Purification, characterization, gene cloning and nucleotide sequencing of D-stereospecific amino acid amidase from soil bacterium: Delftia acidovorans

The D-amino acid amidase-producing bacterium was isolated from soil samples using an enrichment culture technique in medium broth containing D-phenylalanine amide as a sole source of nitrogen. The strain exhibiting the strongest activity was identified as Delftia acidovorans strain 16. This strain produced intracellular D-amino acid amidase constitutively. The enzyme was purified about 380-fold to homogeneity and its molecular mass was estimated to be about 50 kDa, on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was active preferentially toward D-amino acid amides rather than their L-counterparts. It exhibited strong amino acid amidase activity toward aromatic amino acid amides including D-phenylalanine amide, D-tryptophan amide and D-tyrosine amide, yet it was not specifically active toward low-molecular-weight D-amino acid amides such as D-alanine amide, L-alanine amide and L-serine amide. Moreover, it was not specifically active toward oligopeptides. The enzyme showed maximum activity at 40 degrees C and pH 8.5 and appeared to be very stable, with 92.5% remaining activity after the reaction was performed at 45 degrees C for 30 min. However, it was mostly inactivated in the presence of phenylmethanesulfonyl fluoride or Cd2+, Ag+, Zn2+, Hg2+ and As3+ . The NH2 terminal and internal amino acid sequences of the enzyme were determined; and the gene was cloned and sequenced. The enzyme gene damA encodes a 466-amino-acid protein (molecular mass 49,860.46 Da); and the deduced amino acid sequence exhibits homology to the D-amino acid amidase from Variovorax paradoxus (67.9% identity), the amidotransferase A subunit from Burkholderia fungorum (50% identity) and other enantioselective amidases.     

Neurospora crassa invertase. A study of amino acids at the active center.

1. The effects on Neurospora crassa invertase (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26) of a variety of group specific reagnets and other potential inhibitors were determined during a search for an irreversible inhibitor of the enzyme. Aniline, pyridoxal, enzyme substrate and products did not inactivate invertase under reducing conditions. Bromoacetic acid, iodoacetic acid, iodoacetamide, p-chloromercuribenzoate, hydroxylamine and 2-hydroxy-5-nitrobenzyl bromide were also ineffective. Iodine was the only reagent which irreversibly inhibited invertase. 2. Invertase was rapidly inactivated by low concentrations of iodine, indicating specific inhibition. However, the enzyme could not be protected from this inactivation by substrate. It was not reactivated by mercaptoethanol or cysteine. 3. Experiments on the uptake of radioactive iodine demonstrated that invertase is not iodinated under the conditions of iodine inactivation. 4. The sedimentation (S20,w) value of invertase was not altered by iodine inactivation. One-dimensional electrophoresis and finger-printing of tryptic digests revealed no differences between iodine treated and untreated invertase. There was no loss of carbohydrate from this glycoprotein during iodine inactivation. 5. Standard amino acid analyses of iodine-inactivated invertase showed some loss of tyrosine and a trace amount of methionine sulfone. Attempts to demonstrate oxidation of methionine to the sulfone, through modification of the procedure for preparation of samples for analysis, were unsuccessful. However, oxidation of half-cystine was indicated and further loss of tyrosine noted. A hypothesis is advanced that half-cystine is oxidized by iodine to a normally unstable oxidation state which is maintained and protected by its protein invironment and that loss of tyrosine may be an artifact caused by the presence of this residue during acid hydrolysis.     

Human plasma lecithin-cholesterol acyltransferase. An elucidation of the catalytic mechanism

Human plasma lecithin-cholesterol acyltransferase (LCAT) transacylates the sn-2 fatty acid of lecithin to cholesterol forming cholesteryl ester and lysolecithin. Measurement of the phospholipase A2 and transacylase activities of the enzyme using proteoliposome substrates and following selective chemical modification of serine, histidine, and cysteine residues of pure homogeneous LCAT indicated the following catalytic mechanism: HS-Cys-E-Ser-OH + lecithin in equilibrium HS-Cys-E-Ser-O-FA + lysolecithin, HS-Cys-E-Ser-O-FA in equilibrium FA-S-Cys-E-Ser-OH, FA-S-Cys-E-Ser-OH + cholesterol-OH in equilibrium HS-Cys-E-Ser-OH + cholesterol-O-FA, where FA denotes fatty acid. Modification of 2 LCAT cysteine residues with 5,5'-dithiobis-(2-nitrobenzoic acid) or treatment with ferricyanide inactivated the transacylase but not the phospholipase A2 activity. Modification of 1 serine residue with phenylmethanesulfonyl fluoride or 1 histidine residue with diethyl pyrocarbonate inhibited cholesteryl ester formation and phospholipase A2 activity. Proteoliposome substrates protected both activities against chemical inactivation. Lecithin alone protected the phospholipase A2 activity against phenylmethanesulfonyl fluoride inactivation but not the transacylase against 5,5'-dithiobis-(2-nitrobenzoic acid) inactivation. Incubation of native LCAT with arachidonyl-CoA or the lecithin-apo-A-I proteoliposome resulted in acylation of three enzyme sites, only one of which was stable to neutral hydroxylamine after denaturation. Fatty acylenzyme oxy- and thioesters were demonstrable in both cases. No transfer of arachidonic acid from iodoacetamide-modified LCAT to cholesterol occurred, indicating that the fatty-acylated serine residue cannot directly esterify cholesterol. Cholesterol arachidonate was formed upon incubation of phenylmethanesulfonyl fluoride-modified LCAT with arachidonyl-CoA.     

The interaction of alpha-N-(p-toluenesulphonyl)-p-guanidino-L-phenylalanine methyl ester with thrombin and trypsin.

The syntheses are described of p-guanidino-L-phenylalanine and some of its derivatives. alpha-N-(p-Toluenesulphonyl)-p-guanidino-L-phenylalanine methyl ester is an excellent substrate of bovine trypsin (EC 3.4.21.4) (Km 57 micron; kcat. 320s-1 at pH 7.4-8.0) and a very poor substrate of human thrombin (EC 3.4.21.5) (Km 190 micron, kcat. 0.2s-1) and bovine chymotrypsin (EC 3.4.21.1). The ester inhibits thrombin clotting activity. It also inhibits the amidase and esterase activities of human thrombin, this inhibition being of the mixed type. The inhibition constant, K1, of the order of 1 micron, increases with increasing inhibitor concentration. This suggests that the enzyme binds the inhibitor at multiple sites. The importance of the residue at the P1 position [notation of Berger & Schechter (1970) Philos. Trans. R. Soc. London Ser. B 257, 249-264] in determining the selectivity of a substrate or quasi-substrate among trypsin-like enzymes is borne out. p-Guanidino-L-phenylalanine may have a use in the synthesis of selective peptide inhibitors of thrombin.     

The reaction of bovine thrombin with N-butyrylimidazole. Two different reactions resulting in the inhibition of catalytic activity.

N-Butyrylimidazole has been found to be a potent inhibitor of purified bovine thrombin. The rate and extent of inhibition of thrombin by N-butyrylimidazole could be reduced by the presence of benzamidine, a competitive inhibitor, or by the ester substrate, p-tosyl-L-arginine methyl ester. Spectral studies of the reaction of N-butyrylimidazole with thrombin demonstrated the modification of approximately 1 mol of tyrosine/mol of enzyme at maximum inhibition. In addition to the reaction with tyrosine, N-butyrylimidazole also appears to react with a residue at the "active site" as judged by a decrease in the number of active sites available in the modified enzyme for titration with p-nitrophenyl-p'-guanidinobenzoate. The time course of ester hydrolysis by butyrylated thrombin showed a distinct lag phase suggesting partial reactivation of the enzyme under assay conditions. Partial reactivation of the modified enzyme also occurred spontaneously upon standing in 0.5 M NaCl but was much faster in presence of imidazole (0.03 M, pH 7.6). It is suggested that, in addition to reaction with tyrosine, there is a reaction of N-butyrylimidazole with either the histidine and/or serine residue at the active site of thrombin resulting in a derivative unstable under esterase assay conditions such as that described for the reaciton of N-acetylimidazole with trypsin (L. L. Houston and K. A. Walsh (1970), Biochemistry 9, 156).     

A microassay-based procedure for measuring low levels of toxic organophosphorus compounds through acetylcholinesterase inhibition

Using a microtiter plate spectrophotometric system, an assay procedure was developed for the following toxic organophosphorus compounds: 1,2,2-trimethylpropyl ester of methylphosphonofluoridic acid (1, soman); ethyl N,N-dimethylphosphoramidocyanidate (3, tabun); O-ethyl S-[2-[bis(1-methylethyl)amino]ethyl]- methylphosphonothiolate (4, VX); the diethyl 4-nitrophenyl ester of phosphoric acid (5, paraoxon); and bis(1-methylethyl) phosphorofluoridate (6, DFP). The procedure, based on the Ellman assay method, uses inhibition of eel acetylcholinesterase (0.01 unit per well) to carry out the determination of inhibitor concentrations for both a standard curve and the unknown samples on a single 96-well microtiter plate. On a typical plate, samples of both unknowns and standards (a minimum of six concentrations were used per standard curve) were assayed five times per sample, with three control (uninhibited) enzyme activity points included for each sample. The time required for carrying out a single plate was approx 30 min. Sensitivity for the most potent acetylcholinesterase inhibitor tested was 0.4 nM under the conditions used for a typical assay. It should be noted, however, that no attempt was made to optimize the assay procedure for sensitivity.     

The effect of the natural protein inhibitor on H+-ATPase hepatoma 22a mitochondria

The uncoupler-induced inactivation of H+-ATPase in hepatoma 22a and mouse liver mitochondria has been studied. The dependence of this process on delta microH, and pH and ATP was established. The inactivated ATPase could be reactivated at alkaline pH values in the absence of ATP. These data indicate that the inactivation is apparently caused by the natural protein inhibitor. ATP- and pH-dependent decrease of ATPase activity is also observed after Lubrol-WX disruption of mitochondria. It can be proposed that practically all ATPase molecules in hepatoma mitochondria are in a catalytically active complex with the protein inhibitor. At low delta microH this complex is inactivated via reversible pH-dependent and irreversible ATP-dependent rearrangements. The pH-dependent rearrangement of the isolated protein inhibitor from hepatoma mitochondria is also observed.     

Hydrazinopropionic acid: a new inhibitor of aminobutyrate transaminase and glutamate decarboxylase

This paper deals with the synthesis of 3-pyrazolidone and the biochemical action of hydrazinopropionic acid. The latter compound is formed upon alkaline hydrolysis of 3-pyrazolidone. Hydrazinopropionic acid was found in vitro to be a very potent inhibitor of bacterial aminobutyrate transaminase as well as of aminobutyrate transaminase and glutamate decarboxylase from mouse brain. This inhibition was shown to occur despite the presence of high concentrations of pyridoxal phosphate in the incubation media. Injections of 20 mg hydrazinopropionic acid/kg into mice resulted in complete inhibition of aminobutyrate transaminase in brain and approximately 20 per cent inactivation of glutamate decarboxylase. This inhibition could not be prevented or antagonized by administration of pyridoxine to the animals. Addition of pyridoxal phosphate to homogenates of brain from animals treated with hydrazinopropionic acid also failed to reactivate the enzymes. The tentative conclusion reached from these results is that hydrazinopropionic acid has inhibitory action because of its close similarity to GABA with respect to molecular size, structural configuration and molecular charge distribution. This can be demonstrated by comparing a Dreiding model of hydrazinopropionic acid with that representing GABA.     

Kinetics of vanadate dissociation: estimation of the rate by inhibitor inactivation

Vanadate (+5) is a potent inhibitor of a variety of ATPases including dynein ATPase. We describe a method useful for estimating the functional dissociation rate of vanadate from the active site which does not rely on classical physical separation techniques. The method involves spectrophotometrically monitoring the enzymatic activity as the inhibitor dissociates from the enzyme and is inactivated by norepinephrine. Norepinephrine effectively reverses vanadate inhibition by reducing vanadate (+5) to oxovanadium (+4). This reduction by norepinephrine is sufficiently fast for these purposes--addition of vanadate after norepinephrine shows no inhibition of ATPase activity. The mathematical estimation procedure is generally useful for estimation of dissociation rates of other reversible inhibitors which can be quickly inactivated after dissociation from the enzyme. The rate of dissociation of vanadate from dynein with ATP and 2-N3ATP as substrates using this method was estimated to be in the ranges 0.0023-0.0042 and 0.0057-0.0075 s-1, respectively. These rates permit estimation of the rates of vanadate association with dynein by using the reported dissociation constant for vanadate. The results are consistent with the very fast and potent inhibition of dynein ATPase activity observed.     

Propane and propylene formation during the microsomal metabolism of iproniazid and isopropylhydrazine

Both iproniazid and isopropylhydrazine were metabolized to the hydrocarbon products, propane and propylene, with nearly identical Michaelis constants and rates. This reaction appeared to be catalized by microsomal cytochrome P-450. Isonicotinic acid, a product of iproniazid hydrolysis by various amidases, was produced in only very small quantities, suggesting that the other amidase product, isopropylhydrazine, may not be an obligatory intermediate in the pathway of hydrocarbon formation from iproniazid. Hydrocarbon formation from iproniazid was more sensitive to inhibition $\text{in vitro}$ by $\text{bis-p}\text{-}\text{nitrophenylphosphate}$ (used $\text{in vivo}$ as an amidase inhibitor) than was isopropylhydrazine. Iproniazid must be directly metabolized by cytochrome P-450 to yield propane and propylene, presumably via an azo ester intermediate which could give rise to an isopropyl radical, the chemical species presumed to be responsible for the hepatoxicity apparent after administration of large doses of iproniazid $\text{in vivo}$.     

Design and Synthesis of Potent N-Acylethanolamine-hydrolyzing Acid Amidase (NAAA) Inhibitor as Anti-Inflammatory Compounds

N-acylethanolamine-hydrolyzing acid amidase (NAAA) is a lysosomal enzyme involved in biological deactivation of N-palmitoylethanolamide (PEA), which exerts anti-inflammatory and analgesic effects through the activation of nuclear receptor peroxisome proliferator-activated receptor-alpha (PPAR-α). To develop selective and potent NAAA inhibitors, we designed and synthesized a series of derivatives of 1-pentadecanyl-carbonyl pyrrolidine (compound 1), a general amidase inhibitor. Structure activity relationship (SAR) studies have identified a compound 16, 1-(2-Biphenyl-4-yl)ethyl-carbonyl pyrrolidine, which has shown the highest inhibition on NAAA activity (IC₅₀ = 2.12±0.41 µM) and is characterized as a reversible and competitive NAAA inhibitor. Computational docking analysis and mutagenesis study revealed that compound 16 interacted with Asparagine 209 (Asn²⁰⁹) residue flanking the catalytic pocket of NAAA so as to block the substrate entrance. In vitro pharmacological studies demonstrated that compound 16 dose-dependently reduced mRNA expression levels of iNOS and IL-6, along with an increase of intracellular PEA levels, in mouse macrophages with lipopolysaccharides (LPS) induced inflammation. Our study discovered a novel NAAA inhibitor, compound 16, that could serve as a potential anti-inflammatory agent.     

Design,synthesis, and evaluation of postulated transient intermediate and substrate analogues as inhibitors of 4-hydroxyphenylpyruvate dioxygenase

An epoxybenzoquinone, 4-hydroxyphenoxypropionic acid, and 2-hydroxy-3-phenyl-3-butenoic acid derivatives have been designed, synthesized, and evaluated for in vitro inhibition activity against 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) from pig liver by the spectrophotometric enol-borate method. The biological data demonstrated that neither epoxybenzoquinone ester nor 2-hydroxy-3-phenyl-3-butenoic acid is an inhibitor of 4-HPPD. The most potent 4-HPPD inhibitor tested was 3-hydroxy-4-phenyl-2(5H)-furanone with an IC(50) value of 0.5 microM, which may serve as a lead compound for further design of more potent 4-HPPD inhibitors.     

Two molecular forms of penicillin amidase synthesized by Escherichia coli

The Swatek's method was further simplified for the assay of penicillin amidase activity. The absorbance of colour obtained during determination of 6-aminopenicillanic acid was dependent on concentration of 4-dimethylaminobenzaldehyde and on temperature. Antiodies induced in rabbits with one molecular form of penicillin amidase from E. coli PCM 271 (PA-1 or PA-2) did not cross-react with the other amidase form. No differences in substrate specificity on inactivation with SDS and in alkaline medium between the two amidase forms were observed. Concentrated urea inactivated PA-2 irreversibly and PA-1 reversibly. N-Bromosuccinimide inactivated almost completely only PA-1. Two E. coli PCM 271 strain variants were separated by microbial selection. Each of them produced only one amidase form. Also two amidase forms were found in cells of E. coli ATCC 11105, whereas E. coli ATCC 9636 and ATCC 9637 synthesize only PA-1.     

Plant pyruvate dehydrogenase complex: Inactivation and reactivation by phosphorylation and dephosphorylation

Pyruvate dehydrogenase complex activity from spinach leaf mitochondria was inhibited up to 90% within 2 min of incubation with 1 mm ATP at 27 °C. The inhibition was time, temperature and ATP concentration dependent. The inhibition was partially prevented with 3.0 mm dichloroacetate, a known inhibitor of mammalian pyruvate dehydrogenase kinases. Optimum pH for ATP-dependent inactivation was between 8.0 and 9.0 The inactivated complex was reactivated with 10 to 20 mm MgCl₂. Complete reactivation occurs within 10 min after MgCl₂ addition. Reactivation was inhibited by fluoride, a known inhibitor of mammalian pyruvate dehydrogenase phosphatase. Optimum pH for Mg²⁺-dependent reactivation was 8.0. It is concluded that the inactivation and reactivation process of pyruvate dehydrogenase complex from spinach leaf mitochondria is due to phosphorylation and dephosphorylation.     

Target cell-directed inactivation and IL-2-dependent reactivation of LAK cells

In a previous study, we demonstrated that human natural killer cells (NK) lost their lytic activity after interaction with a sensitive target. The loss of NK activity also led to the loss of antibody-dependent cellular cytotoxicity (ADCC), prompting us to postulate that NK and ADCC activities may result from a common lytic mechanism. In this study, we examined whether nonadherent lymphocytes cultured 7 days in the presence of IL-2 (lymphokine-activated killer (LAK) cells) could also be inactivated and, subsequently, be reactivated in the presence of IL-2. We tested three populations of effector cells (EC): cells isolated from freshly drawn blood and tested immediately, cells cultured with IL-2 for 18 hr, and LAK cells. Once they have interacted with K562, all three cell populations lost greater than 90% of their NK-like lytic activity (NK-CMC) but only 80% of ADCC. However, when we treated the three cell types with antibody-coated K562, they lost 90-99% of NK-CMC and 90-97% of ADCC. In these inactivated effector cells we also observed: (i) a reduction in membrane expression of C-reactive protein; and (ii) a decrease in the expression of Leu-11a when EC were inactivated with antibody-coated K562. The loss of lytic activity against K562 was accompanied by a concomitant loss of activity against other LAK-sensitive targets as well as against antibody-coated targets (ADCC). In competitive inhibition experiments the inactivated effector cells failed to inhibit normal NK-CMC and ADCC activities mediated by fresh NK cells. As we have shown previously, this target-directed inactivation was not due to cell death or to lack of conjugate formation. Inactivated LAK cells regained their lytic potential when cultured with IL-2 and this effect was time dependent. By 72 hr, LAK cells inactivated with K562 regained 99% NK-CMC and 82% ADCC, whereas LAK cells inactivated with antibody-coated K562 regained only 80% NK-CMC and 70% ADCC. When we treated the effector cells with emetine, a potent inhibitor of protein synthesis, we could still inactivate the effector cells with K562 and with antibody-coated K562 but could not reactivate them with IL-2.     

Purification and properties of the nuclease inhibitor of Aspergillus oryzae and kinetics of its interaction with crystalline nuclease O.

A nuclease inhibitor found in the mycelia of Aspergillus oryzae has been purified 158,000-fold by ammonium sulfate precipitation, chromatography on Sephadex G-75, DEAE-Sephadex A-50 and Bio-Gel p-60 columns, preparative disc electrophoresis on acrylamide gel, and electrofocusing in ampholite. The purified inhibitor is nearly homogeneous as judged by disc electrophoresis. It shows a typical ultraviolet absorption curve for protein, and the inhibitory activity is inactivated by chymotrypsin. The inhibitor and nuclease O (EC 3.1.4.9, a crystalline enzyme from the mycelia of the same organism) form a stable enzyme inhibitor complex. The molecular weights of nuclease O, the inhibitor and the enzyme inhibitor complex are estimated to be 46,000, 22,000 and 73,000 respectively, by Sephadex G-100 gel filtration. The isoelectric points of the enzyme and the inhibitor are 10.0 and 4.09, respectively, as determined by electrofocusing in ampholite. The inhibition is noncompetitive, and the inhibitor constant (K1) is 3.2 X 10(-12) M, whereas the Michaelis constant (Km) for DNA is 2.2 X 10(-8) M. The inactive enzyme-inhibitor complex is reactivated by chymotrypsin through inactivation of the inhibitor. The reactivated enzyme can be inactivated again by the inhibitor, which shows that desensitization of the enzyme does not occur by the action of chymotrypsin.     

Structural inhibition and reactivation of Escherichia coli septation by elements of the SOS and TER pathways.

The inhibition of cell division caused by induction of the SOS pathway in Escherichia coli structurally blocks septation, as deduced from two sets of results. Potential septation sites active at the time of SOS induction became inactivated, while those initiated during the following doubling time were active. Penicillin resistance increased in wild-type UV light-irradiated cells, a behavior similar to that observed in mutants in which structural blocks were introduced by inactivation of FtsA. Potential septation sites that have been structurally blocked by either the SOS division inhibitor, furazlocillin inhibition of PBP3, or inactivation of a TER pathway component, FtsA3, could be reactivated one doubling time after removal of the inhibitory agent in the presence of an active lon gene product. Reactivation of potential septation sites blocked by the presence of an inactivated FtsA3 was significantly lower when the lon protease was not active, suggesting that Lon plays a role in the removal of inactivated TER pathway products from the blocked potential septation sites.     

The protein phosphatase inhibitor okadaic acid induces morphological changes typical of apoptosis in mammalian cells.

Okadaic acid, a specific and potent inhibitor of protein phosphatases 2A and 1, was tested for its effect on the morphology of a number of cell types: freshly isolated rat hepatocytes in suspension or in primary culture, the human mammary carcinoma cell line MCF-7, the human neuroblastoma cell line SK-N-SH, rat pituitary adenoma GH3 cells, and rat promyelocytic IPC-81 cells. All the cell types reacted within a few hours to okadaic acid in the concentration range 0.1 to 1 microM with profound morphological alterations. Among the changes noted were: condensation of chromatin, shedding of cell contents via surface bleb formation, redistribution and compacting of cytoplasmic organelles, formation of cytoplasmic vacuoles, and hyperconvolution of the nuclear membrane. In some cells nuclear fragmentation was noted. In addition, cells growing as monolayers rounded up and detached from the substratum. The treated cells had no swollen mitochondria and retained the ability to exclude trypan blue until the final stage of dissolution, supporting the hypothesis that the changes were apoptotic rather than necrotic. In hepatocytes the action of okadaic acid was mimicked by another phosphatase inhibitor, microcystin, and was accompanied by shrinkage of the cell volume, as judged by Coulter counter analysis. The action of phosphatase inhibitor was not abolished by protein synthesis inhibitors, Ca(2+)-depleted medium, or phorbol ester. Although hepatocyte DNA replication was very sensitive to inhibition by okadaic acid, DNA fragmentation was less pronounced in response to okadaic acid than other agents inducing the morphological appearance of apoptosis.     

Ferulic acid inhibits endothelial cell proliferation through NO down-regulating ERK1/2 pathway

The aim of this study was to determine the antiproliferative mechanism of ferulic acid (FA) on serum induced ECV304 cell, a human umbilical vein endothelial line. The results suggest that FA significantly suppressed ECV304 cells proliferation and blocked the cell cycle in G0/G1 phase. Treatment of the cells with FA increased nitric oxide (NO) production and inactivated the extracellular signal-regulated kinase (EERK1/2), and the NO donor, sodium nitroprusside, inhibited both ECV304 cells proliferation and phosphorylation of ERK1/2. However, the NO synthase inhibitor, Nomega-nitro-L-arginine methyl ester, caused ECV304 cells proliferation. PD 98059, the inhibitor of ERK1/2, had no effect on the NO production. These results indicate that NO suppressed ECV304 cells proliferation through down-regulating ERK1/2 pathway. Moreover, the inhibition of cell cycle progression was associated with the decrement of cyclin D1 expression and phosphorylation of retinoblastoma protein (pRb) by increment of p21 level. The findings not only present the first evidence that FA is a potent inhibitor on ECV304 cells proliferation, but also reveal the potential signaling molecules involved in its action.     

Trypsin-like hatching protease from mouse embryos: evidence for the presence in culture medium and its enzymatic properties

Enzymatic properties of a protease involved in hatching of mouse embryos were examined. A trypsin-like protease, which most efficiently hydrolyzed t-butoxycarbonyl-Leu-Ser-Thr-Arg-4-methylcoumaryl-7-amide, was demonstrated in culture medium of mouse hatching embryos. The enzyme was strongly inhibited by diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, leupeptin, antipain, N alpha-tosyl-L-lysyl-chloromethane, soybean trypsin inhibitor, and Trasylol, but not or weakly inhibited by p-chloromercuribenzoic acid, EDTA, E-64, pepstatin, chymostatin, and bestatin, suggesting a trypsin-like serine proteinase. The protease activity in the medium gradually elevated during the course of hatching, whereas the embryo-associated activity showed no significant change. Furthermore, pyroglutamyl-Leu-argininal, the strongest inhibitor for the enzyme among peptidyl argininals, all of which are potent trypsin inhibitors, showed the strongest inhibition toward hatching. Thus, a trypsin-like protease secreted from hatching embryos into the culture medium may participate in mouse hatching, probably as a hatching enzyme.