Tosyl Lysine Chloromethyl Ketone Research Articles

Purification and biochemical characterization of a fibrin(ogen)olytic metalloprotease from Macrovipera mauritanica snake venom which induces vascular permeability.

In the present study, a novel fibrin(ogen)olytic metalloprotease from Macrovipera mauritanica snake venom was purified and characterized in terms of enzyme kinetics and substrate specificity. The purified enzyme [termed snake venom metalloprotease-Macrovipera mauritanica (SVMP‑MM)] was composed of a single polypeptide with an apparent molecular weight of 27 kDa, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminus of the enzyme was composed of NH(2)-QRFAPRYIEL-COOH, as determined by N-terminal sequencing. The Aα- and the Bβ-chains of fibrinogen were completely cleaved by SVMP-MM within 20 and 480 min, respectively. However, the γ-chain was much more resistant to digestion by the enzyme. The enzyme also exhibited proteolytic activity, cleaving the α-α polymer of cross-linked fibrin, but did not effectively digest the γ-γ polymer. To determine the kinetic parameters for SVMP-MM, a fluorescence-quenching peptide (termed o-aminobenzoic acid-HTEKLVTS-2,4-dinitrophenyl‑NH(2)) containing a K-L sequence for SVMP-MM cleavage was designed and synthesized. The optimal pH and temperature for the enzyme activity were found to be 5.5 and 37˚C, respectively, when the fluorogenic substrate was synthesized and used as a substrate. Among the various divalent cations tested, Ni(2+) and Cu(2+) showed strong inhibitory effects on enzyme activity, with an average of 69.6% inhibition. The enzyme activity was also inhibited by treatment with 1,10-phenanthroline, ethylenediaminetetraacetic acid and glycol-bis-(2‑aminoethylether)-N,N,N',N'-tetra-acetic acid, but not with aprotinin, tosyl-lysine chloromethyl ketone and tosyl-phenylalanyl chloromethyl ketone, suggesting that SVMP-MM is a metalloprotease and not a serine protease. The enzymatic parameters, including the K(M), k(cat), and k(cat)/K(M) values were estimated to be 0.015 mM, 0.031 sec(-1), and 20.67 mM(-1)sec(-1), respectively. SVMP-MM induced vascular permeability by digesting type IV collagen. The results obtained in our study demonstrate that SVMP-MM is a fibrin(ogen)olytic P-I class metalloprotease, which can induce a hemorrhagic reaction in vivo.

Purification and partial characterization of a chymotrypsin-like serine fibrinolytic enzyme from Bacillus amyloliquefaciens FCF-11 using corn husk as a novel substrate

A non-toxic, direct-acting fibrinolytic enzyme, FCF-11, from a newly isolated Bacillus amyloliquefaciens FCF-11 was purified, characterized and assayed both in vitro and in vivo for its thrombolytic potential. Corn husk was used as for the first time as the sole carbon/nitrogen source for enzyme production. The molecular weight of the purified enzyme was 18.2 kDa and purification increased its specific activity 443.5-fold with a recovery of 17 %. Maximal activity was attained at a temperature of 40 °C and pH of 8.0. Additionally the isoelectric point of this protein was 10 ± 0.2. Tosyl lysine chloromethyl ketone, phenylmethylsulphonyl fluoride, soybean trypsin inhibitor, and aprotinin highly repressed this activity. The presence of ethylenediaminetetraacetic acid, and two metalloprotease inhibitors, 2,2'-bipyridine and o-phenanthroline, didn't affect the enzymatic activity. Furthermore, it was found to exhibit a higher specificity for the chromogenic substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like serine protease. Its apparent K(m) and V(max) for the synthetic substrate N-Suc-Phe-pNA were 0.45 mM and 8.26 μmoles/mg/min, respectively. FCF-11 showed direct action upon blood clots in vitro and prolonged the blood clotting time to 4.1-fold, suggesting this enzyme be a beneficial thrombolytic agent especially, with regard with low molecular weight and non specificity to other plasma proteins. FCF-11 could not degrade collagen and was non-cytotoxic to HT29 cells or mammalian erythrocytes. Further, enzyme at a dose of 2 mg/kg was devoid of toxicity as well as hemorrhagic activity on BALB/c mouse model, supporting its suitability for the development of a better and safer thrombolytic drug.

Purification of a Trypsin-Like Enzyme and Cloning of Its Gene from Chinese Ground Beetle (E upolyphaga sinensis )

An insect trypsin-like enzyme with similar biochemical properties to psychrophilic analogs was purified from Chinese ground beetle (Eupolyphaga sinensis) for the first time. The purified trypsin-like enzyme was designated as E. sinensis trypsin-like enzyme (ESTL) with molecular weight of 22.8 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The maximal activity of ESTL was observed at pH 9.5, and the temperature optimum of ESTL was observed at 45C by its amidolytic effect on the substrate benzoyl-L-Arg-p-nitroanilide. The analysis of inhibitors showed that specific inhibitor of serine proteases (phenylmethanesulfonyl fluoride) and trypsin inhibitors (tosyl–lysine chloromethyl ketone, bovine pancreatic trypsin inhibitor and benzamidine) inactivated ESTL almost completely. Over the range of tested temperature of 10–30C, the catalytic efficiency (kcat/Km) of the ESTL was about five times than that of bovine trypsin. The N-terminal sequencing of ESTL revealed the following sequence: I1VGGSTTTIQ10NFPYQVSL, and the complementary DNA (cDNA) of ESTL was cloned by rapid amplification of cDNA ends. Practical Applications In recent years, increasing attention has been drawn to the psychrophilic (cold-adapted) enzymes. Psychrophilic trypsins with high activity may be interesting for several industrial applications of enzymes, such as in certain food-processing operations that require low-processing temperatures. For example, cod trypsin is already used in food production and cosmetics. In this article, a trypsin-like enzyme [Eupolyphaga sinensis trypsin-like enzyme (ESTL) ] was purified from Chinese ground beetle by a relatively simple method, and the complementary DNA (cDNA) of this enzyme was cloned by rapid amplification of cDNA ends. This enzyme was characterized for molecular and enzymatic properties, as demonstrated that it had high activity at low and moderate temperature compared with the mammalian trypsins, and maintained good thermal stability compared with psychrophilic trypsins. ESTL has potential interest in food industry.

A Novel Hatching Enzyme from Starfish Asterias amurensis: Purification, Characterization, and Cleavage Specificity

Hatching enzyme (HE) is of importance to degrade egg membrane to let the larvae be free. HE was purified and characterized from starfish blastula. The specific activity and the purification ratio of the purified HE with 110.9kDa of molecular weight were 449.62U/mg and 7.42-fold, respectively. Its optimal pH and temperature for activity were pH 8.0 and 30°C, respectively. This enzyme was relatively stable in the range of pH 4.0-6.0 and 30-40°C. This enzyme was inhibited by ethylene diamine tetraacetic acid (EDTA) and ethylene glycol tetraacetic acid, and also done moderately by Leupeptin, tosyl-lysine chloromethyl ketone, tosyl-phenylalanine chloromethyl ketone, and phenyl-methanesulfonyl fluoride. Zn(2+) ion activated HE activity strongly and recovered the EDTA-pretreated activity more than did Ca(2+), Mg(2+), and Cu(2+). Based on the results above, the starfish HE was classified as a zinc metallo- and trypsin-like serine protease. The values of Km, Vmax, and Kcat of the starfish HE on dimethyl casein were 0.31mg/ml, 0.17U/ml, and 122.70s(-1), respectively, whereas 1.09mg/ml, 0.12U/ml, and 771.98s(-1) on type I collagen. Therefore, the starfish HE could be a potential cosmeceutical because of its strong cleavage specificity on type I collagen.

Characterization of Acidic Protease from Aspergillus niger BCRC 32720

An acid protease from the broth of a 24 h cultivated Aspergillus niger BCRC 32720 was purified to electrophoretical homogeneity by CM Sepharose FF and Sephacryl S-100 HR chromatographs. The specific activity, purification fold, and yield were 23.29 kU/mg, 2.5, and 24.2%, respectively. Molecular mass (M) and N-terminal amino acid sequence were 47.5 kDa and SKGSAVTT, whereas the pH and temperature optima were at 2.5 and 50 °C, respectively. It was stable at pH 2.0-4.0 or ≤40 °C and activated by Fe(2+) and cysteine, but partially inhibited by phenylmethanesulfonyl fluoride and tosyllysine chloromethyl ketone and highly inhibited by Ag(+), Sn(2+), Fe(3+), Sb(3+), and pepstatin A. It was considered to be an aspartic protease.

Purification and Characterization of a Trypsin-Like Protease from Flatfish (Paralichthys olivaceus) Intestine

A trypsin-like protease was purified from the intestine of flatfish (Paralichthys olivaceus) by gel filtration and anion-exchange chromatography. The molecular weight was estimated to be 29.6 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Flatfish protease had maximal activity at 70C and pH 7.5 using N-α-benzoyl-dl-arginine-ρ-nitroanilide as substrate. It was stable to heat treatment up to 50C and to pH ranges between 7.0 and 10.0. It was activated by calcium ion and completely inhibited by mercury ion and known serine-protease inhibitors, such as phenylmethylsulfonyl fluoride, tosyl lysine chloromethyl ketone and benzamidine. Flatfish trypsin could be an alternative to commercial trypsins in a wide variety of applications in the food, detergent and pharmaceutical industries. In the food industry, trypsin is used to produce protein hydrolysates, extract flavor, tenderize meat and improve oil extractions. It is also used in detergent, bating of leather and in diabetes diagnosis and therapy. Moreover, it can break down tumors and cancer cells in cancer therapy.

Partial Purification and Characterization of Fibrinolytic Enzymes from Yellow Mealworm

Two proteins with fibrinolytic activity were partially purified from yellow mealworm (Tenebrio molitor) by ammonium sulfate precipitation between 30 and 70% saturation, gel filtration on Sephacryl-S200-HR, ion exchange chromatography on DEAE-Sepharose-FF and metal chelate on Cu–HiTrap–IMAC–FF, but the enzymes had not been completely separated from each other. The two partially purified fibrinolytic enzymes were designated as TMFE-I and TMFE-II (Tenebrio molitor fibrinolytic enzyme) with molecular weights of 27.5 and 24.9 kDa by SDS-PAGE individually. The partially purified solution of TMFE-I and TMFE-II was considerably stable in the range of pH 5–10 and characterized by pH optimum of the enzymatic activity at 8.0. Thermal stability of TMFE was excellent at 45°C and below. The K M value was 0.26 mM for amidolysis of Bz–Arg–pNA. According to inhibitor analysis by fibrin plate method, phenylmethylsulfonyl fluoride and tosyl-lysine chloromethyl ketone inactivated TMFE almost completely, but trans-(epoxysuccinyl)-l-leucylamino-4-guanidinobutane (E-64) and EDTA had little effect on their fibrinolytic activity. According to metal ion analysis by fibrin plate method, the effect of metal ions on activity of TMFE showed a great difference. Na+, K+ and Zn2+ had little effect on the activity of TMFE. Mg2+ and Cu2+ showed inhibition effect on the fibrinolytic activity of TMFE, but Ca2+ increased the fibrinolytic activity of TMFE at final concentration varying from 0 to 30 mM.

Studies on activity, distribution, and zymogram of protease, α-amylase, and lipase in the paddlefish Polyodon spathula

A series of biochemical determination and electrophoretic observations have been conducted to analyze the activities and characteristics of protease, α-amylase, and lipase of paddlefish Polyodon spathula. The results obtained have been compared with those of bighead carp (Aristichthys nobilis) and hybrid sturgeon (Huso dauricus ♀×Acipenser schrenki Brandt ♂), in order to increase available knowledge of the physiological characteristics of this sturgeon species and to gain information with regard to its nutrition. Further, a comparative study of enzymatic activity, distribution, and characterization between commercial feed-reared paddlefish (CG) and natural live food-reared (NG) paddlefish was conducted. Results showed that higher proteolytic activity was observed in the pH range 2.5-3.0 and at a pH of 7.0 for paddlefish. Levels of acid protease activity of paddlefish were similar to that of hybrid sturgeon, and significantly higher than that of bighead carp. The inhibition assay of paddlefish showed that the rate of inhibition of tosyl-phenylalanine chloromethyl ketone was approximately 2.6-fold that of tosyl-lysine chloromethyl ketone. There was no significant difference observed for acid protease activity between PG and CG groups, whereas the activity of alkaline protease, α-amylase, and lipase in the PG group were significantly lower than those in the CG group. The substrate sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis further showed that there were certain types of enzymes, especially α-amylase, with similar molecular mass in the paddlefish and hybrid sturgeon. It can be inferred that acid digestion was main mechanism for protein hydrolysis in paddlefish, as reported for other fishes with a stomach. This indicates that the paddlefish requires higher alkaline protease, α-amylase, and lipase activity to digest natural live food.

Taurine Biosynthesis by Neurons and Astrocytes

The physiological roles of taurine, a product of cysteine degradation and one of the most abundant amino acids in the body, remain elusive. Taurine deficiency leads to heart dysfunction, brain development abnormalities, retinal degradation, and other pathologies. The taurine synthetic pathway is proposed to be incomplete in astrocytes and neurons, and metabolic cooperation between these cell types is reportedly needed to complete the pathway. In this study, we analyzed taurine synthesis capability as reported by incorporation of radioactivity from [(35)S]cysteine into taurine, in primary murine astrocytes and neurons, and in several transformed cell lines (human (SH-SY5Y) and murine (N1E-115) neuroblastoma, human astrocytoma (U-87 MG and 1321 N1), and rat glioma (C6)). Extensive incorporation of radioactivity from [(35)S]cysteine into taurine was observed in rat glioma cells as well as in primary mouse astrocytes and neurons, establishing the presence of an intact taurine synthesis pathway in these cells. Interestingly, exposure of cells to cysteine or cysteamine resulted in elevated intracellular hypotaurine without a corresponding increase in taurine levels, suggesting that oxidation of hypotaurine limits taurine synthesis in cells. Consistent with its role as an organic osmolyte, taurine synthesis was stimulated under hypertonic conditions in neurons.

Trypsin from the Processing Waste of the Lane Snapper (Lutjanus synagris) and Its Compatibility with Oxidants, Surfactants and Commercial Detergents

A trypsin from the viscera of the lane snapper (Lutjanus synagris) was purified by heat treatment, fractionation with ammonium sulfate and affinity chromatography. The molecular weight of the enzyme was estimated to be 28.4 kDa (SDS-PAGE). The purified enzyme was capable of hydrolyzing the specific substrate for trypsin benzoyl-arginine-p-nitroanilide (BApNA) and was inhibited by benzamidine and tosyl lysine chloromethyl ketone (TLCK), synthetic trypsin inhibitors and phenylmethylsulfonyl fluoride (PMSF), which is a serine-protease inhibitor. The enzyme exhibited maximal activity at pH 9.0 and 45 degrees C and retained 100% of the activity after incubation at the optimal temperature for 30 min. At a concentration of 10 mM, activity was slightly activated by Ca(2+) and inhibited by the following ions in decreasing order: Cd(2+) > Hg(2+) > Cu(2+) > Zn(2+) > Al(3+). The effects of Ba(2+), K(1+) and Li(1+) proved to be less intensive. Using 1% (w/v) azocasein as substrate, the enzyme revealed high resistance (60% residual activity) when incubated with 10% H(2)O(2) for 75 min. The enzyme retained more than 80% activity after 60 min in the presence of different surfactants (Tween 20, Tween 80 and sodium choleate). The alkaline protease demonstrated compatibility with commercial detergents (7 mg/mL), such as Bem-te-vi, Surf and Ala, retaining more than 50% of initial activity after 60 min at 25 degrees C and 30 min at 40 degrees C. The thermostability and compatibility of this enzyme with commercial detergents suggest a good potentiality for application in the detergent industry.

Identification and partial characterization of selected proteolytic enzymes in the digestive system of giant freshwater PrawnMacrobrachium rosenbergii(De Man) Postlarvae

Biochemical assays and substrate SDS-PAGE were conducted to partially characterize and identify various types of proteases present in the digestive tract of PL₁₅ giant freshwater prawn (Macrobrachium rosenbergii). Casein hydrolytic assay of the enzyme extracts showed major proteolytic activities at pH 3.0, 6.0 and 9.0, while assay of preincubated enzyme extracts with phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor produced a 33.17% reduction in alkaline protease activity. When specific inhibitors tosyl-lysine chloromethyl ketone and tosyl-phenylalanine chloromethyl ketone were used, they resulted in a reduction in activity of proteases in the enzyme extracts by 82.41% and 55.03%, respectively, confirming the presence of trypsin and chymotrypsin, while ethylenediamine tetraacetic acid produced protease activity reduction in 33.92% showing the presence of metalloproteases in the digestive tract of the prawn. Further characterization of the alkaline proteases using SDS-PAGE technique, after incubating the extract in the presence or absence of specific inhibitors, produced six bands corresponding to molecular masses of between 13.48 and 136.1 kDa; two trypsin bands of 13.48 and 36.4 kDa, three chymotrypsin bands in the range of 23.0-73.4 kDa and one for metalloprotease of 136.1 kDa, all of which were identified from a zymogram. This study suggests that protein digestion in M. rosenbergii is initiated by an acid protease followed by a combination of action of alkaline proteases: trypsin, chymotrypsin and metalloproteases.

Up-regulation of Nicotinic Receptors by Nicotine Varies with Receptor Subtype

Recent evidence suggests that in addition to alpha4beta2 and alpha3-containing nicotinic receptors, alpha6-containing receptors are present in midbrain dopaminergic neurons and involved in the nicotine reward pathway. Using heterologous expression, we found that alpha6beta2, like alpha3beta2 and alpha4beta2 receptors, formed high affinity epibatidine binding complexes that are pentameric, trafficked to the cell surface, and produced acetylcholine-evoked currents. Chronic nicotine exposure up-regulated alpha6beta2 receptors with differences in up-regulation time course and concentration dependence compared with alpha4beta2 receptors, the predominant high affinity nicotine binding site in brain. The alpha6beta2 receptor up-regulation required higher nicotine concentrations than for alpha4beta2 but lower than for alpha3beta2 receptors. The alpha6beta2 up-regulation occurred 10-fold faster than for alpha4beta2 and slightly faster than for alpha3beta2. Our data suggest that nicotinic receptor up-regulation is subtype-specific such that alpha6-containing receptors up-regulate in response to transient, high nicotine exposures, whereas sustained, low nicotine exposures up-regulate alpha4beta2 receptors.

CKAP4/p63 Is a Receptor for the Frizzled-8 Protein-related Antiproliferative Factor from Interstitial Cystitis Patients

Antiproliferative factor (APF) is a low molecular weight sialoglycopeptide that is secreted by bladder cells from interstitial cystitis patients and is a potent inhibitor of both normal bladder epithelial and bladder carcinoma cell proliferation. We hypothesized that APF may produce its antiproliferative effects by binding to a transmembrane receptor. This study demonstrates that cytoskeleton-associated protein 4/p63 (CKAP4/p63), a type II transmembrane receptor, binds with high affinity to APF. The antiproliferative activity of APF is effectively inhibited by preincubation with anti-CKAP4/p63-specific antibodies, as well as by short interfering RNA knockdown of CKAP4/p63. Immunofluorescent confocal microscopy showed co-localization of anti-CKAP4/p63 and rhodamine-labeled synthetic APF binding in both cell membrane and perinuclear areas. APF also inhibits the proliferation of HeLa cervical carcinoma cells that are known to express CKAP4/p63. These data indicate that CKAP4/p63 is an important epithelial cell receptor for APF.

Staphylococcus aureus Operates Protein-tyrosine Phosphorylation through a Specific Mechanism

Protein phosphorylation on tyrosine has been originally characterized in animal systems and has been shown to be involved in several fundamental processes including signal transduction, growth control, and malignancy. It has been later demonstrated to occur also in a number of bacteria, and recent data suggest that it may participate in the control of bacterial pathogenicity. In this work, we provide evidence that the gram-positive human pathogen Staphylococcus aureus harbors a protein-tyrosine kinase activity. This activity is borne by a protein, termed Cap5B2, whose phosphorylating capacity is expressed only in the presence of a stimulatory protein, either Cap5A1 or Cap5A2, that enhances its affinity for the phosphoryl donor ATP. In fact, the last 27/29 amino acids of the C-terminal domain of either polypeptide are sufficient for stimulating Cap5B2 activity. The stimulation of Cap5B2 by Cap5A1 involves essentially three amino acid residues in a helix of Cap5A1 (Asp202, Glu203, and Asp205) and three residues in a helix (helix 7) of Cap5B2 (Glu190, Lys192, and Lys193), thus suggesting helix-helix interaction between these two proteins. This type of helix-helix interaction resembles the interaction required for the activation of MinD ATPase by MinE protein in the process of septum-site determination, MinD sharing sequence similarity with Cap5B2. Such activation mechanism is described here in a gram-positive bacterial tyrosine kinase, and differs from the activation mechanism previously proposed for gram-negative bacteria. Therefore, it appears that S. aureus, and possibly other gram-positive bacteria, utilizes a specific molecular mechanism for triggering protein-tyrosine kinase activity.

Do Alkylating Agents Modify the Histidine Residue of the Desensitized Butyrylcholinesterase?

Tosylphenylalanine chloromethyl ketone (TPCK) and tosyllysine chloromethyl ketone (TLCK) are irreversible modifiers of histidine which is located in the catalytic triad of chymotrypsin and trypsin, respectively. The effects of TPCK and TLCK on the histidine in the catalytic triad of the desensitized butyrylcholinesterase (BChE), prepared from human serum by heating at 45 degrees C for 24 h, were investigated in detail. It is found that these reagents do not modify, but reversibly inhibit the desensitized enzyme as a function of time. Just as it is for the native enzyme, TPCK is a hyperbolic mixed-type inhibitor of the desensitized BChE with Ki, alpha and beta values of 0.017 +/- 0.003 mM, 3.942 +/- 1.125 and 0.524 +/- 0.070, respectively. However, TLCK is the pure competitive inhibitor of the desensitized BChE with a Ki value of 0.008 +/- 0.000 mM, while it is hyperbolic mixed-type inhibitor of the native form. These findings show that the conformation of the active site cavity of desensitized BChE is different from that of the native enzyme.

Phosphatidylinositol 3-kinase in angiotensin II-induced hypertrophy of vascular smooth muscle cells

Activation of 4E-binding protein 1 (4E-BP1) by growth factors regulates protein synthesis in vascular smooth muscle cells. The interaction between G protein-coupled receptors and activated 4E-BP1 is unclear. We examined phosphadityl inositol (PI) 3-kinase in angiotensin II-induced 4E-BP1 phosphorylation in cultured rat vascular smooth muscle cells. Angiotensin II time and dose dependently stimulated phosphorylation of 4E-BP1 through the angiotensin AT 1 receptor. Pretreatment with wortmannin or 2-(4-Morpholinyl)-8-phenyl-4 H-1-benzopyran-4-one (LY294002), a PI 3-kinase inhibitor, suppressed angiotensin II-induced phosphorylation, but a mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases (ERK) kinase-1 (MEK-1) inhibitor, 2′-Amino-3′-methoxyflavone (PD98059), and a p38 MAPK inhibitor, 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1 H-imidazole (SB203580), had no effect. With regard to the involvement of mammalian target of rapamycin (mTOR) and p70 S6 kinase, angiotensin II-induced phosphorylation was abolished by pretreatment with rapamycin, but not by tosylphenylalanine chloromethyl ketone or tosyllysine chloromethyl ketone. Ca 2+ was involved, since intracellular Ca 2+ chelation inhibited angiotensin II-induced phosphorylation while a Ca 2+ ionophore, A23187, stimulated phosphorylation. Thus, angiotensin II induces the phosphorylation of 4E-BP1 via the PI 3-kinase/mTOR pathway, but not via ERK or p70 S6 kinase.

Jasmonic acid-induced necrosis and drastic decreases in ribulose-1,5-bisphosphate carboxylase/oxygenase in rice seedlings under light involves reactive oxygen species

Summary Leaves and leaf sheaths of rice ( Oryza sativa L.) seedlings responded to 100 μmol/L jasmonic acid with necrotic lesions and browning, respectively. By SDS-PAGE and immunoblot analysis, light-, dose- and time-dependent drastic reductions in ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) were found in jasmonate-treated leaves and leaf sheaths. Cycloheximide, free radical scavengers, a metal chelator, and protease and photosystem II inhibitors significantly reversed decrease in RuBisCO protein. Polypeptides were induced in light-incubated jasmonate-treated leaf sheaths. Surprisingly in dark incubated leaves only, induction was blocked by cycloheximide, indicating de novo synthesis. Photosystem II inhibitors, the metal chelators 1,10-phenanthroline and EDTA, and a protease inhibitor, tosyl-lysine chloromethyl ketone, also blocked polypaptide induction, whereas free radical scavengers were only partially effective. Results indicate that jasmonate action in rice may be mediated through inhibition or damage of photosynthesis via production of reactive oxygen species and leading to necrotic lesion formation.

Inhibition of NF-kappaB by IkappaB prevents cytokine-induced NO production and promotes enterocyte apoptosis in vitro.

Nuclear factor-kappaB (N-kappaB) plays a key role in gut inflammation. NF-kappaB up-regulates proinflammatory genes encoding cytokines, adhesion molecules, and inducible nitric oxide synthase (iNOS). However, NF-kappaB has also been shown to up-regulate protective or anti-apoptotic factors. We utilized an adenoviral vector carrying a super-repressor form of the inhibitor of NF-kappaB, IkappaB, to examine the effects of NF-kappaB inhibition on cytokine-induced nitric oxide production and apoptosis in rat small intestinal epithelial cells (IEC-6). Chemical inhibitors of NF-kappaB, including pyrrolidine dithiocarbamate (PDTC), tosyl-lysine-chloromethylketone (TLCK), genistein, and N-acetyl-leu-leu-norleucinal (n-LLnL) were also utilized. Treatment of AdIkappaB-transfected cells with cytomix [1000 U/mL IFN-gamma, 1 nM IL-1beta, and 10 ng/mL tumor necrosis factor alpha (TNFalpha)] or TNFalpha-containing cytokine combinations resulted in inhibition of cytokine-induced nitrite production and a marked increase in apoptosis compared to control cells. Apoptosis occurred independently of nitric oxide (NO) production since exogenous sources of NO did not inhibit apoptosis. Inducible NOS and clAP were down-regulated in AdIkappaB-transfected cells treated with cytomix. TLCK and LLnL treatment also induced apoptosis in cytomix-treated cells, while PDTC and genistein did not. Thus, although NF-kappaB up-regulates various pro-inflammatory genes, it may also have protective or anti-apoptotic effects in enterocytes. NF-kappaB appears necessary for upregulating cIAP in IEC-6 cells upon cytokine exposure.

PURIFICATION AND CHARACTERIZATION OF PROTEASES FROM HEPATOPANCREAS OF CRAWFISH (PROCAMBARVS CLARKII)

Four trypsin-like enzymes (CP-I, II, III and IV in order of elution on DEAE-Sepharose chromatography), purified from the hepatopancreas of crawfish, were inhibited by protease inhibitors such as phenyl methyl suifonyl fluoride (PMSF), soybean trypsin inhibitor (SBTI), aprotinin and tosyl lysine chloromethyl ketone (TICK). The molecular weights of CP-I, II, III and IV were determined to be 35.0, 41.2, 37.9 and 39.5 kDa, respectively, using sodium dodecyl sulfate polyacryl-amide gel electrophoresis (SDS-PAGE), These proteases had optimal esterase activity at pH 8.0–8.5 and showed the highest activity at 60–70C. Crawfish proteases were rich in acidic amino acids. Activation energies for hydrolysis of tosyl arginine methyl ester (TAME) by these proteases were 6.98 – 8.34 kcal/mole. Unlike other serine proteases, the activities of CP-I and CP-II were activated by mercury while CP-HI and IV were inhibited.

N-Bromoacetylethanolamine Phosphate as a Probe for the Identification of a Liver Microsomal Glucose-6-Phosphate Transporter Peptide in Rats and Ehrlich Ascites Tumor-Bearing Mice

Hepatic microsomal glucose-6-phosphatase is a multicomponent system composed of substrate/product translocases and a catalytic subunit. Previously we (Foster et al. (1996) Biochim. Biophys. Acta 12, 244–254) demonstrated that N-bromoacetylethanolamine phosphate (BAEP) is a time-dependent, irreversible inhibitor of glucose-6-phosphate hydrolysis in intact but not disrupted microsomes. We proposed that BAEP manifests its inhibitory effect by binding with a glucose-6-phosphate translocase protein of the glucose-6-phosphatase system. Here we provide additional evidence that BAEP inhibits glucose-6-phosphate transport in microsomal vesicles and utilize [32P]BAEP as an affinity label in the identification of a glucose-6-phosphate transport protein. In this study, we identify 51-kDa rat and mouse liver microsomal proteins involved in glucose-6-phosphate transport into and out of microsomal vesicles by utilizing (1) an Ehrlich ascites tumor-bearing mouse model, which displays a decreased sensitivity to the time-dependent inhibitory effect of BAEP, and (2) another glucose-6-phosphate translocase inhibitor, tosyl-lysine chloromethyl ketone, in conjunction with [32P]BAEP as an affinity label.