Satoru Makisumi

Interactions of derivatives of guanidinophenylalanine and guanidinophenylglycine with Streptomyces griseus trypsin.

The rates of hydrolysis of the ester, amide and anilide substrates of p-guanidino-L-phenylalanine (GPA) by Streptomyces griseus trypsin (S. griseus trypsin) were compared with those of arginine (Arg) substrates. The specificity constant (kcat/km) for the hydrolysis of GPA substrates by the enzyme was 2-3-times lower than that for arginine substrates. The kcat and Km values for the hydrolysis of N alpha-benzoyl-p-guanidino-L-phenylalanine ethyl ester (Bz-GPA-OEt) by S. griseus trypsin are in the same order of magnitude as those of N alpha-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt), although both values for the former when hydrolyzed by bovine trypsin are higher by one order of magnitude than those for the latter. The specificity constant for the hydrolysis of Bz-GPA-OEt by S. griseus trypsin is much higher than that for N alpha-benzoyl-p-guanidino-L-phenylglycine ethyl ester (Bz-GPG-OEt). As with the kinetic behavior of bovine trypsin, low values in Km and kcat were observed for the hydrolysis of amide and anilide substrates of GPA by S. griseus trypsin compared with those of arginine substrates. The rates of hydrolysis of GPA and arginine substrates by S. griseus trypsin are about 2- to 62-times higher than those obtained by bovine trypsin. Substrate activation was observed with S. griseus trypsin in the hydrolysis of Bz-GPA-OEt as well as Bz-Arg-OEt, whereas substrate inhibition was observed in three kinds of N alpha-protected anilide substrates of GPA and arginine. In contrast, no activation by the amide substrate of GPA could be detected with this enzyme.

Some properties of trypsin-like proteases extracted from the seaweedCodium fragile and their purification

Two enzymes were purified from the green algaCodium fragile, collected in 1988 at Fukuoka, Japan, by batch-wise ion-exchange extraction, affinity chromatography and ion-exchange high-performance liquid chromatography (HPLC) using Boc-Ala-Ala-Pro-Arg-pNA as a substrate. The molecular weights of the enzymes were estimated as 38 000 and 39 000, using gel filtration HPLC. The enzymes had the same optimal pH range of 7 to 9 for both activities, and an exclusively hydrolyzed peptide bond on the carboxyl-terminal side of theL-arginine of peptidep-nitroanilides. The ratios of the enzymatic activity for X-Arg-pNA to X-Lys-pNA were larger than 100. The enzymes exhibited 30 times higher activity toward Boc-Ala-Ala-Pro-Arg-pNA when compared with trypsin. The activities were strongly inhibited by diisopropyl phosphofluoridate (DFP), and partially inhibited by phenylmethylsulfonyl fluoride (PMSF), benzamidine, leupeptin and antipain. The isolated enzymes were presumed to be trypsin-like serine protease from their primary substrate specificities and inactivation behavior.

A new serine protease which preferentially recognizes p-guanidino-L-phenylalanyl residue in ascitic plasma from Ehrlich ascites tumor-bearing mice.

A new enzyme which hydrolyzes anilide substrates of p-guanidino-L-phenylalanine in preference to those of arginine was found in the ascitic plasma from Ehrlich ascites tumor-bearing mice. The activity of this enzyme on N alpha-benzyloxycarbonyl-p-guanidino-L-phenylalanine p-nitroanilide was strongly inhibited by diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride but not by sulfhydryl-reactive reagents and metal chelating agents. Peptide substrates containing p-guanidino-L-phenylalanine were hydrolyzed by this enzyme much faster than those containing arginine. These results suggest that this enzyme is a different type of serine protease from trypsin and thrombin. This enzyme was also found in the human gastric and colon cancer cells and their surrounding ascitic plasmas.

Absolute configuration of Nδ-benzoyl-γ-hydroxyornithine from Vicia pseudo-orobus

Abstract A pair of diastereoisomers of N δ-benzoyl-γ-hydroxy- l -ornithine was synthesized. By comparison with the two synthetic compounds, the natural