Tosylphenylalanine Chloromethyl Ketone Research Articles

Effects of inhibitors on the protease profiles and degradation of activated Cry toxins in larval midgut juices of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)

Midgut juice plays an important role in food digestion and detoxification in insects. In order to understand the potential of midgut juice of Cnaphalocrocis medinalis (Guenée) to degrade Bt proteins, the enzymatic activity of midgut juice and its degradation of Bt proteins (Cry2A, Cry1C, Cry1Aa, and Cry1Ac) were evaluated in this study through protease inhibitor treatments. The activities of total protease in midgut juices were significantly inhibited by phenylmethylsulfonyl fluoride (PMSF), tosyl-L-lysine chloromethyl ketone (TLCK), pepstatin A and leupeptin. The enzymatic activity of chymotrypsin was significantly inhibited by PMSF, and enzymatic activity of trypsin was significantly inhibited by ethylenediaminetetraacetic acid (EDTA), PMSF, tosyl phenylalanine chloromethyl ketone (TPCK), TLCK and trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64). EDTA could significantly inhibit the degradation of Cry2A by C. medinalis. EDTA, PMSF, TPCK, and TLCK could inhibit the degradation of Cry1C and Cry1Aa. EDTA, PMSF, TPCK, TLCK, and E-64 could inhibit the degradation of Cry1Ac. Our results indicated that some protease inhibitors hindered various enzymatic activities in the larval midgut of C. medinalis, which may reduce the insect's ability to degrade Bt toxins. These findings may aid the application of protease inhibitors in the management of this insect pest in the future.

Proteoglycans affect monomer infiltration in the etch-and-rinse bonding technique

ObjectivesTo identify proteoglycans (PGs) and collagen fibrils (CF) within human dentin by means of a dual immunofluorescent labeling technique and to investigate the monomer infiltration of two etch-and-rinse adhesives to tosyl-phenylalanine chloromethyl-ketone (TPCK)-treated trypsin (TRY)-pretreated dentin. MethodsThirty-micrometer sections of middle coronal dentin were obtained and etched with 37% phosphoric acid gel for 15s. After preconditioning with or without TRY digestion, the sections were subjected to dual immunofluorescent labeling and observed with a confocal laser scanning microscope (CLSM). Demineralized dentin matrixes treated with or without TRY were observed with field emission scanning electron microscope (FE-SEM). Two etch-and-rinse adhesives, Adper Single Bond 2 (SB) and Prime & Bond NT (PBNT), were applied to the dentin surfaces that were pretreated with or without TRY. The thickness of the hybrid layers was evaluated under confocal micro-Raman spectroscopy and analyzed with a two-way ANOVA. ResultsGreen and red fluorescence was used to represent the PGs and the CF that were colocalized in the same section with different distributions. PGs were localized in the lumens of the dentin tubules and in peritubular dentin, while the type-I collagen fibrils were localized in intertubular dentin and peritubular dentin. After preconditioning with TRY digestion, the red fluorescence decreased or disappeared, the organic filaments in the lumens of the dentin tubules disappeared, the tubules were enlarged, and the hybrid layer thickness for adhesives bonded to the TRY-pretreated dentin surfaces were significantly increased (p<0.001 for both SB and PBNT). SignificanceThe dual immunofluorescence labeling methodology can be used to study the human dentin matrix without decalcifying the entire dentin fragment. Proteoglycans were localized in the lumens of the dentin tubules and in peritubular dentin, which could depress the infiltration of the adhesive resin monomers. The use of TRY digestion increased the thickness of the hybrid layer created by the tested two-step etch-and-rinse adhesive.

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.

Regulated intramembrane proteolysis contributes to control of the osmoprotective transcription factor, NFAT5

Nuclear Factor of Activated T‐Cells 5 (NFAT5, also TonEBP or OREBP) increases transcription of osmoprotective genes in response to hypertonicity. Regulated intramembrane proteolysis (RIP) is involved in a number of intracellular and extracellular signaling pathways. The present studies were aimed at testing for a possible role of RIP in controlling NFAT5 activity. RIP is activated by sheddases and intramembrane cleaving proteases (iCLiPs). We studied the effects of inhibitors of those proteases on NFAT5 transcriptional and transactivating activities, using luciferase reporters in HEK293 cells. We screened inhibitors of sheddases and iCLiPS, namely ADAM, MMP, β‐secretase, γ‐secretase, SPP, zinc metalloprotease, and rhomboid. Zinc metalloprotease inhibitor (1, 10 phenanthroline monohydrate) and rhomboid inhibitor (tosyl phenylalanine chloromethyl ketone: TPCK) inhibit hypertonicity induced NFAT5 transcriptional and transactivating activity in a dose dependent manner. Further, zinc metalloprotease inhibitor, but not rhomboid inhibitor, reduces hypertonicity‐induced increase of NFAT5 protein abundance. We conclude that RIP contributes to hypertonicity‐induced increase of NFAT5 activity. The research is funded by The Intramural Research Program of NHLBI, NIH.

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.

High glucose promotes intracellular lipid accumulation in vascular smooth muscle cells by impairing cholesterol influx and efflux balance

High glucose promotes macrophage-derived foam cell formation involved in increased influx or reduced efflux of lipids. The aim of this study is to investigate the influence of hyperglycaemia on foam cell transformation of vascular smooth muscle cells (VSMCs) and possible mechanisms contributing to these effects. The results showed that high glucose increased the expression of CD36, a regulator of lipid influx, and suppressed the expression and activity of the adenosine triphosphate-binding cassette (ABC) transporter ABCG1, a regulator of cholesterol efflux to high-density lipoprotein, in a dose- and time-dependent manner. However, cholesterol efflux to lipid-free apoAI was not impaired. VSMCs exposed to high glucose readily developed into lipid-loaded cells, as demonstrated by Oil Red O staining and cholesterol content analysis. In addition, high glucose-induced down-regulation of ABCG1 was reversed by nuclear factor-kappaB (NF-kappaB) inhibitors BAY 11-7085 and tosyl-phenylalanine chloromethyl ketone and by the antioxidant N-acetyl-L-cysteine (NAC). This reversal was accompanied by reduced cellular lipid content. Also, NAC and NF-kappaB inhibitors can effectively block the high glucose-induced activity of NF-kappaB binding to DNA and/or peroxide production. These results suggested that hyperglycaemia-induced foam cell formation in VSMCs was related to the imbalanced lipid flux by increasing CD36-mediated modified low-density lipoprotein uptake and reducing ABCG1-regulated cellular cholesterol efflux. Moreover, this effect was associated with increased oxidative stress and activated NF-kappaB pathway signalling.

Digestive peptidases and proteinases in the midgut gland of the pink shrimpFarfantepenaeus paulensis(Crustacea, Decapoda, Penaeidae)

Proteases from the midgut gland of the Farfantepenaeus paulensis juveniles were assessed. Enzyme activity was determined using protease substrates and inhibitors. The effect of pH, temperature and calcium on proteolytic activity was assayed. Caseinolytic activity was analysed in substrate-sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Trypsin, chymotrypsin and leucine aminopeptidase activity was detected. Proteolytic activity was strongly inhibited by the specific trypsin inhibitors. Tosyl-phenylalanine chloromethyl ketone inhibited 59.3% of chymotrypsin activity. The greatest trypsin-like activity occurred at pH 8.0 and 45 °C. Chymotrypsin-like activity reached maximal values at alkaline pH (7.2–9.0) and 55 °C. CaCl2 did not increase trypsin-like activity, but rather inhibited it at concentrations of 30 (20%), 50 (30%) and 100 mM (50%). The substrate-SDS-PAGE zymogram revealed eight proteinase bands. Two possibly thermal-resistant (85 °C, 30 min) chymotrypsin isoforms were found, which were inhibited by phenyl-methyl-sulphonyl-fluoride. Aminopeptidase activity of enzyme extracts (Arg, Leu, Lys, Phe and Val) and the recommended concentrations of these essential amino acids in penaeid shrimp diets were positively correlated (P<0.05). Beause protein digestion involves the combined action of different enzymes, adequate knowledge of shrimp digestion and enzyme characteristics is required for the assessment of the digestive potential of different feed sources and development of in vitro digestibility protocols.

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.

Abstract 5523: High Glucose Promotes Intracellular Lipid Accumulation by Impairing the Balance between CD36 and ATP-Binding Cassette Transporter G1 in Vascular Smooth Muscle Cells

Objective: Foam cell formation is a characteristic of atherosclerotic lesions. It’s known that high glucose promotes macrophage-derived foam cell formation involved in increased influx or reduced efflux of lipids. The aim of this study is to investigate the influence of hyperglycemia on foam cell transformation of vascular smooth muscle cells (VSMCs) and possible mechanisms contributing to these effects. Methods and Results: The results showed that high glucose in cultured human aortic SMCs increased the mRNA and protein expressions of CD36, a regulator of lipid influx, and suppressed the mRNA and protein expressions of ATP binding cassette (ABC) transporters ABCG1, a regulator of cholesterol efflux to HDL, in a dose- and time-dependent manner. However, the ability of cholesterol efflux to lipid-free apoAI was not impaired. VSMCs exposed to high glucose were easily developed into lipid-loaded cells as demonstrated by oil red O staining. Meanwhile, it had a maximum 2.3-fold increase in accumulation of esterified cholesterol compared to VSMCs cultured in normal glucose. Additionally, there was no change found in either liver X receptor (LXR)α or LXRβ, suggesting that high glucose-induced down-regulation of ABCG1 was LXR-independent. Down-regulation of ABCG1 induced by high glucose was almost totally reversed by the NF-κB inhibitors BAY 11–7085, tosyl-phenylalanine chloromethyl-ketone (TPCK) and by the antioxidant N-acetyl-L-cysteine(NAC). This reversal was accompanied by reduced intracellular lipid content. Furthermore, we also demonstrated that high glucose enhanced the binding of nuclear proteins extracted from human VSMCs to the NF-κB regulatory elements. This effect was abrogated by NAC and NF-κB inhibitors. Conclusions: These results suggested that hyperglycemia-induced foam cell formation in VSMCs was related to the imbalanced lipid flux by increasing CD36 mediated modified LDL uptake and reducing ABCG1 regulated intracellular cholesterol efflux. Moreover, this effect was associated with activated NF-κB pathway signaling.

Inhibiting NF-κ B increases cholesterol efflux from THP-1 derived-foam cells treated with AngII via up-regulating the expression of ATP-binding cassette transporter A1

Objective To study the role of nuclear factor-kappa B (NF-κB) in cholesterol efflux from THP-1 derived-foam cells treated with Angiotensin II (Ang II). Methods Cultured THP-1derived-foam cells were treated with Ang II or preincubated with tosyl-phenylalanine chloromethyl-ketone (TPCK) NF-κB inhibitor. The levels of activated NF-κB in the cells were examined by sandwich ELISA. Cellular cholesterol content was studied by electron microscopy scanning and zymochemistry via fluorospectrophotometer and cholesterol efflux was detected by scintillation counting technique. ABCA1 mRNA and protein were quantified by RT-PCR and Western blotting. Results Addition of TPCK to the cells before Ang II stimulation attenuated the response of NF-κB p65 nuclear translocation induced by Ang II and showed no peak in foam cells group and caused a reduction in cholesterol content and an increase in cholesterol efflux by 24.1%( P < 0.05) and 41.1%( P < 0.05) respectively, when compared with Ang II group. In accordance, the ABCA1 mRNA and protein were increased by 30% and 19%( P < 0.05) respectively, when compared with Ang II group. Conclusion Ang II can down-regulate ABCA1 in THP-1 derived-foam cells via NF-κB, which leads to less cholesterol efflux and the increase of cholesterol content with the consequence of the promotion of atherosclerosis.

Treatment with tosylphenylalanine chloromethyl ketone after the onset of collagen‐induced arthritis reduces joint inflammation and NF‐κB activation

Treatment with tosylphenylalanine chloromethyl ketone after the onset of collagen‐induced arthritis reduces joint inflammation and NF‐κB activation

High glucose decreases the expression of ATP-binding cassette transporter G1 in human vascular smooth muscle cells

Objective ATP-binding cassette transporters(ABC) Al and Gl play an important role in mediating cholesterol efflux and preventing macrophage foam cell formation. In this study, we examined the regulation of ABC transporters by high glucose in human vascular smooth muscle cells(VSMCs), the other precursor of foam cells. Methods Incubation of human VSMCs with D-glucose(5 to 30 mM) for 1 to 7 days in the presence or absence of antioxidant and nuclear factor(NF)- κ B inhibitors, the expressions of ABCA1 and ABCG1 were analyzed by real time PCR and Western blotting. Results High glucose decreased ABCG1 mRNA and protein expression in cultured VSMCs, whereas the expression of ABCA1 was not significantly decreased. Down-regulation of ABCG1 mRNA expression by high glucose was abolished by antioxidant N-acetyl-L-cysteine(NAC) and NF- κ B inhibitors, BAY 11-7085 and tosyl-phenylalanine chloromethyl-ketone(TPCK). Conclusion High glucose suppresses the expression of ABCG1 in VSMCs, which is the possible mechanism of VSMC derived foam cell transformation.

Isolation and Characterization of a Novel Elastase Inhibitor, AFLEI from Aspergillus flavus

A novel elastase inhibitor from Aspergillus flavus (AFLEI) was isolated, and biochemical properties of AFLEI were examined. Column chromatography using diethylaminoethyl (DE) 52-Cellulose and Sephadex G-75 was used to purify the inhibitor. The final preparation was found to be homogeneous as indicated by a single band after disc polyacrylamide gel (PAGE) and isoelectric focusing electrophoreses. AFLEI had a molecular weight of 7,525.8 as determined by TOF-MS (time of flight mass spectrometry). The elastolytic activity of elastases from A. flavus, A. fumigatus and human leukocytes were inhibited by AFLEI. However, this activity from porcine pancreas elastase, trypsin, chymotrypsin, thrombin, and Ac1-Proteinase from snake venom was not affected by AFLEI. The fibrinogenase activity of the elastase from A. flavus was inhibited by AFLEI. AFLEI was inhibited by alpha2-macroglobulin. However, ethylenediaminetetraacetic acid (EDTA-2Na), benzamidine, chymostatin, tosyl phenylalanine chloromethyl ketone (TPCK) and dithiothreitol (DTT) did not show any inhibitory effect on the elastase inhibitory activity of AFLEI.

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.

Studies on Trypsin-modified Bovine and Human Lens Acylpeptide Hydrolase

Acylpeptide hydrolase removes the N -acetylated amino acids from the peptide substrates but not from intact proteins. Cleavage between amino acid residues 203–204 of the native acylpeptide hydrolase results in the formation of a 55kDa truncated active enzyme in the bovine lens, in vivo. In this study we explored the hydrolytic properties of the truncated enzyme using lens β- and γ-crystallins as substrates. SDS–PAGE analysis indicated that the βB2-crystallin was cleaved by truncated acylpeptide hydrolase into several protein fragments (10–26kDa). No cleavage of the γ-crystallins was observed under similar conditions. Both the acylpeptide hydrolase activity and the protease activity of the 55kDa enzyme were completely inhibited by diisopropylfluorophosphate, p -chloromercuribenzoate and ebelactone, and moderately inhibited by N -tosyl phenylalanine chloromethyl ketone. SDS–PAGE analysis followed by fluorography of (3H) diisopropylfluorophosphate labeled human lens acylpeptide hydrolase preparation showed the presence of the 55kDa truncated form of the enzyme, as observed in the bovine lens. The peptide (d)-AIKGDQFL-NH2—the amino acid sequence 200–207 of the native bovine acylpeptide hydrolase with an in vivo cleavage site of native protein—was hydrolysed by the lens protease(s) suggesting that the in vivo generation of the 55kDa acylpeptide hydrolase may be mediated through a proteolytic processing. The protease(s) responsible for the cleavage of this peptide was inhibited by diisopropylfluorophosphate and p -chloromercuribenzoate.

Chloromethyl ketones are insulin-like stimulators of lipid synthesis in locust fat body.

N alpha-tosyl-L-lysine chloromethyl ketone (TLCK) stimulates lipid synthesis in locust fat body in vitro, and is able to reverse the inhibitory effects of AKH-I on lipid synthesis. Effective stimulatory concentrations of TLCK were in the range of 0.2-1.0 mM. Similar stimulatory effects were also achieved with phenylalanine chloromethyl ketone (PheCK) and leucine chloromethyl ketone (LeuCK), but not with tosyl-phenylalanine chloromethyl ketone (TPCK), dansyl-glu-gly-arg-CK, chloroacetone, chloroacetic acid, chloroacetamide, chloroacetaldehyde, chloroacetyl-L-leucine or acetylated or fluorescamine-labelled TLCK, PheCK, and LeuCK. The level of stimulation caused by TLCK was dependent on incubation time, so that after a 5-h preincubation of fat body tissue with TLCK the stimulated rate was severalfold higher than the control. TLCK also increased the rate of uptake of trehalose and uridine, but not glucose, deoxyglucose or glycine. Increasing concentrations of bovine serum albumin (BSA) in the incubation medium caused a reduction in the rate of TLCK-stimulated acetate uptake, such that levels of uptake were no higher with 1% BSA than in the controls. A range of more specific protease and kinase inhibitors was tested, but none caused stimulation; thus the mode of action of TLCK on the stimulation of acetate uptake has yet to be identified. Elucidation of the mode of action of TLCK may facilitate the development of novel compounds for insect pest control.

Tosylphenylalanine chloromethyl ketone inhibits TNF-alpha mRNA synthesis in the presence of activated NF-kappa B in RAW 264.7 macrophages.

Serine proteinase inhibitors such as N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) were shown to inhibit production of tumour necrosis factor-alpha (TNF-alpha) in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. The proteinase inhibitors were also reported to inhibit activation of the transcription factor nuclear factor-kappa B (NF-kappa B) by blocking the signalling pathway for stimuli-induced phosphorylation of the inhibitory subunit (I kappa B alpha) and thus preventing its degradation. In RAW 264.7 cells TPCK and TLCK significantly suppressed LPS-induced increase in TNF-alpha mRNA, induction of nuclear kappa B-binding activity and degradation of I kappa B alpha. TPCK and TLCK effectively blocked TNF-alpha mRNA synthesis even when they were added after LPS stimulation. In these cells, however, the inhibitory modes of the two inhibitors were found to be different: while addition of TLCK suppressed I kappa B alpha degradation and reduced NF-kappa B activity, a comparable decrease in the nuclear kappa B-binding activity or in I kappa B alpha degradation was not observed in cells treated with TPCK. Our results show that TPCK inhibits LPS-induced TNF-alpha mRNA synthesis in the presence of activated NF-kappa B and suggests that mechanisms other than NF-kappa B activation are involved in the transcriptional regulation of the TNF-alpha gene.

Histidine Modification of Human Serum Butyrylcholinesterase

The effects of histidine-modifying reagents on human serum butyrylcholinesterase (BChE) were investigated. The commercially available enzyme was further purified by chromatography on a Sepharose CI-6B column prior to use. In the modification studies, we found that the histidine-specific reagents tosylphenylalanine chloromethyl ketone (TPCK) and tosyllysine chloromethyl ketone (TLCK) did not modify the enzyme; however, they inhibited the enzyme reversibly. The kinetic parameters of enzyme inhibition calculated were α = 10.8, β = 0.26, andKi= 0.016 mmfor TPCK. TLCK inhibition gave similar kinetic behavior, with α = 41.6, β = 0.065, andKi= 0.039 mm. Tosyllysine, an analog of TLCK, did not inhibit the enzyme. Removal of TPCK and TLCK by dialysis resulted in significant reactivation of the enzyme. From kinetic studies, it was found that the inhibitions were hyperbolic mixed-type inhibitions. We concluded that the reagents competed with substrate for hydrophobic binding sites and inhibited the enzyme reversibly. On the other hand, in the modification studies with diethyl pyrocarbonate (DPC), it was observed that inactivation of the enzyme was irreversible and time-dependent. In the protection studies, the activity of the enzyme was partially protected from inactivation by DPC even at a 50 mmconcentration of butyrylthiocholine. The results indicate that DPC modifies some essential histidine side chains in BChE, including the functional histidyl residue found at the active site.

The Serine Protease Inhibitors, Tosylphenylalanine Chloromethyl Ketone and Tosyllysine Chloromethyl Ketone, Potently Inhibit pp70s6k Activation

pp70(s6k) is a mitogen-regulated serine/threonine kinase involved in the G1 to S phase transition of the cell cycle. We have analyzed its regulation in several cell lines by a variety of agonists and have found that pp70(s6k) activation by all stimuli tested is completely blocked by the serine protease inhibitors tosylphenylalanine chloromethyl ketone (TPCK) and tosyllysine chloromethyl ketone (TLCK). TPCK inhibition of the pp70(s6k) signaling pathway resembles that of the immunosuppressant rapamycin; however, we demonstrate that their methods of inhibition differ. We find that TPCK and TLCK are not general signaling inhibitors since the activation of the mitogen-activated protein kinase pathway is not abrogated. The demonstration that these protease inhibitors prevent signaling via the pp70(s6k) pathway will help in understanding the variety of physiological processes that TPCK and TLCK have been shown to effect.