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International audiencePreviously, we found three enzyme fractions containing activities for the hydrolysis of royal jelly proteins from honeybee queen larvae. In this study, we identified a honeybee chymotrypsin-like protease (HCLPase) by LC-MS/MS and expressed it as a recombinant protein in Escherichia coli. The protease had an estimated molecular weight of around 26 kDa and showed high specificity for succinyl-Ala-Ala-Pro-Phe p-nitroanilide as a proteolytic substrate. Furthermore, the protease had an optimal pH of 9, and the activity was markedly inhibited by phenylmethylsulfonyl fluoride but not tosyl phenylalanyl chloromethyl ketone, both of which are irreversible inhibitors of chymotrypsin-like serine proteases. These results suggested that this recombinant protease, HCLPase, was a chymotrypsin-like serine protease with different characteristics from mammalian chymotrypsin

Ethyl alpha-D-glucoside (alpha-EG) is normally contained in Sake, which has been taken by Japanese people since ancient times. In this study, the intestinal absorption of alpha-EG was investigated using rat everted intestinal sac. Furthermore, the alpha-EG hydrolytic activity in rat intestine was compared with disaccharides hydrolytic activities, and the effects of alpha-EG on disaccharides hydrolysis were examined using crude enzyme preparation from rat intestinal acetone powder. Glucose liberated from alpha-EG was detected in a serosal solution of everted rat intestinal sac, but it was only less than 4% of absorbed intact alpha-EG. alpha-EG absorption into small intestinal tissue was reduced by elimination of sodium ion from the mucosal solution or under the presence of phlorizin. The hydrolytic activity for alpha-EG was detected in crude enzyme preparation from rat intestinal acetone powder, but it showed a low value as compared to those for disaccharides. alpha-EG showed mixed type inhibition for maltose and sucrose hydrolysis, but inhibitory concentrations of alpha-EG required for 50% inhibition for the maltose and sucrose hydrolysis were higher than those of arabinose and acarbose. In conclusion, a small amount of alpha-EG was hydrolyzed and most of it was absorbed via SGLT1 as an intact form in the rat small intestine, and the inhibitory effect of alpha-EG on disaccharides hydrolysis was weak.

SummaryThe objective of this study was to examine how transient high plasma homocysteine (Hcy) levels affect the metabolism of Hcy, the activity and expression of S -adenosylhomocysteine (SAH) hydrolase which catalyzes both SAH hydrolysis and SAH synthesis. Wistar ST rats (males) were cannulated in the right jugular vein for intravenous infusion of physiological saline or DL -Hcy solutions (15 and 30 mg/mL) for 1 h at 1.1 mL/h/rat. The content of S -adenosylmethionine (SAM), SAH-synthetic activity of SAH hydrolase and the expression of SAH hydrolase mRNA in liver extracts showed no significant difference in the Hcy infused groups as compared to the Control group. On the other hand, the contents of hepatic SAH in the Hcy infused groups were dose-dependent and significantly higher than that of the Control group. Thus, this study showed that hepatic SAH increased without any increase in the SAH-synthetic activity and the expression of SAH hydrolase mRNA under transient high plasma Hcy levels after intravenous infusion of Hcy. Key Words plasma homocysteine, S -adenosylhomocysteine, S -adenosylhomocysteine hydrolase, methionine metabolism Homocysteine (Hcy) is an intermediate of the methionine metabolic pathway, and it occupies a branch point of two metabolic pathways, the remethylation pathway to methionine and the transsulfuration one to cysteine. These two pathways are coordinated by S -adenosylmethionine (SAM), which acts as an allosteric inhibitor of the methylentetrahydrofolate reductase (EC 1.1.1.68) and as an activator of cystathionine ␤ -synthase (EC 4.2.1.22; CBS) ( 1 ). SAM is converted to S -adenosylhomocysteine (SAH) by some transmethylation reactions, and then SAH is hydrolyzed by SAH hydrolase (EC 3.3.1.1), a reversible enzyme which catalyzes SAHhydrolytic reaction to Hcy and adenosine, and SAHsynthetic reaction from Hcy and adenosine. In general, equilibrium of the SAH hydrolase reaction favors the direction of SAH synthesis, while the SAH-hydrolytic reaction predominates under physiological conditions. Predominant SAH hydrolysis is maintained by rapid removal of Hcy. However, if it is difficult for sufficient Hcy to be removed from this cycle due to CBS deficiency or inhibition of methionine synthase, it means that the methionine cycle can not maintain its balance. The balance of the methionine cycle is maintained intricately by methionine metabolites. Therefore disturbed methionine metabolism induces several diseases, e.g. hyperhomocysteinemia.An increase of plasma Hcy is caused by nutritional deficiencies or genetic mutations. Vitamin B 6 (B 6 ) deficiency ( 2 , 3 ) and folic acid deficiency ( 4 ) were reported as the factors affecting Hcy accumulation. Furthermore, the relationship between elevated plasma Hcy and accumulated SAH was observed in several studies using rats ( 5 , 6 ). Elevated plasma Hcy and accumulated SAH are likely to be caused by disturbed methionine metabolism. We reported in our previous study that the elevation of plasma Hcy and accumulation of hepatic SAH were observed in B 6...

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