An in-gel digestion method of chymotrypsin to improve sequence coverage of membrane protein by mass spectrometry / 生物工程学报

In recent years, mass spectrometry has been widely used to study membrane protein structure and function. However, the application of mass spectrometry to study integral membrane protein is limited because there are many hydrophobic amino acids in the trans-membrane domain of integral membrane protein to cause low sequence coverage detected by LC-MS/MS. Therefore, we used vitamin K epoxide reductase (VKORC1), a human integral membrane protein, as a model to optimize the digestion conditions of chymotrypsin, and developed an in-gel digestion method of chymotrypsin to improve sequence coverage of membrane protein by mass spectrometry. By exploring the effects of calcium concentration, pH value and buffer system on the percentage of sequence coverage, number of total detected and types of unique peptide, and the size of unique peptide, sequence coverage and peptide diversity could be considered under condition of Tris-HCl buffer with 5-10 mmol/L calcium ion concentration and pH value 8.0-8.5. This method could make the sequence coverage of membrane protein to reach more than 80%. It could be widely used in the study of membrane protein structure and function, identification of interaction site between membrane proteins, and identification of binding site between membrane protein and small molecular drug.

The Effect of Bortezomib on Expression of Inflammatory Cytokines and Survival in a Murine Sepsis Model Induced by Cecal Ligation and Puncture

PURPOSE: Although the proteasome inhibitor known as bortezomib can modulate the inflammatory process through the nuclear factor-kappa B signaling pathway, the immunomodulatory effect of pre-incubated bortezomib has not been fully evaluated for inflammation by infectious agents. Therefore, we evaluated the effect of bortezomib on the expression of inflammatory cytokines and mediators in macrophage cell lines and on survival in a murine peritonitis sepsis model. MATERIALS AND METHODS: Bortezomib was applied 1 hr before lipopolysaccharide (LPS) stimulation in RAW 264.7 cells. The cecal ligation and puncture (CLP) experiments were performed in C57BL/6J mice. RESULTS: Pre-incubation with bortezomib (25 nM or 50 nM) prior to LPS (50 ng/mL or 100 ng/mL) stimulation significantly recovered the number of viable RAW 264.7 cells compared to those samples without pre-incubation. Bortezomib decreased various inflammatory cytokines as well as nitric oxide production in LPS-stimulated cells. The 7-day survival rate in mice that had received bortezomib at 0.01 mg/kg concentration 1 hr prior to CLP was significantly higher than in the mice that had only received a normal saline solution of 1 mL 1 hr prior to CLP. In addition, the administration of bortezomib at 0.01 mg/kg concentration 1 hr before CLP resulted in a significant decrease in inflammation of the lung parenchyma. Collectively, pretreatment with bortezomib showed an increase in the survival rate and changes in the levels of inflammatory mediators. CONCLUSION: These results support the possibility of pretreatment with bortezomib as a new therapeutic target for the treatment of overwhelming inflammation, which is a characteristic of severe sepsis.

Kazal-type serine proteinase inhibitors in the midgut of Phlebotomus papatasi

Sandflies (Diptera: Psychodidae) are important disease vectors of parasites of the genus Leishmania, as well as bacteria and viruses. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2). Analyses of expression profiles indicated that PpKzl1 and PpKzl2 transcripts are both regulated by blood-feeding in the midgut of P. papatasi and are also expressed in males, larva and pupa. We expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells) that was applied to in vitro studies to assess serine proteinase inhibition. Recombinant PpKzl2 inhibited α-chymotrypsin to 9.4% residual activity and also inhibited α-thrombin and trypsin to 33.5% and 63.9% residual activity, suggesting that native PpKzl2 is an active serine proteinase inhibitor and likely involved in regulating digestive enzymes in the midgut. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sandfly midgut. Thus, characterising serine proteinase inhibitors may provide new targets and strategies to prevent transmission of Leishmania.

Early electron transfer in cytochrome c oxidase occurs by a chymotrypsin type relay / La transferencia temprana de electrones en el citocromo c oxidasa ocurre por un relé tipo quimotripsina

Evidence suggests that when ferrocytochrome c (the substrate) reduces cytochrome c oxidase (COX), electrons from the former enter the latter via Trp-104. What is still to be determined is the method by which electrons are transferred from ferrocytochrome c to Trp-104 and the method by which electrons arriving at Trp-104 are moved on to cua, the first of the enzyme's four redox centres to be reduced. To shed light on this process, we used the computer to create and analyse an enzyme-substrate complex formed from the published structure of the two proteins. It was found that the front haem edge of ferrocytochrome c was in close proximity to Trp-104 of COX and that inclusive of Trp-104, only nine amino acid residues from COX lie along a broad channel stretching from Trp-104 to the enzyme's CuA centre. Six of the nine residues, Trp-104, Tyr-105, His-102 Trp-106, Asp-158 and Glu-198, had the ideal chemical properties and were properly aligned to facilitate electron transfer. Here we propose that the reduction of Trp-104 and the subsequent reduction of CuA occur by a hydride/hydrogen ion relay system similar to that seen at the active site of chymotrypsin.

La evidencia sugiere que cuando el ferrocitocromo C (el sustrato) reduce el citocromo c oxidasa (COX), los electrones del primero entran a este último vía Trp-104. Lo que queda aún por determinar es el método por el cual los electrones son transferidos del ferrocitocromo c al Trp-104, así como el método mediante el cual los electrones que llegan al Trp-104 son trasladados al cua, el primero de los cuatro centros de reducción-oxidación (redox) de la enzima a ser reducidos. A fin de arrojar luz sobre este proceso, usamos la computadora para crear y analizar un complejo de sustrato enzimático formado a partir de la estructura publicada de las dos proteínas. Se halló que el borde frontal del hemo del ferrocitocromo c estaba muy cercano al Trp-104 del COX, incluyendo el Trp-104. Sólo nueve residuos de aminoácidos de COX se encuentran a lo largo de un ancho canal que se extiende desde Trp-104 hasta el centro CuA de la enzima. Seis de los nueve residuos, Trp-104, Tyr-105, His-102 Trp-106, Asp-158 y Glu-198, poseían las propiedades químicas ideales y estaban alineados adecuadamente para facilitar la transferencia de electrones. En este trabajo proponemos que la reducción de Trp-104 y la subsiguiente reducción de CuA ocurre mediante un sistema de relé iónico hidrógeno-hidruro, similar al que se observa en el sitio activo de la quimotripsina.

Kinetics of alpha-chymotrypsin catalyzed hydrolysis of 4-nitrophenyl acetate in ethanolamine surfactants.

The kinetics of alpha-chymotrypsin (alpha-CT) catalyzed hydrolysis of 4-nitrophenyl acetate has been studied in aqueous solution of alkyldimethylethanolammonium bromide (cetyl, dodecyl, decyl) surfactants at concentrations below and above their critical micelle concentration. From Michaelis-Mcnten kinetics, the catalytic rate constant kcat and the Michaelis constant KM have been determined. The bell-shaped profiles of alpha-CT activity with increasing surfactant concentrations indicate the interaction between the micelle-bound enzyme and substrate.

Enhancement of catalytic activity of enzymes by heating in anhydrous organic solvents: 3D structure of a modified serine proteinase at high resolution.

For the first time, it is demonstrated that exposure of an enzyme to anhydrous organic solvents at optimized high temperature enhances its catalytic power through local changes at the binding region. Six enzymes, namely, proteinase K, wheat germ acid phosphatase, alpha-amylase, beta-glucosidase, chymotrypsin and trypsin were exposed to acetonitrile at 70 degrees C for three hr. The activities of these enzymes were found to be considerably enhanced. In order to understand the basis of this change in the activity of these enzymes, proteinase K was analyzed in detail using X-ray diffraction method. The overall structure of the enzyme was found to be similar to the native structure in aqueous environment. The hydrogen bonding system of the catalytic triad remained intact after the treatment. However, the water structure in the substrate binding site underwent some rearrangement as some of the water molecules were either displaced or completely absent. The most striking observation concerning the water structure was the complete deletion of the water molecule which occupied the position at the so-called oxyanion hole in the active site of the native enzyme. Three acetonitrile molecules were found in the present structure. All the acetonitrile molecules were located in the recognition site. Interlinked through water molecules, the sites occupied by acetonitrile molecules were independent of water molecules. The acetonitrile molecules are involved in extensive interactions with the protein atoms. The methyl group of one of the acetonitrile molecules (CCN1) interacts simultaneously with the hydrophobic side chains of Leu 96, Ile 107 and Leu 133. The development of such a hydrophobic environment at the recognition site introduced a striking conformation change in Ile 107 by rotating its side chain about C alpha-C beta bond by 180 degrees to bring about the delta-methyl group within the range of attractive van der Waals interactions with the methyl group of CCN1. A similar change had earlier been observed in proteinase K when it was complexed to a substrate analogue, lactoferrin fragment.

Serum butyrylcholinesterase of non-human primate shows amine sensitive aryl acyl amidase and metallopeptidase activities and characteristics similar to those of the human serum enzyme.

Butyrylcholinesterase (BChE) was purified from monkey serum and the catalytic activities were examined. The enzyme has a molecular mass of approximately equal to 74 kDa as seen by SDS-gel electrophoresis. Monkey serum BChE also exhibits an amine sensitive aryl acylamidase (AAA) and a metallocarboxypeptidase activity. The tyramine activation of the aryl acylamidase activity and the metal chelator inhibition of the peptidase activity were characteristics similar to those of the human enzyme. Studies on 65Zn2+ binding and zinc chelate Sepharose chromatography showed that monkey serum BChE and human serum BChE have similar characteristics. Limited alpha chymotrypsin digestion of monkey serum BChE followed by Sephadex gel chromatography cleaved the enzyme into a 36 kDa fragment exhibiting peptidase activity. However the 20 kDa fragment corresponding to cholinesterase and aryl acylamidase activity was not detectable possibly due to the unstable nature of the fragment. Immunological studies showed that a polyclonal antibody against human serum BChE cross reacted with monkey serum BChE. The identical nature of the catalytic activities of human serum BChE and monkey serum BChE supports the postulate that all three catalytic activities co-exist in the same enzyme. This is the first time that purification and characterisation of the monkey serum BChE which has the highest sequence identity and immunological identity with that of human serum BChE, is being reported.

Partial purification and characterization of a novel murine factor that augments the expression of class I MHC antigens on tumor cells

A soluble factor which augments the expression of major histocompatibility complex class I (MHC I) antigens on a number of murine tumor cell lines, has been isolated from the culture supernatants of mixed lymphocyte reaction of spleen cells derived from C57B1/6, Balb/c and Swiss mice. The factor, termed MHC-augmenting factor (MHC-AF) has been partially purified by Sephadex G-100 column chromatography and reverse phase HPLC. MHC-AF activity is associated with an 18 kDa molecule. MHC-AF activity was resistant to pH 2.0 treatment and partially purified MHC-AF preparations did not have any activity in L929 cell/vesicular stomatitis virus (VSV) interferon bioassay system. Antibodies to IFN-gamma did not block the activity of MHC-AF. These results indicate that a MHC-AF distinct from IFN-gamma, is produced by mouse spleen cells undergoing a mixed lymphocyte reaction.

Colonic proteinase: increased activity in patients in patients with ulcerative colitis

In order to study the colonic intraluminal proteinase-antiproteinase imbalance under inflammatory conditions, we determined proteolytic activity (PA), alpha-1-antitrypsin and the activities of trypsin, chymotrypsin and neutrophil elastase in feces from patients with ulcerative colitis (UC) comparing the results with a control group. A fecal sample was obtained from each of 25 patients with ulcerative colitis and 10 control subjects were studied. The severity of the disease was assessed by the Truelove index. Proteolytic activity was measured lesing azocasein as proteolytic substrate. The fecal concentration of alpha-1-antitrypsin was measured by radial immunodiffusion and the activities of the enzymes were measured using specific substrates. We found an increase in fecal PA, alpha-1-antitrypsin and neutrophil elastase in patients with UC and the correlation between the severity of the disease and the PA was statistically significant (r = 0.62, P < 0.05). We conclude that elevated colonic proteinase activity could contribute to the pathophysiology of ulcerative colitis.

Plasmodium falciparum invades human red cells via a parasite produced glycosidase.

This report describes that P. falciparum produces a neuraminidase like activity on invasion into erythrocytes in culture on the basis of biochemical and immunological investigations. This activity in turn modifies the surface glycoprotein receptors of red cells and may be of help in the inhibition of further invasion by merozoites. The characterization of this enzyme activity may help elucidate the mechanism of cerebral malaria.

The nature of the unhydrolysed fraction of alpha-globulin, the major protein component of Sesamum indicum L. hydrolysed by alpha-chymotrypsin.

Alpha-globulin, the high molecular weight protein fraction from sesame (Sesamum indicum L.) seed, was hydrolysed by alpha-chymotrypsin. The hydrolysate was resolved into two fractions, the hydrolysed part and the unhydrolysed part of alpha-globulin using gel filtration on Sepharose 6B-100. The unhydrolysed alpha-globulin residue was characterized for its sedimentation coefficient, subunit composition, fluorescence emission spectrum, secondary structure, and other biophysical properties. The results indicated a decrease in the size of the protein molecule upon hydrolysis to a very small extent. The effect of hydrolysis products on hydrolysis of native alpha-globulin as well as on a standard substrate, casein, was also investigated. The results indicated that the hydrolysis products contribute to the resistance of alpha-globulin to proteolysis by alpha-chymotrypsin to the extent of 40%.

Further studies on proteinases and alpha 2-macroglobulin activity in diabetic plasma.

Loss of chymotrypsin binding capacity of alpha 2-macroglobulin in diabetic plasma on in vitro incubation, could be partially prevented by phenylmethyl sulphonyl fluoride and pepstatin A. Prior ten-fold dilution of plasma with 0.02 M phosphate buffer (pH 7.0) completely arrested the process. The phenomenon could not be reactivated by Ca2+, lecithin or bovine serum albumin. Diabetic plasma, like normal plasma, exhibited maximal hydrolytic activities on H-D-Pro-Phe-Arg-p-nitroanilide, H-D-Val-Leu-Arg-p-nitroanilide and H-D-Ile-Pro-Arg-p-nitroanilide. The hydrolytic activities were not significantly diminished on incubation of plasma at 37 degrees C for 12 hr, unlike alpha 2-macroglobulin activity. On gel chromatography on Sephadex G-200, part of the proteolytic activity in diabetic plasma coeluted with alpha 2-macroglobulin in the VO region. A second activity peak (absent in normal plasma) was eluted with a Ve/V0 value of 1.40. Possible role of free proteinases in diabetic plasma in the inactivation of alpha 2-macroglobulin is discussed.

Influência do regime alimentar na evoluçäo tardia da pancreatite aguda. Estudo experimental em ratos / Influence of diet composition on the evolution of acute pancreatitis. Experimental study in rats

Com a finalidade de estudar o efeito de regimes dietéticos diversos sobre a recuperaçäo das funçöes do pâncreas exócrino pós pancreatite aguda (PA), dois lotes de ratos submetidos, durante três semanas, a dietas isoprotéicas, diferindo apenas no teor de gordura (normo e hiperlipídica), foram distribuídos em grupos controles e com pancreatite aguda moderada, induzida com taurocolato de sódio a 1%. Amostras de sangue para dosagem de amilase sérica e fragmentos de tecido pancreático, para determinaçäo de enzimas triptolíticos, amilase, proenzimas e nucleotídeos foram colhidos nos grupos controles e nos com pancreatite após um, quatro, sete e 15 dias da induçäo da doença. Os resultados foram comparados estatisticamente por ANOVA, entre grupoos sob o mesmo regime alimentar e pelo teste "t" de Student, entre grupos correspondentes, sob regimes alimentares diversos (p <0.05). Os autores verificaram que, nas condiçöes experimentais utilizadas, a pancreatite agravou-se progressivamente até o quarto dia pós-PA, independentemente do regime alimentar prévio. A recuperaçäo funcional no órgäo manifestou-se a partir do sétimo dia pelo aumento de RNA/DNA, mas foi incompleta durante o período deste estudo. No décimo quinto dia pós-PA, ocorreu a normalizaçäo de parâmetros histopatológicos e bioquímicos, para os grupos com ambas as dietas. Os autores concluíram que, nas condiçöes experimentais deste estudo, o agravamento da doença, traduzido pela capacidade de síntese do pâncreas exócrino, foi progressivo até o quarto dia e independeu do teor de gordura na dieta

Stability and kinetic behaviour of some enzymes in surfactant environment.

The interaction of alpha-chymotrypsin, invertase, alcohol dehydrogenase and alkaline phosphatase with some ionic and non-ionic surfactants, viz. sodium dodecyl sulphate, dioctyl sodium sulphosuccinate, hexadecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide and Triton X-100, has been examined by studying the effect of varying surfactant concentrations on enzyme activities as well as by determining the time-dependent inactivation and the time-independent inhibition. The kinetic parameters, Km and Vmax, for alpha-chymotrypsin-catalysed reaction in presence of sodium dodecyl sulphate were evaluated. Anionic surfactants markedly decreased enzyme activity, whereas cationic surfactants were less effective. Nonionics showed no effect. This change in enzyme activity was also dependent on the nature of enzyme.

Bethanechol-induced restricted stimulation of pancreatic juice secretion in mice

La liberación ("out-put") de enzimas y el volumen de secreción pancreática fueron estudiados "in vivo" en ratones, bajo estimulación con betanecol. La curva dosis-respuesta para ambos parámetros se ajustó a un patón bifásico, implicando que el flujo de jugo pancreático participa también en el fenómeno de estimulación restringida

Estudio in vivo de la secreción de calcio por el páncreas exocrino del raton: relación con la secreción enzimática / In vivo study of the calcium secretion by the exocrine pancreas of mice: relationship with enzymatic secretion

La secreción de calcio y su relación con la secreción de quimiotripsina (QT) por el páncreas del ratón, in vivo, fueron determinadas en jugo pancreático puro colectado bajo tres condiciones de estimulación: secretina 32 mU/gm; secretina 32 mU/mg más colecistocinina 16 mU/gm y secretina 32 mU/gm más betanecol 0.2 ug/gm. En las muestras obtenidas se determinó actividad de quimotripsina y calcio secretado en 30 minutos luego de la estimulación. La secreción de calcio y QT fue mayor en ratones tratados con secretina más CCK y secretina más betanecol. En estos dos últimos grupos, entre ambos parámetros se estableció una correlación positiva. Las rectas obtenidas no fueron diferentes de una recta única y la extrapolación al origen indica que existe calcio en el jugo pancreático aún ausencia de secreción enzimática. Los resultados obtenidos concuerdan con la existencia de al menos dos mecanismos diferentes de secreción de calcio por el páncreas exocrino del ratón