α-Fetoprotein impairs activation of natural killer cells by inhibiting the function of dendritic cells.

α-Fetoprotein (AFP) is a tumour-associated antigen in hepatocellular carcinoma (HCC). The biological properties of AFP have been identified in its regulatory effects on immune responses of T cells and B cells. However, AFP effects on natural killer (NK) cells are still unclear. In this study, we examined the immunoregulation of AFP on NK activity. The cytolytic activity against K562 cells and Huh7 cells of NK cells co-cultured with AFP-treated dendritic cells (DCs) (AFP-DCs) was lower than that with albumin-treated DCs (Alb-DCs). Direct addition of AFP to NK cells did not alter the cytolytic activity of NK cells. Adding AFP inhibited the interleukin (IL)-12 production of DCs after stimulation with lipopolysaccharide (LPS) [Toll-like receptor (TLR)-4 ligand], or Poly(I:C) (TLR-3 ligand), but not IL-18 production. The mRNAs of IL-12p35 and IL-12p40 were significantly inhibited in AFP-DCs compared with Alb-DCs, but those of TLR-4 or TLR-3 were not. Transwell experiments revealed that soluble factors derived from DCs played roles in inhibition of the ability of activating NK cells by AFP-DCs. Adding the neutralizing antibody of IL-12 to NK cells co-cultured with Alb-DCs resulted in a decrease of cytolytic activity to the levels of NK cells co-cultured with AFP-DCs. Adding IL-12 to NK cells co-cultured with AFP-DCs resulted in an increase of cytolytic activity to the levels of NK cells co-cultured with Alb-DCs. These demonstrated that the impairment of IL-12 production from AFP-DCs resulted in inhibition of the ability of the activation of NK cells by DCs, and thus suggests a role of AFP in HCC development.

Synthesis and the stereoselective enzymatic hydrolysis of flurbiprofen-basic amino acid ethyl esters.

Ethyl esters of flurbiprofen L-arginine (FP-Arg-OH), flurbiprofen L-lysine (FP-Lys-OH) and flurbiprofen p-guanidino-L-phenylalanine (FP-GPA-OH) were synthesized and then the release of flurbiprofen enantiomers from these derivatives in the presence of trypsin (Tp), carboxypeptidase B (CPB) and carboxypeptidase Y (CPY) were examined in order to evaluate their availability as prodrugs for flurbiprofen (FP). The ester bonds of the three racemic FP derivatives were hydrolyzed by Tp at about 3 to 20 times the rates of N-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt), a specific substrate for Tp. (R)-FP was released faster than (S)-FP by either CPB or CPY from both FP-Arg-OH and FP-Lys-OH. On the other hand, FP-GPA-OH was not hydrolyzed at all by CPB and the hydrolysis rate of this compound by CPY was very slow. (S)-Flurbiprofen L-arginine ethyl ester ((S)-FP-Arg-OEt) was separated from (R)-FP-Arg-OEt by high-performance liquid chromatography. A comparison of the kinetic parameters for the tryptic hydrolysis of the two optically active FP-Arg-OEt diastereomers and those of Bz-Arg-OEt suggested that the orientation of the scissile bond in each diastereomer to the catalytic center of Tp is more favorable than that of Bz-Arg-OEt. However, no significant difference was found between the kinetic parameters for the two diastereomers, suggesting that the orientational difference between (S)-FP and (R)-FP in the diastereomers does not have any effect on the tryptic hydrolysis of the ester bond.(ABSTRACT TRUNCATED AT 250 WORDS)

Conformational effect on the fragmentations of peptide derivatives in field desorption mass spectrometry.

Fragmentations of N-benzyloxycarbonyl-protected tri-peptide ethyl esters containing proline at the P2 site were compared with those of the corresponding peptide derivatives containing no proline in field desorption mass spectrometry. The fragment ion [M-107]+ due to a loss of the benzyloxy group from a molecular ion was observed in the field desorption mass spectra for the peptides containing no proline, while it was not found in the peptides containing proline at all. These results suggest that the conformational difference of the peptide derivatives attributable to the existence of proline has an effect upon fragmentations in the field desorption ionizing process.

The conformation of the lysyl side chain of substrates at the active center of trypsin.

In order to study the conformation of the side chain of lysine substrates bound to the active center of trypsin, two lysine analogs, cis- and trans-2,6-diamino-4-hexenoic acids (4,5-dehydrolysines) were synthesized and kinetic parameters for the hydrolysis of benzoyl methyl esters and phenylthiazolones of these analogs by this enzyme were compared with those of the corresponding lysine derivatives. The derivatives of cis-4,5-dehydrolysine were hydrolyzed much more slowly than those of lysine, owing largely to the small kcat values for the former. On the other hand, the derivatives of the trans-isomer were hydrolyzed at about the same rates as those of lysine and the values of both Km and kcat of the former are also similar to those of the latter. These results indicate that the conformation of the side chain of the lysine derivatives hydrolyzed by trypsin is such that the beta- and epsilon-carbons are in a trans-like conformation, as suggested by X-ray crystallographic studies of inhibitor-trypsin complex.

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.

A new beta-naphthylamide substrate of p-guanidino-L-phenylalanine for trypsin and related enzymes.

N alpha-Benzyloxycarbonyl-p-guanidino-L-phenylalanine beta-naphthylamide (Z-GPA-beta NA) was synthesized and the susceptibility of this compound to trypsin and related enzymes was compared with that of N alpha-benzyloxycarbonyl-L-arginine beta-naphthylamide (Z-Arg-beta NA). Both Z-GPA-beta NA and Z-Arg-beta NA were rapidly and almost completely hydrolyzed by trypsin and pronase. Z-Arg-beta NA was hydrolyzed slowly by thrombin, while Z-GPA-beta NA was not susceptible to this enzyme at all. The rate of hydrolysis of Z-GPA-beta NA by papain was slower than that of Z-Arg-beta NA. Neither beta-naphthylamide substrate was hydrolyzed by alpha-chymotrypsin. The specificity constant (kcat/Km) for the hydrolysis of Z-GPA-beta NA by trypsin was somewhat larger than that for the hydrolysis of Z-Arg-beta NA. Contributions of the benzene ring in the side chain of Z-GPA-beta NA to good binding of this substrate to the specificity site of this enzyme and to the poor fit of the scissile bond in the substrate molecule to the active serine residue are presumed from comparison of the individual kinetic parameters (Km and kcat) for the two beta-naphthylamide substrates. Z-GPA-beta NA was ascertained to be a useful substrate in the study of the binding and catalytic specificities of various trypsin-like enzymes.

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.

4,4'-Bis[8-(phenylamino)naphthalene-1-sulfonate] binding to human thrombins: a sensitive exo site fluorescent affinity probe.

The binding of the fluorescent probe 4,4'-bis[8-(phenylamino)naphthalene-1-sulfonate] (bis-ANS) to human alpha- and gamma-thrombins was investigated. Bis-ANS binds in a 1:1 complex to both forms of the enzyme, with Kd = 14.8 +/- 2.2 microM and 5.8 +/- 1.0 microM for alpha- and gamma-thrombin, respectively, at pH 7.0 [25 mM tris(hydroxymethyl)aminomethane, 0.15 M NaC1]. Fluorescence changes upon complexation included a considerable (approximately 30-nm) blue shift in the fluorescence emission maximum as well as a dramatic increase in the fluorescence emission intensity: a 70-fold enhancement was observed with alpha-thrombin vs. a approximately 220-fold enhancement with gamma-thrombin. Proflavin was not displaced upon bis-ANS binding. The unknown thrombin effectors ATP, Ca(II)ATP, Co(III)ATP, phosphate, and pyrophosphate bound with enhancement of the fluorescence of the bis-ANS-alpha-thrombin complex. The two inhibitors benzamidine and p-chlorobenzylamine as well as heparin caused decreases in bis-ANS-thrombin fluorescence: valerylamidine had no effect on the fluorescence of the bis-ANS-thrombin complex. Kinetic measurements with two chromogenic substrates, S-2238 and S-2160, indicated that bis-ANS acts as a partial noncompetitive inhibitor of thrombin amidase activity. The kinetic evidence combined with the ligand binding results suggests that bis-ANS does not overlap the catalytic site. The fluorophore ANS complexed with equal affinity to both alpha- and gamma-thrombins (Kd = 24 +/- 4 microM); however, the gamma-thrombin-ANS complex emission at 470 nm was enhanced 26% more than that for the alpha form.

Peritoneal macrophages of guinea pig possibly lack LTC4 synthetase.

Peritoneal cells and adherent cells of mice and rats synthesized LTC4 and LTB4 when stimulated with A23187 in vitro. On the other hand, neither peritoneal cells nor adherent cells of guinea pigs generated LTC4, D4, and E4, but did the lower amounts of LTB4. Only generation of LTB4 was potentiated by simultaneous addition of 10 microM A.A. in this species. Enzyme solutions which were extracted from peritoneal cells of these three species were capable of converting DNCB to a colored product in the presence of glutathione and then these potencies were in the following order; guinea pig greater than mouse greater than rat. On the other hand, the potencies of converting LTA4 to LTC4 in the presence of glutathione were in the following order; mouse greater than rat much greater than guinea pig approximately equal to 0. These results suggest that macrophages of guinea pigs lack "LTC4 synthetase" and also this enzyme is different from usual GSH S-transferases.

Kinetics of hydrolysis of amide and anilide substrates of p-guanidino-L-phenylalanine by bovine and porcine trypsins.

The rates of hydrolysis of N alpha-benzoyl-p-guanidino-L-phenylalaninamide (Bz-GPA-NH2) and N alpha-substituted p-nitroanilides (pNA) of GPA (benzyloxycarbonyl(Z)-GPA-pNA, benzoyl(Bz)-GPA-pNA and acetyl(Ac)-GPA-pNA) by bovine and porcine trypsins were compared with those of arginine (Arg) substrates. The amide type substrates of GPA were hydrolyzed as fast as those of Arg by the two enzymes with much the same kcat/Km values, though significant differences were found between the kcat and Km values of GPA derivatives and those of Arg derivatives. The kinetic behavior of porcine trypsin toward GPA substrates was almost the same as that of the bovine enzyme. The ratio of the kcat value for Bz-GPA-OEt to that for Bz-GPA-NH2 was much larger than that for the ester to amide substrates of arginine, suggesting that the conformational change of the active site of trypsin induced by a benzene ring in the side chain of Bz-GPA-OEt specifically increases the velocity of the deacylation process of the ester substrate. Remarkably low values of both kcat and Km were found for the tryptic hydrolysis of Z-GPA-pNA and Ac-GPA-pNA, as well as on that of Bz-GPA-pNA (Tsunematsu, H., et al. (1983) J. Biochem. 94, 123-128). Z-GPA-pNA is the best substrate for the two trypsins among the three N alpha-substituted anilide substrates of GPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and evaluation of 19F labeled sulfonyl fluorides as probes of protease structure: alpha-chymotrypsin.

Active site Ser-195-fluorine-labeled derivatives of alpha-chymotrypsin were prepared from a series of N-(trifluoromethylphenyl)-fluorosulfonylphenyl carboxamides whose synthesis is described. The six new 19F spin labels varied in the position of the -CF3 substituent (o-, m-, and p-) and the fluorosulfonyl substituent (m- or p-). The chemical shifts of these covalently bound analogs of "tosyl-chymotrypsin" were each uniquely sensitive to their environment in the catalytic center as evidenced by differences in resonance line position for each label. Upon titrating these derivatives with the reversible competitive inhibitor, indole, a downfield shift was observed (with all but one label), which could be fit in each case to an apparent dissociation constant for indole binding. Indole binding to the p-sulfonyl derivatives was essentially unaltered from that for the native enzyme, while the m-sulfonyl derivatives required some additional free energy of binding to saturate the enzyme. The results are consistent with a partial embedding of the phenylsulfonyl moiety in the aromatic specificity pocket.

Hydrolysis of phenylthiazolones of p-guanidinophenylalanine and arginine by trypsin and related enzymes.

The phenylthiazolone (PTA) of p-guanidinophenylalanine (GPA) was synthesized and the susceptibility of this compound to trypsin and related enzymes was compared with that of the PTA of arginine (Arg). Both PTA-GPA and PTA-Arg were almost completely and rapidly hydrolyzed by trypsin and pronase. PTA-Arg was hydrolyzed rapidly by thrombin, whereas PTA-GPA was less susceptible to this enzyme. The rates of hydrolysis of the two PTAs by alpha-chymotrypsin and papain were fairly slow. The specificity constant (kcat/Km) for the hydrolysis of PTA-GPA by trypsin was about 4 times larger than that of PTA-Arg. Thus, PTA-GPA, as well as PTA-Arg, behaves as a specific internal thioester substrate for trypsin and is the best one in the series of PTA substrates examined so far. However, PTA derivatives of GPA and Arg were hydrolyzed with kcat/Km values smaller than those of N alpha-benzoyl ethyl esters of L-GPA and L-Arg by factors of 4 and 17, respectively.

Kinetics of hydrolysis of Na-benzoyl-p-guanidino-L-phenylalanine p-nitroanilide by trypsin.

A new chromogenic substrate, Na-benzoyl-p-guanidino-L-phenylalanine p-nitroanilide (Bz-GPA-pNA), was synthesized. This compound is a good substrate for bovine trypsin (Km = 1.56 X 10(-5) M, kcat = 0.081 s -1, at pH 8.2) and was hydrolyzed as fast as Na-benzoyl-L-arginine p-nitroanilide (Bz-Arg-pNA) with much the same kcat/Km values. But the values are two orders of magnitude smaller than those for ester substrates, Na-benzoyl-p-guanidino-L-phenylalanine ethyl ester (Bz-GPA-OEt) and Na-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt). Substrate activation behavior was observed on tryptic hydrolysis of this new substrate in a substrate concentration range higher than about 5.0 X 10(-4) M in analogy with the trypsin-Bz-Arg-pNA system.

Interactions of derivatives of guanidinophenylglycine and guanidinophenylalanine with trypsin and related enzymes.

Ethyl N-benzoyl-p- and m-guanidino-DL-phenylglycinates (DL-Bz-p-GPG-OEt and DL-Bz-m-GPG-OEt), and ethyl N-benzoyl-p-guanidino-L- and D-phenylalaninates (L-Bz-p-GPA-OEt and D-Bz-p-GPA-OEt) were synthesized. The ester of the racemic p-guanidinophenylglycine derivative was completely hydrolyzed by trypsin, pronase, alpha-chymotrypsin, and thrombin, though hydrolysis by the latter two enzymes was much slower. Papain hydrolyzed this ester substrate stereospecifically at a moderate rate and left the ester derivative of the D-enantiomer unaltered. Optical resolution of DL-Bz-p-GPG-OEt with papain gave N-benzoyl-p-guanidino-L-phenylglycine (L-Bz-p-GPG-OH) and the ester of the D-enantiomer of this amino acid derivative. On the other hand, DL-Bz-m-GPG-OEt was completely hydrolyzed by pronase and was stereospecifically hydrolyzed by papain, but was unaffected by trypsin, alpha-chymotrypsin, and thrombin. The trypsin-catalyzed hydrolysis of N alpha-benzoyl-L-arginine p-nitroanilide (L-Bz-Arg-pNA) was inhibited competitively by this ester. The specificity constant (kcat/Km) for L-Bz-p-GPG-OEt was about 57 times smaller than that for a specific ester substrate, ethyl N alpha-benzoyl-L-argininate (LO-Bz-Arg-OEt), while the value for the D-enantiomer of the former is about 14 times larger than that for the D-enantiomer of the latter. L-Bz-p-GPA-OEt has a specificity constant comparable to that for L-Bz-Arg-OEt. The value for the former is about 51 times larger than that for L-Bz-p-GPG-OEt. This suggests that the existence of the beta-methylene group in L-Bz-p-GPA-OEt is important in relation to the higher susceptibility of the ester to trypsin-catalyzed hydrolysis. In contrast with the L-enantiomer, D-Bz-p-GPA-OEt was found to be as competitive inhibitor for the hydrolysis of L-Bz-Arg-pNA. A significant difference was found between the stereospecificities of hydrolysis of the ester substrates of the two amino acid derivatives by trypsin.

Kinetics of hydrolysis of phenylthiazolones of arginine, homoarginine, norarginine, and canaavanine by trypsin.

Phenylthiazolones (PTAs) of arginine and its homologs and analogs, homoarginine, norarginine (alpha-amino-gamma-guanidinobutyric acid), canavanine, and gamma-hydroxyarginine, were prepared. A steady-state kinetic analysis of the trypsin [EC 3.4.21.4]-catalyzed hydrolysis reactions was carried out and the kinetic parameters for these internal thioesters were compared with those for normal linear ester substrates. PTA-gamma-hydroxyarginine was so labile that hydrolysis by the enzyme could not be followed. PTA-arginine has a specificity constant (Kcat/Km) comparable to that for the Nalpha-unblocked arginine ester substrate, though the value is about 0.1% of that for a specific ester substrate, Nalpha-tosylarginine methyl ester. PTA derivatives of canavanine and homoarginine were hydrolyzed with Kcat/Km walues of the same order of magnitude as that for PTA-arginine. However, PTA-noraginine was much less susceptible to tryptic hydrolysis that PTA-homoarginine, while the linear esters of norarginine are known to be more susceptible than those of homoarginine.