enzymes of polysaccharide synthesizing enzyme were seen to decrease remarkably. In the damaged liver, the glycoprotein fraction decreased more strikingly than the acid mucopolysaccharide fraction. Moreover, the decrease of fructose-l,6-diphosphatase activity seems also to effect on the inhibition of polysaccharide synthesis. In these respects, in the sever hepatic damage, the synthesis of glycoprotein which is essential for liver cell seems to be inhibited.

The purification of arylsulfatases A and B

Abstract Arylsulfatases A and B (arylsulfate sulfohydrolase, EC 3.1.6.1) are isoenzymatical. Both enzymes are located mainly in the liver, though they are widely distributed in mammalian tissues [rabbit liver (1), ox liver (2), human kidney (3), human brain (4), and human liver (5)]. These enzymes are essential for the metabolism in mucolipid and mucopoly-saccharide. Therefore, the study of arylsulfatases A and B is very important. Worwood (6) recently reported an enzyme purification method for rat liver arysulfatases A and B. However, their purified enzyme showed the presence of several bands of inactive protein in addition to the active enzyme protein in disc electrophoresis. At present, there is no method for purification from rat liver except Worwoods method. Therefore, we studied a new method of enzyme purification.

The effects of hydrocortisone and glycyrrhizine on the enzyme releases of arylsulfatase and hyaluronidase from lysosomes of liver.

Hydrocortisone and glycyrrhizine act as both stabilizers and labilizers of the lysosomes of liver. The effect of both agents on the lysosomes is changeable according to the duration of their administration.

A polyhydric phenol sulfokinase and chronic liver injury

SummaryThe sulfokinase, that transfers activated sulfate from PAPS (3′-phosphoadenosine-5′phosphosulfate) to 4nitrocatechol sulfate was studied.1.It is thought that the enzyme plays an important role in detoxication by the sulfation of polyhydric phenols in liver.2.The enzyme had an optimum at pH 8.0 in Tris-acetate buffer. Km was 0.105 x 10-3M. The rate of conjugation was linear within 1 min.3.The main distribution of the sulfokinase was found in the liver, lungs, spleen and various other organs in the rat. The activity of the enzyme in rat liver was the highest in soluble fraction. In chronic hepatic injury caused by carbon tetrachloride injection, the activity of the enzyme gradually decreased to below about onehalf of the control value.

The binding rate of sialic acid to serum α1-acid glycoprotein in patients with chronic hepatic injury

SummaryThe level of serum orosomucoid, the amount of sialic acid binding to it, and the binding rate of sialic acid to orosomucoid in normal individuals and in patients with chronic hepatitis and liver cirrhosis were determined by affinity column chromatography, SDS electrophoresis and the method of Warren. Although no apparent changes in the level of serum orosomucoid or the amount of sialic acid binding to it in each group were seen, the binding rate of sialic acid to orosomucoid in chronic persistent hepatitis and chronic active hepatitis showed a remarkable increase, compared with normal individuals and patients with liver cirrhosis. Furthermore, the binding rate of sialic acid in liver cirrhosis was the lowest in each group.

Degradation of arylsulfate by hepatic microsomes.

The enzyme liberated by some treatments and the changes in arylsulfatase C activity in chronic hepatic damage were investigated in rat liver. 1. The enzyme activity liberated by ultrasound was the highest in the conditions studied. 2. Arylsulfatase C was assayed using p-nitrophenyl sulfate in 0.25 M Tris/acetate buffer as substrate. It is shown that this method can be used to measure arylsulfatase C activity in a mixture of arylsulfatases A and B. 3. The enzyme is mainly located in the microsomal fraction in rat liver. In toxic hepatic damage, the enzyme activity decreases from the early stage; decreasing markedly in chronic hepatic damage. The activity seems to reflect damage to the microsomes and therefore arylsulfatase C activity can be a good indicator of injury to liver microsomes.

The effects of vitamin E on the indicator enzymes of organella membranes in the injured liver.

SummaryAcute and chronic liver damage was caused by the administration of either galactosamine or carbon tetrachloride. Consequently, the rats with damaged livers were killed after vitamin E was administered. The livers were removed and were homogenated. Indicator enzymes (5′-nucleotidase, arylsulfatase, cytochrome C oxidase and glucose-6-phosphatase) of organella membranes were measured in the homogenates of the normal and damaged livers. The effects of vitamin E resulted in the stabilizing of the impaired membranes of plasma, lysosome, mitochondria and microsome; (1) the abnormal decrease of 5′-nucleotidase activity and glucose-6-phosphatase activity, and the abnormal increase of arylsulfatase activity, which induced galactosamine or carbon tetrachloride, and (2) the abnormal decrease of cytochrome C oxidase activity induced by galactosamine-HCl, were normalized.